The Cleavage Mode of the Cre Recombinase

A.C. Shaikh

A thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy

Graduate Department of Molecular and Medical Genetics University of Toronto

O Copyright by A.C. Shaikh, 2000.

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A.C. Shaikh The Cleavage Mode of the Cre Recombinase Doctor of Philosophy 2000 Graduate Department of Molecular and Medical Genetics University of Toronto

Abstract

The Cre protein, encoded by the bacteriophage P 1, and the Flp protein,

encoded by the 2 p M plasmid found in budding yeast, belong to the fntegrase family

of site-specific recombinases. Cre and Flp recombinases each bind specifically to

recombination targets comprised of inverted repeats of their cognate DNA binding

sites, the lox and FRT syrnmetry elements, respectively, that flank a central 8 bp

spacer region. Like al1 integrases, both recombinases cleave one strand of the target

site through a nucleophilic attack of the scissile phosphate by a conserved catalytic

tyrosine residue. Cleavage generates a covalent attachrnent of the tyrosine to the 3'-

phosphate and a free S'-OH end. Association of two recombinase-bound target sites

that have been cleaved into a synaptic complex provides the framework for the

exchange of the crossing (cleaved) strands. The 3'-covalent linkage is subsequently

destroyed by the nucleophilic attack of the incoming %end and the DNA strands

made continuous through a recombinase-mediated Iigation that results in the

formation of a Holliday intermediate. This Holliday junction is resolved into linear

DNA products through a second set of recombinase-mediated cleavages, strand

exchanges and ligations.

An important issue in the chemistry of the recombination reaction is the

location of the recombinase molecole that provides the catalytic nucleophile in the

synapse. The mode of cleavage by a recombinase has been denoted as either

occumng in cis, where the cleaving monomer is bound adjacent to the scissile

phosphate. or in tram, where the cleaving monomer is bound elsewhere in the

synaptic cornplex. Studies of a number of integrases have shown that the LInt and

XerC/D proteins cleave in CU, while the Flp recombinase cleaves in t r m s .

1 used half-site complementation to show that Cre cleaves its lox target site in

trans. Following publication of this report, the crystal structure of the Cre synapse

showed cis-cleavage by Cre. To resolve this discrepancy and to answer whether my

use of conditionally active sites and Cre proteins biased the results in favor of tram-

cleavage. 1 constructed novel recombinases, Fre and Clp. that were functional

chimeras of the Cre and Flp proteins. 1 showed that these chimenc proteins had

altered binding specificities compared to their respective native recombinase and

designed novel specific target sites for these chimeric proteins. 1 used hybrid

recombination sites that combined the target sites of a chimeric and native

recombinase to test the mode of cleavage by Cre and Flp in conditions that did not

exclude either cis- or tram-cleavage from occumng. 1 found that consistent with

previous reports, Cre cleaved in cis and Flp cleaved in ms .

Table of Contents

Page

Abstract

Table of Contents

List of Figures

List of Tables

List of Abbreviations

Chapter 1 General Introduction.

1. Introduction

2. Consewative Site-Specific Recombination

a) The Resolvuse/l~tvertase Family

b) The Integrme Family

3. Biological Role of Cre Recombinase

4. The Cre Recombinase

5. The fox Target Site

6. Biological Role of Flp Recombinase

7. The Flp Recombinase

ii

iv

vii

X

xi

8. The FRT Target Site

9. Steps in Cre- and Flp-Mediated Recombination

a) DNA Binding

i. Model of Cre Interaction with the lox Site

ü. Model of Flp Interaction with the FRT Site

b) DNA Bending

c) Strand Cleavage

d ) Synupsis

e ) Ligation und Strond Exchange

f) Resolution

IO. Thesis Outline 1-44

Chapter II The Cre Recombinase Cleaves the lox Site in T m .

1. Introduction

2. Materials and Methods

3. Results

4. Discussion

11-2

11-3

II- 14

11-24

Chapter III Cis- and Trans-Cleavage Modes of Cre and Flp Recombinases Using Functional Chimeras of Cre and Flp.

1. Introduction

2. Materials and Methods

3. Results

4. Discussion

Chapter IV Discussion and Future Experiments.

1. Discussion

2. Future Experiments

References

List of Figures

Figure Page

1-1. The effect of general and site specific recombination on Pl plasrnid maintenance.

1-2. Peptide maps of Cre and Flp recom binase.

1-3. The Cre recombinase.

1-4. The lox and FRT target sites.

1-5. The 2 pM plasmid of Saccharomyces cerevisiae.

1-6. Steps in Cre-mediated recom bination.

1-7. Models of binding of Cre to the lox site.

1-8. Tertiary structure mode1 of binding of Cre to the lox site.

1-9. Model of binding of P X and P l 3 peptides of Flp to the FRT site.

1-10. Mode1 of the asymmetry of bending and cleavage witbin a Cre dimer.

1-1 1 Cleavage modes between recombining sites.

1-12. Conformatioas of the eatalytic domains within a Cre dimer.

1-13. Cross-core and synaptic interfaces within a Cre synapse.

- vi i -

I l The lox sites used in this study. 11-29

11-2. Covalent attacbment of various Cre proteins to the X25 haif-site, 11-31

11-3. Rationale of the complementation test. II-33

11-4. Trans-complementation by Cre and CreHis. 11-35

11-5. Effect of unlabeled competitor on binding by Cree II-37

1 Influence of full-lox site on cleavage of half-fox site by Cre proteins.

11-7. Assembly of synaptic complexes from half-lox sites.

11-8. Assem bly of synaptic complexes from full- and half-lox sites.

111-1. Complementation tests of the mode of cleavage using hybrid target sites and cbimeric proteins.

111-2. Peptide maps of proteins used in this study and the purification of Fre and Clp.

111-3. The lox- and FRT-based substrates used in this study.

111-4. Binding of Cre- and Flp-derived proteins to the ArkZ su bstrate.

111-5. Binding of Cre- and Flp-derived proteins to the lox and FRT substrates.

111-6. Binding of Cre- and Flp-derived proteias to the ArkP su bstrate.

- viii -

111-7. Binding of Cre- and Flpderived proteins to the ArkF substrate.

111-8. Binding of Cre- and Flpderived proteins to the ArkL su bstrate.

111-9. Covalent attachment of Cre and Fre to the ArkP substrate.

111-10. Covalent attachment of Cre and Clp to the ArkF su bstrate. 111-62

111-1 1. Covalent attachment of Flp and Fre to the ArkL su bstrate. UI-64

111-12. Conformations of the catalytic domains in the Cre-Fre mixed dimeric cornplex. In-66

111-13. Conformations of the catalytic domains in the Cre-Clp mixed dimeric cornplex. 111-68

III-14. Conformations of the catalytic domains in the Flp-Fre mixed dimeric cornpiex. 111-70

List of Tables

Table

11-1. Cornparison of the activities of Cre and CreHis on the half-sites.

Page

List of Abbreviations

bp: base pair(s)

nt : nucleotide(s)

kbp : kilobase pairs

PCR : polymerase chain reaction

kDa : kiloDalton

SDS : sodium dodecyl sulphate

PAGE : polyacrylamide gel electrophoresis

BSA : bovine serum albumin

Note : The nomenclature of mutant proteins is defmed as follows: the number o f the

amino acid in the protein sequence is flanked on the lefi side by a single letter that

specifies the arnino acid present in the wild-type protein and on the right side by a

single letter that designates the amino acid introduced by mutation. For example,

Y324C means that the tyrosine normally present at position 324 in the protein has

been replaced by a cysteine.

Chapter 1

General Introduction.

The integrase family of conservative site-specific recombinases includes the

Cre protein of the bacteriophage Pl and the Flp protein of the 2 p M plasmid of

Saccharomyces cerevisiae (Blakely and Sherratt, 1996, Nunes-Düby et al., 1998).

The Cre protein resolves multimers of the Pl plasmid that &se fiom general

recombination, thereby ensuring faithfbl partition of the plasmid in the lysogenic life

cycie of the phage (Austin et al., 199 1). Flp is involved in the maintenance of the 2

pM plasmid in yeast by facilitating a plasmid amplification (Futcher, 1986).

A remarkable degree of sirnilarity exists between the Cre and Flp systems. The

compact target sites coupled with the lack of requirement for accessory DNA or

protein components make both proteins ideal for engineering controlled and accurate

rearrangernents of DNA in higher eukaryotic systems (Golic. 199 1, Sauer and

Henderson, 1990, Sadowski, 1993, 1995).

In this chapter, I begin with reviews of conservative site-specific

recombination systems. A more detailed account of the Cre and Flp systems follows

with particular emphasis on Cre since it constitutes the primary focus in Chapters II

and III of this thesis.

2. Conse ~ a t i v e Site-Specific Recombina tion

Conservative site-specific recombinases are involved in a number of biological

processes including facilitation of the stability of bacterial replicons (Austin et al.,

1 99 1 ), plasmid amplification (Futcher, l986), integration and excision of

bacteriophagr chromosomes (Landy, 1989), altemating expression of different sets of

genes (Van de Pune and Goosen, 1992) and the resolution of linked CO-integrate

structures (Shenatt, 1989, Stark et al., 1992). These recombinases are characterized

by the formation of covalent protein-DNA complexes upon DNA strand cleavage by

the recombinase. The formation of this covalent intermediate is analogous to a DNA-

topoisomerase reaction where the energy of the phosphodiester bond is conserved in

the protein-DNA linkage for a subsequent protein-mediated rejoining of DNA strands

(Sadowski, 1993). As a result, conservative site-specific recombinases require no net

input of fkee energy for activity. A conservative site-specific recombination event

involves two reciprocal strand cleavage and rejoining events which occur with no

gain or loss of nucleotides (hence, "conservative"). Unlike general recombination

processes, strand exchange by conservative site-specific recombinases requires

relatively little sequence homology between the two recombining DNAs. The

minimal target site of a conservative site-specific recombinase contains two inverted

recombinase-binding sites. These sites flank the sites of cleavage across which strand

exchange occurs. Some target sites include binding sites for accessory proteins

- 1 4 -

required for recombination (Van de Putte and Goosen, 1992, Sadowski, 1993).

Conservative site-specific recombinases are divided into two categories by

sequence homology and shared chemistry of the recombination process. The

resolvase/invertase family includes the Tn3 type resolvases and the GinRiin

invertases (Stark et al., 1992, Van de Putte and Goosen, 1992). The second family is

the integrase farnily typified by the archetypical Int protein fkom phage A and the Cre

and Flp recombinases (Landy, 1993, Sadowski, 1995).

a) The Resolvasdnvertase Family

The resolvase/invertase farnily mernben are exempli fied by the Tn3, Tn2 1,

Tn552 and the y 6 resolvases, and the Gin and Hin invertases (Stark et al., 1992).

Members of this class either resolve CO-integrate DNA structures that result from

transposition or alter the expression of genes through an inversion of a DNA segment

(Van de Pune and Goosen, 1992). The members share only 13% overall protein

sequence identity. The members of the resolvase subclass exhibit 30% identity and

invertases have a 60% sequence similarity (Stark et al., 1992, Van de Putte and

Goosen, 1992). Additiondly, the resolvases c m be divided further based on protein

sequence homology and target site consensus. The Tn3 and y6 resolvases can be

differentiated from the resolvases in Tn2 1 and Tn552 transposons (Stark et al., 1992).

- 1-5 -

The memben of these subclasses are able to fùnctionally substitute for recombinases

belonging to the same group (Stark et ai., 1992). Similarly, members of the invertase

sub-family are functionally interchangeable (Van de Putte and Goosen, 1992).

Resolvases bind to a res site that is comprised of three similar sub-sites. each

of them a specific binding site for the recombinase. Site 1 is the locus of the DNA

cross-over event and sites II and III facilitate the assembly of a protein-DNA structure

that is required for recombination (Stark et al.. 1992: Soultanas and Halford, 1995).

Invertase-mediated recombination is initiated by the binding of the invertase protein

to specific inverted repeats of DNA. This is followed by the assembly of an

"invertasorne" complex in which a recombinational enhancer DNA element in cis is

occupied by an accessory protein FIS (Factor for Inversion Stimulation) and forms a

multi-protein-DNA complex with the invertase-bound DNA (Van de Putte and

Goosen? 1992. Stark et al., 1992).

The recombinase-DNA complexes are assembled into higher order synaptic

complexes through protein-protein and protein-DNA interactions (Stark et aL , 1992).

In this synaptosome, the recombinase cleaves al1 four DNA strands through double

strand breaks in the DNA. The resulting DNA ends have a two nucleotide protrusion

at the 3'-OH end and a recessed S'-phosphate end that is covalently attached to a

-1-6-

conserved serine residue of the recombinase. This phosphoserine linkage is

subsequently ligated to the 3'-OH end of the DNA break in a protein-dependent

manner (Stark et al., 1992). Strand exchange proceeds through the two base pair

overlap region of the DNA break and is thought to be facilitated by protein-mediated

DNA-DNA interactions between the recombining sites in the higher order complex

that rotates one pair of DNA ends through t 80° (Rice and Steitz, 1994, Yang and

Steitz, 1995). Therefore, in addition to being responsible for the chemistry of the

recombination process, the recombinase acts also in the formation and stabilization of

the protein-DNA scaffold where the actual exchange of strands between two

recombining sites occurs.

Resolvases and invertases have strict topological requirements. Substrates

must be supercoiled and the target sites must be arranged on the substrate in a specific

orientation (Stark et al., 1992). Presumably, an additional function of the higher

order assembly is to discem the synapsis topology required for efficient

recombination. Consistent with their function to resolve CO-integrates, resolvases

require their sites to be in direct orientation (Stark et al., 1992). Similarly, as the

invertases catalyze an inversion of DNA segments, the target sites must be in an

inverted orientation with respect to each other (Van de Putte and Goosen, 1992).

b) The Integrase Family

The integrase family of conservative site-specific recombinases contains over

100 member (Nunes-Duby et al., 1998). The best characterized members include the

Int protein fiom phage A, the Cre protein fiom bacteriophage P l , the XerC and XerD

proteins of Escherichia coli and the Flp protein from budding yeast. Recombinases

of this type bind specifically to DNA target sites that flank a central cross-over

region. The integrase binding sites are arranged as inverted repeats around the strand

exchange region. However, the complexity of the target site varies with respect to the

organization and location of the binding sites and the inclusion of additional binding

sites for accessory proteins that are required for some integrase-mediated

recombination reactions (Sadowski, 1993, 1995).

Through protein-protein and protein-DNA interactions, integrases assemble a

higher order synaptic complex where the actual exchange of strands occurs. In

contrast to the resolvase/invertase farnily of proteins, the integrases cany out

recombination through two sequential sets of reactions that consist of protein-

mediated events of cleavage, strand exchange and ligation. The fint leads to the

formation of a Holliday intermediate and the second resolves this X-structure to give

recombinant products. The hallmark of an integrase-type reaction is a protein-

mediated cleavage event of one strand of the target site that generates a covalent

- 1-8 -

attachent of the protein to the 3'-phosphate and free S'-OH (Sadowski, 1993). The

first strand exchange event proceeds across a 6 to 8 base pair spacer region and is

driven by a nucleophilic attack of the fiee S'-OH end of one cleaved target site to the

3'-covalent linkage at the nick of the partner site. The second set of resolution

reactions also proceeds in a similar protein-dependent fashion (Guo et al., 1997,

Sadowski, 1993,1995).

Some integrases, Iike AInt and the XerC/D proteins, have a specific topology

requirement for recombination (Stark et ai., 1992, Arciszewska and Sherratt, 1995),

while others, such as Cre and Flp, do not (Sadowski, 1993). Integrases cm carry out

both inter- and intra-motecular recombination events. Recombinase-mediated intra-

molecular excision events are accomplished with directly onentated sites and

inversion of DNA segments occur when the sites are inversely orientated (Landy,

1993, Sadowski, 1993).

Members of the integrase fàmily of conservative site-specific recombinases

differ greatly in their amino acid sequence. Despite this, the recent crystal structures

of ÀInt, XerD, HP 1 and Cre proteins (Kwan et al., 1997, Hickman et al., 1997,

Subramanya et al., 1997, Guo et al., 1997, Gopaul and Van Duyne, 1999, Nunes-

Düby et al., 1998) show that the core structure of the COOH-terminal domains of

- 1-9 -

these proteins is structurally conserved. In fact, al1 integrase members contain four

absolutely conserved residues that are involved in catalyzing the breaking and

rejoining of DNA strands (Nunes-Düby et al., 1998). In Cre, these residues are

Arg173, His, 289, Arg292 and Tyr324. Each of these residues has been assigned

functions in the recombination pathway through various biochemical studies of

different integrases. Mutation of these residues in an integrase abolishes both in vivo

and in vitro recombination. Alteration of the Arg173 residue in Cre gives a cleavage

defective phenotype (Gopaul et al. , 1998). The equivalent Arg 173 position in Flp has

been found to be critical for strand ligation and is implicated in protein binding to the

target site (Friesen and Sadowski, 1992, Abremski and Hoess, 1992). Both His289

and Arg292 are important in activating the scissile bond for cleavage and ligation

(Prasad el al., 1987. Parsons et al., 1988, Zhu and Sadowski. 1995). The tyrosine

residue is the main catalytic actor involved in the strand cleavage that leads to a

covalent intermediate with the DNA (Evans et al., 1990, Chen et al., 1992). In

addition, a cornparison of the protein sequence of a number of integrase-type proteins

reveals that a small40-50 amino acid region in the COOH-terminal domains of these

proteins is somewhat conserved (about 30%, Nunes-Düby et al., 1998). Coupled

with the presence of the conserved residues in this region and the apparent structural

conservation in the COOH-terminal portions of the solved integrases, this result

emphasizes that a cornmon motif is employed in the catalytic domains of integrase

As this thesis is primarily concemed with the Cre and Flp recombinases, 1

focus on these proteins in the following sections, with particular emphasis on the Cre

system.

3. Biological Role of Cre Recombinase

Lysogenic bacteriophage P 1 exists as a circular DNA molecule and is present

in its Escherichia coli host in one or two copies per cell. Even with such a smail copy

number. c u h g of the host cells carrying the P 1 plasmid is rare (Austin et al. 198 1 ).

Wihen the P 1 plasmid is duplicated during replication, general recombination between

the newly replicated P 1 plasmid and the original P 1 plasmid may lead to a dimer of

the P 1 plasmid. This dimer plasmid cannot be equally partitioned during ce11 division

and as a result, only one daughter ce11 will contain a copy o f the Pl plasmid (Austin et

al., 198 1 ), as depicted in Figure 1- 1 a. This curing occurs in only 1 in 10 000 cells,

indicating that an efficient process ensuring effective Pl partitioning is involved.

One component of this mechanism involves the Cre-fox recombination system. As

shown in Figure 1- 1 b, dimeric P 1 DNA can be separated into single copies of P 1

DNA by the action of Cre at its target lox sites present in P 1 DNA. These can be

faithfully partitioned between daughter cells during ce11 division.

4. The Cre Recombinase

The Cre recombination system consists of the Cre protein and its cognate

target site, l m , with no ancillary DNA sequences or protein factors. The Cre gene of

the Pl plasmid encodes a 343 arnino acid protein with a predicted molecular weight

of 38.5 kDa (Abremski and Hoess, 1984). As a member of the integase farnily of

site-specific recombinases, Cre accomplishes recombination through four absolutely

conserved catalytic residues: Argl72, His289, k g 2 9 2 and Tyr324.

The Cre protein can be partially proteolyzed into two discrete peptides: a 13.5

kDa NHz-terminal peptide, Cre 13, and a 25 kDa COOH-terminal peptide, Cre25

(Hoess et al., 1990). The Cre 13 domain contains the fmt 1 1 8 arnino acid residues of

Cre, while the remainder of the protein resides in the larger COOH-terminal fragment

(Figure 1-2a). The secondary structure assignent of the Cre recombinase h m the

crystal structure of a Cre synaptic complex shows that this proteolytic division in the

protein occurs in the middle of the E helix (amino acid residues 11 1-126) (Figure 1-3)

(Guo el al., 1997). It is important to note that the division of the NH,- and COOH-

terminal domains of Cre in this assignment occun at amino acid residue 130 in the

intervening region between the E and F helices. However, the original designation of

the NH,- and COOH-terminal domains of Cre was used in developing recombinantly-

derived Cre peptides, in subsequent assays of their activity (Hoess et al., 1990, M.Sc.

- 1-12 -

Thesis, A.C. Shaikh-1997) and in the construction of the Cre and Flp chimeras

presented in Chapter III. Therefore, unless otherwise noted, the amino acid 1 18-1 19

dividing mark will be used here in denoting the two portions of the Cre protein.

Cre25 contains al1 four integrase catalytic residues. The COOH-terminal

peptide retains binding specificity for the lox target site. but is deficient in al1 other

steps of the recombination process (Hoess et al., 1990, M.Sc. Thesis, A.C. Shaikh-

1 997). The NH2-terminal domain of Cre shows no activity at al1 in biochemical

assays, but in combination with the COOH-terminal peptide, a Crel3-dependent

stimulation of Cre25 activities is observed in vitro (M.Sc. Thesis, A.C. Shaikh-1997).

The DNA sequences encoding each of the peptides of Cre have been separately

cloned and the peptides purified from a recombinant source (Hoess et al., 1990.

M.Sc. Thesis, A.C. Shaikh-1997).

5. The lox Target Site

The lox site (Figure Ma) is the cognate DNA target site for the Cre

recombinase and is present as a single copy in the Pl plasmid. The 34 base pair site

consists of two identical 13 base pair inverted repeats, the lox symmetry elements.

that are separated by an A-T nch, 8 base pair core region (Hoess and Abremski,

1984). Each symmetry element is a specific site for Cre binding and thus, a fully

- 1-13 -

occupied lox site has two monomers of Cre bound to it. Cre binding to the lux site

occurs with high affinity and is highly CO-operative (Hoess and Abremski, 1984.

Ringrose et al., 1998, Guo et al., 1997). (A more detailed description of the

interaction of Cre with its target site is presented in Section 9a : i.)

The only asymmetry in the fox site is present in the core and may be

responsible for the bias in bottom strand cleavage and exchanges observed with Cre

(Hoess et al.? 1987). Cre-mediated cleavage occurs on each strand of the core, one

base pair away from the core-symmetry element junction. Hence, the effective cross-

over region between two synapsed lox sites in a Cre-mediated recombination event is

6 base pairs (Hoess et al., 1987). Cleavage by Cre at the scissile bond generates a

covalent attachment of the conserved tyrosine to the 3'-phosphate and a fiee 5'-OH

end. Recent evidence has shown that in addition to facilitating the interaction with

the NH,-terminal region of the Cre protein, the sequence of the core region was

integral in the recombination process. The first two positions on the right side of the

lox core were essential for the generation of the Holliday intermediate in the first

strand exchange reaction, while the remaining four positions at the left side of the

core were required for the resolution step (Lee and Saito, 1998).

In vitro, Cre does not require supercoiling of the substrate, and is equally

- 1-14 -

capable of catalyzing both inversion and excision-type reactions. In addition, Cre

functions eficiently in both inter- and intra-molecular recombination events (Hoess

and Abremski, 1990, Sadowski, 1993).

6. Biological Roie of Flp Recombinase

About 100 copies of an autonomously replicating plasmid, the 2 p M plasmid.

exist in most strains of Sacchromyces cerevisioe yeast (Sadowski, 1995). While the

plasmid confers no apparent advantage to its host, it is highly stable. The 2 pM

plasmid is diagramed in Figure 1-5 and is a circular double stranded DNA of 63 18 bp

in size. Two 599 bp inverted repeats serve to divide the plasmid into two regions'

Large (L) and Small (S). A total of five known open reading fiames exist within

these two regions. Rep 1 and Raf are the products encoded by their respective genes

in L. while Rep2 and the largest coding sequence, Flp are present in S. The fifth

open reading frame has no known assigned gene product associated with it. The

ongin of replication, (Ori), contains an ARS (autonomous replicating sequence) at the

junction of one of the inverted repeats and the L region of the plasmid. This Ori

region is activated only once dunng the ce11 cycle. Two isofonns of the plasmid

exist, the A and B form. Removal of the open reading h e , Flp, or the removal of

inverted repeat sequences, prevented the switch from one isofonn to another. Further

investigation by Broach et al., (1 WU), showed that Flp-mediated recombination was

- 1-15 -

site-specific and occurred within a 65 bp region of the 599 bp inverted repeats of the

plasmid. This region was later termed the FRT site (Flp Recognition Target,

McLeod et al., 1986).

The Futcher mode1 explains the role of Flp in 2 p M plasmid amplification.

Briefly, Flp-rnediated recombination between the inverted repeats facilitates multiple

rounds of 2 FM replication fiom one firing of the Ori region in the plasmid. This

amplification generates a multimeric fom of the 2 p M plasmid which is resolved into

single copies of the plasmid by further action by the Flp recombinase (Sadowski,

1995. Futcher. 1986).

7. The Flp Recombinase

The largest open reading h e in the 2 PM plasmid is the Flp gene, which

encodes a 423 arnino acid protein with a predicted molecular weight of 45 kDa. Like

al1 integrases, Flp contains four absolutely conserved catalytic residues: Argl9 1.

His3 05. Arg308 and Tyr343. A Flp-mediated recombination event requires no

accessory proteins or CO-factors in vitro (Sadowski, 1995).

Flp can be divided into two distinct domains through a carefully regulated

digestion of the intact protein with Proteinase K (Pan et al., 199 1). A 13 kDa , NH,-

- 1-16 -

terminal peptide, P l 3, and a 32 kDa, COOH-terminal peptide, P32, are generated by

this process (Figure 1-2b). A 2 1 Da, COOH-terminai fiagrnent results fiom further

digestions of the P32 peptide. Both of these COOH-terminal fragments of Flp retain

the DNA binding specificity of the intact protein and include al1 four of the catalytic

residues (Pan et al., 199 1 ). P32 additionally has cleavage and ligation activity and

can also resolve Holliday intermediates. However, the COOH-terminal peptide of Flp

is incapable of full recombination (M.Sc. Thesis, A.C. Shaikh- 1997, Pan et al., 199 1 ).

The smaller P 13 fragment of Flp is thought to have a weak non-specific DNA

binding activity, but no other catalytic activities (M.Sc. Thesis, A.C. Shaikh-1997,

Pan and Sadowski, 1993% Panigrahi and Sadowski, 1994). Combination of Pl3 with

the COOH-terminal peptide effects a stimulation of P32-dependent activities (M.Sc.

Thesis, A.C. Shaikh-1997). The DNA sequences encoding each of the peptides of

Flp have been separately cloned and the peptides purified fiom a recombinant source

(M.Sc. Thesis, A C Shaikh- 1997).

8. The FRT Target Site

The 48 base pair Flp Recognition Target (FRT) site is present in each inverted

repeat of the 2 FM plasmid (Broach el al., 1982, Meyer-Leon et al., 1984). The 8

base pair core region is flanked by three symrnetry elements, a, b, c (Figure 1-4b).

Each element is 13 base pairs long and is the site of binding for one Flp monomer

- 1-17 -

(Andrews et al., 1987). Hence, a filled FRT site has three Flp monomers bound to it.

(A more detailed description of the interaction of Flp with its target site is presented

in Section 9a : ii.) Symmetry elements a and b are inverted repeats that are separated

by an 8 bp core region and differ fiom each other at a position one base pair away

from the core-symrnetry element junction. Elements b and c are directly oriented

with respect to each other. As element c has been shown to be dispensable in both NI

vivo and in vitro Flp-mediated reactions (Proteau et al., 1986, Sadowski, 1995). most

DNA substrates used in assays of Flp recombination lack this third symmetry element.

Hence, the minimal FRT site is analogous in both size and organization to the lox

target site of the Cre recombinase system. The FRT core region is A-T rich and the

site has two poly-pyrimidine tracts, one on each DNA strand, that radiate fiom the

core into the symmetry elements. While important for recombination? the exact role

of these tracts is not known (Sadowski, 1995). The major asyrnmetry in the FRT site

occurs in the core region and it is believed that this sequence asymmetry influences

the directionality of the recombination reaction by directing parallel alignment of two

recombining FRT sites (Huffman and Levene, 1999).

Flp-mediated recombination between two paired FRT sites commences with

concerted cleavages at the core-symmetry element junctions on both sides of the core.

A Flp cleavage event establishes a 3'-phosphotyrosine covalent linkage between the

- 1-18 - 3'- phosphoryl end of the scissile phosphodiester bond and the conserved tyrosine

residue of the cleaving protein. Flp-mediated strand exchange occurs between the 8

base pair core regions of the two recombining FRT sites. Flp is competent in both

inter- and intra-molecular recombination reactions and can perfonn both inversion

and excision reactions (Sadowski, 1995, H u h a n and Levene, 1999).

9. Steps in Cre- and Flp-Mediated Recombination

The use of purified Cre and Flp proteins in a variety of in vitro assays has been

instrumental in dissecting the entire recornbination process of these two recombinases

into distinct steps. Both Cre and Flp have been found to act similarly in some of

these steps and coupled with the shared chemistry and the similarity in their

respective target sites, a working mode1 of Cre- and Flp-mediated recombination has

evolved (Figure 1-6). The recent crystal structures of Cre-DNA complexes (Guo et

al.. 1997, 1999, Gopaul et al., 1998) also provide a structural insight to the

recombination reaction as a whole. In the following section, steps in the Cre-

mediated recombination pathway will be described with references to the Flp system

where warranted.

a) DNA Binding

Both Cre and Flp bind their cognate lox and FRT sites, respectively, in a

sequence-specific manner with hi& affinity. Flp has been observed to bind the FRT

site in an ordered fashion by occupying the b element first, then the a, and finally the

c site. The step-wise binding of Flp to the FRT site results in three distinct protein-

DNA complexes that c m be detected in non-denaturing polyacrylamide gels

(Andrews et al., 1987, Beatty and Sadowski, 1988). The high cooperativity of

binding observed between Cre monomers makes defïning the order of Cre binding to

the lox site difficult (Hoess et ai., 1984, Ringrose et al., 1998). As with Flp, the

binding of one and two Cre monomers to the site can be detected by non-denahiring

PAGE (Hoess and Abremski, 1984, Wierzbicki et al., 1987). Footprinting studies of

fülly-bound lox and FRT sites have shown that both Cre and Flp completely cover

their respective target sites (Andrews et al., 1987, Hoess et a/. , 1990). Both Cre and

Flp can generate higher order complexes with their target sites that represent the

assembly of more than one protein-bound target site. These synaptic complexes are

dependent on protein-protein and protein-DNA interactions and are essential

components of the recombination process (Amin et al., 1990, 1 99 1, Hoess et al.,

1990, Wierzbicki et al., 1987). The first crystal structure of the Cre recombinase was

of a Cre synapse (Guo et al., 1997).

- 1-20 -

A more detailed description of the interaction of Cre and Flp with their

respective cognate sites follows.

i. Mode1 of Cre Interaction with the lox Site

Examining a monomer Cre-DNA complex with the neocarzinotatin chernical

probe, Hoess and Abremski (1 984) demonstrated that the entire 13 base pairs of the

Zox symmetry element and the first 4 positions of the core region were protected by

Cre. Therefore, two Cre monomers bound to the lox site would in effect cover the

entire target site. Later hydroxy-radical footprinthg studies of Cre and Cre25 dimer

complexes showed that while Cre protected the entire site, the protection afforded by

the smaller COOH-terminal Cre peptide was limited to the syrnmetry elements alone

(Hoess et al.. 1990). The difference in the protection pattern suggested that the NHZ-

terminai region of Cre was positioned over the fox core region. 1 did footprinting

studies with a single lox element-containing substrate to compare the binding of Cre

and the Cre25 peptide (M.Sc. Thesis, A.C. Shaikh-1997). Contacts in the 4 base pair

region just proximal to the core-element junction were found to be important for

binding of intact Cre, but not the COOH-terminal peptide. This observation divided

the lox site into a 4 base pair NH,-terminal binding region, the core-proximal site, and

a 9 base pair COOH-terminal binding region, the core-distal site. The division and

organization of the iox site into discrete binding regions for the two dornains of the

- 1-2 1 -

Cre protein were analogous to the designation of similar binding elements in the FRT

site for the NH2- and COOH-terminal regions of Flp (Panigrahi and Sadowski, 1994,

see next section).

Since the substrate that 1 used contained only a single symmetry element,

which was bound by a single monomer of Cre, the differences in the positioning of

the domains of Cre suggest that conformational changes in the Cre protein are

induced by communication between adjacently bound Cre monomers that reposition

the NH,- and COOH-terminal domains of Cre over the lox site.

The initial interaction between a Cre monomer and the site involves the

placement of the NH,- and COOH-terminal domains of Cre over the core-proximal

and core-distal elements of the lox site, respectively. Protein-protein interactions

between Cre monomers bound across the core of the lox site appears to induce a

conformational change in the protein that extends the Cre25 and Crel3 domains over

the core-proximal and core regions, respectively. as suggested by the footprinting

assays done by Hoess et ai., (1990)(M.Sc. Thesis, A.C. ShaiWi- 1997). Evidence for

such an alteration in the Flp protein has also been documented (Panigrahi and

Sadowski, 1994). Furthemore, 1 have hypothesized that the stimulatory effects of

Cre 1 3 and P 13 on COOH-terminal domain activities are the result of conformational

- 1-22 -

changes in the COOH-terminal domains of Cre and Flp that are induced by the NH,-

terminal domains (M.Sc. Thesis, A C . Shaikh- 1997).

The crystal structure of the Cre synaptic complex (Guo et al., 1997) provides

support for the mode1 presented above. In the structure, contacts are observed

behveen COOH-tenninal domain residues and the entire lox symrnetry element and

the first 2 base pairs of the core region in both the major and minor grooves of the

DNA. In addition, the NH,-terminal B and D helices are arranged over both the core-

proximal and core-regions of the fox site with three direct contacts to two major

grooves of the DNA. The crystal structure illustrates drarnatic protein-protein

interactions between Cre monomers in the synapse. The NHZ-terminal A and E

helices of opposing Cre monomers bound at the same lox site or between paired lox

sites form cross-core and synaptic interfaces in the synapse. The combination of both

cross-core and synaptic interfaces generates a network of protein-protein interactions

between the NH,-termini of the monomers in the synapse. In addition, the COOH-

terminal N helix is donated between Cre monomers in the synapse to form another set

of cross-core and synaptic interfaces (Guo et al., 1997). These protein-protein

interactions may effect a conformational change in the bound Cre monomers and

reposition the NH,- and COOH-terminal domains of the protein over the target site.

The cartoon in Figure 1-7 is a composite view depicting the interaction of the NH2-

and COOH-terminal domains of Cre with the lox site. as suggested fkom the

biochemical data and the crystal structure.

Of the five helices in the NH2-terminal dornain of Cre, only the B and D

helices contact the DNA directly in the major groove. A third helix. E, comprises the

interface region between Cre monomers dong with the A helix (Guo et al., 1997).

The E helix of a cleaving monomer of Cre is positioned over the core region where it

interacts with the phosphates of the bent DNA backbone and forms a cross-core

interface. Asyrnmetry in the Cre dirner is evident with the position of the E helix of

the non-cleaving monomer, as it is organized between the synapsed lox sites in the

structure and forms a synaptic interface.

The COOH-terminal domain of Cre is comprised of nine helices and five P

strands and makes the most contacts with lox site, as would be expected for the

primaty binding determinant of the protein. The helices, P strands and bop regions

of this domain interact with the major and minor grooves of the DNA, as well as the

DNA backbone (Guo et al , 1997, 1999, Gopaul el al., 1998). The J helix makes the

only direct major groove contact of the COOH-terminal domain, between kg259 and

a guanine base one position to the lefi of the margin of the core proximal-core distal

regions. Surprisingly, most of the direct contacts with the DNA occur in the minor

- 1-24 -

groove (Guo et ai., 1997). The f3 sheet between the 1 and J helices forms three

contacts with the DNA, while the looped region between the J and K helices rnakes

one. Interestingly, an absolutely conserved lysine residue (Cao and Hayes, 1999),

Lys201 in Cre, is present in the loop region between P strands 2 and 3 and makes two

direct minor groove contacts to the positions at either side of the core-symrnetry

element junction (Guo et al., 1997). It has been postulated that this consenred lysine

is another catalytic residue (Cao and Hayes, 1999). The overall effect of the

interacting domains of Cre is that the entire lox symmetry element DNA is enveloped

by Cre in a clamp-like fashion (Figure 1-8), with additional electrostatic interactions

behveen Cre and the DNA adding to the stability of the entire protein-DNA complex

(Guo et ai., 1 997, 1 999).

ii. Mode1 of Flp Interaction with the FRT Site

The interactions of Flp with the FRT site have been studied by such methods

as nuclease footprinting, methylation protection and interference, mutational analysis

of the FRT site and UV cross-linking of Flp and its peptides to the target site. These

types of experiments showed that Flp protected the entire FRT site and contacted both

the major and minor grooves of each symmetry element (Andrews et al.. 1985, Beatty

and Sadowski, 1 988, Panigrahi et ai., 1992). At present, there is no crystallographic

data for the Flp/FRT interaction of the type which is available for the Cre/iox

- 1-25 -

interaction. Pan and Sadowski (l993a) showed that the COOH-terminal peptide P32

bound specifically to the FRT site while P l 3 exhibited only weak non-specific

binding. Cross-linking studies of the Flp peptides to a single FRT element showed

that the P32 peptide contacted the outer 9 base pairs of the symrnetry element, the

core-distal region, while the NH2-terminal domain of Flp cross-linked solely to the

core- proximal site, the fmt 4 base pairs of the symmetry element directly adjacent to

the FRT core (Panigrahi and Sadowski, 1994). Combined reactions. containing both

the P 13 and P32 peptides in the same cross-linking assays, demonstrated that P 13

stimulated cross-linking of P32 to the core-distal site and extended the P32 cross-

linking region to the include the core-proximal site as well (Panigrahi and Sadowksi,

1994). These results were extended to the mode1 shown in Figure 1-9, where the P 13

and P32 peptides separately contact their respective core-proximal and core-distal

sites in the FRT element. The interaction of the two domains of Flp induces a

conformational change in the COOH-terminal peptide that rearranges the domain

over the entire 13 bp of the syrnmetry element (Panigrahi and Sadowski, 1994, M.Sc.

Thesis, A.C. Shaikh- 1997).

Mutational analysis of the FRT site also demonstrated that positions in both

the core-proximal and core-distal regions of the FRT element were important for Flp

binding. Alterations at any of the fint three positions of the core-proximal region of

- 1-26 -

the FRT a element resulted in a drarnatic reduction of Flp binding and Flp-mediated

recornbination (Senecoff et d, 1988). In the core-distai region of the FRT element, a

change in the guanine base of the bottom strand of the FRT b element, two positions

to the lefi of the core-proximal - core-distal junction, has been shown to reduce Flp binding to the site (Senecoff et al., 1988). While the interaction behveen specific

residues of Flp and certain base positions of the FRT element have yet to be

detennined, the conserved lysine residue in Flp (Lys2231 may rnake direct contacts

with the DNA in the core-distal region of the FRT site, just as the equivalent residue

in Cre does with the lox site (Guo et al., 1997). Indeed, the sirnilar organization of

the binding elements in the lox and FRT sites and the interacting domains of Cre and

Flp coupled with the structural conservation observed in the crystal structures of the

solved integrase members, suggest that the interaction of Flp with the FRT site should

be analogous to the Cre-lox interface (Guo et al., 1997, Gopaul and Van Duyne,

1999).

b) DNA Bending

Both Cre and Flp induce bends in their target sites upon binding DNA

(Schwartz and Sadowski, 1990, Guo et al, 1997). Circular permutation experiments

were used to deduce that Flp generates three distinct bends in the FRT site (Schwartz

- 1-27 -

and Sadowski, 1990). Binding of a Flp monomer to a FRT symmetry element forms

a type 1 bend of 60'. A type II bend of greater than 144" results fiom cross-core

interactions between two Flp monomers bound to FRT symmetry elements a and b of

the same site. Finally, protein-protein interactions between Flp monomers bound to

elements b and c generate the least characterized type 111 bend (Schwartz and

Sadowski, 1989, IWO) . Flp bending of the FRT site is independent of synapsis and

through a number of elegant studies utilizing cyclic permutation gel shifl assays,

Leutke and Sadowski (1995) determined that the center of the FLP-induced type II

bend was located in the center of the FRT core region.

The various crystal structures of Cre-DNA complexes provide a definite

description of Cre-induced bending in the lox site. Upon binding the l m symmetry

element. a slight 25" bend is formed in the site that does not alter the structure of the

DNA appreciably (Guo et al., 1997, 1999). However, the entire Cre synaptic

cornplex demonstrates a severe 10ZO bend that is located in the center of the paired fox

sites and this arranges the synapsed sites into a square planar isomer of a Holliday

j unction.

A closer examination of the Cre-induced bend in the fox site reveals that the

deformation in the DNA is a result of a kink in core region generated mostly by

- 1-28 -

interactions between amino acid residues in both the m- and COOH-terminal regions of Cre and the sugar-phosphate backbone of the DNA (Guo et al., 1999).

These contacts serve to expose T-A and G-C steps in the core region to the solvent

which in mm facilitates water-mediated interactions with Cre residues that fiirther

stabilize the bent protein-DNA complex (Guo et al, 1997, 1999). In addition, the

bend in the DNA forces a compression of the minor groove and opening of the major

groove towards the center of the lox site. Within synapsed sites where cleavage has

occurred, this effects a separation of the DNA strands in the core region leaving the

5' OH DNA end of the crossing strand un-stacked and positioned for sirand uptake.

The continuous strand and the 3'-covalent linkage remain stabilized by both protein-

phosphate interactions and the interface between the Cre monomers (Guo et al., 1997,

1999).

The asymrnetry in the bend direction correlates to the asyrnmetry of cleavage

within a Cre dimer. The strand cleaved by Cre and exchanged in later stages of the

recombination reaction is at the opposite end of the core from the Cre-induced kink in

the DNA (Guo et al., 1997, 1999). That is to Say, if the kink occurs at the left side of

the Zox core region, Cre-mediated cleavage is predisposed to the occur at the scissile

bond at the right side of the core and vice versa (Figure 1-10). Hence, the asyrnmetry

in the bend location is either involved in activating the opposite cleavage site or

- 1-29 -

inactivating the adjacent scissile phosphate (or both). However, the exact

determinants in the protein-DNA complex that impose the bending directionality are

st il1 unknown (Guo et al., 1 999).

The role of bending in Cre- and Flp-mediated recombination has been inferred

h m the Crellox crystallographic structures and documented by a number of

biochemical studies. Firstly, the CO-crystal structures of Cre and lox illustrate that the

DNA strands in the crosssver region are separated to position the crossing strand for

a strand exchange event, while at the sarne time the continuous, un-cleaved strands

are fixed in the synaptic complex. Hence, the Cre-induced bend in the lox sites favors

the exchange of incoming S'-OH DNA ends. In the Flp system, DNA bending is

thought to facilitate the separation of DNA strands in the core region (Zhu and

Sadow-ski, 1998) and just as in the Cre system, the protein-induced bend may prevent

straight re-ligation of the cleaved strand and favor strand exchange.

The establishment of the type 11 bend appears to be critical for successful Flp-

mediated recombination. Flp variants defective in this type of bend and the P32

peptide which forms only a type 1 bend, are al1 defective in recombination (Leutke

and Sadowski, 1995, Schwartz and Sadowski, 1990, Kulpa et al., 1993). While less

characterized in this respect, the Cre system shows similar phenotypes with regards to

- 1-30 -

bending defects. A Cre variant, G3 14R, is the equivalent Cre protein to a known Flp

variant with a bending defect, G328R (Schwartz and Sadowski, 1989). This altered

Cre protein is defective in cleavage, synapsis and recombination, but can be

complemented in m n s by another Cre variant, Y324C. that is also defective in

cleavage, synapsis and recombination, to restore the fint two catalytic activities (A.C.

Shaikh, unpublished results). As this is the sarne phenotype observed with Flp

G328R (Dixon et al., 1995), this Cre variant may represent a putative bending-

de ficient Cre protein. Sirnilarly, because of the parallels between the COOH-terminal

domains of Cre and Flp, the inactivity of Cre25 in recombination may be because the

smaller COOH-terminal fiagrnent cannot induce the bend that is necessary to permit

recombination (Hoess et al., 1990, M-Sc. Thesis, A C Shaikh- 1997).

In addition to describing the bend generated in the target site, the crystal

structure of the Cre synapse implies a relationship between the bending activity of

Cre and the activities of cleavage and synaptic complex formation. Introduction of a

4 bp bulge on one of the strands in the core of the site is thought to bend the FRT site.

This bulge stimulates cleavage of the strand in which it resides. In fact, a bending-

and cleavage- defective Flp variant (W60S) is able to cleave the bulged substrate (Lee

et a/. , 1997). Similar experiments using bulged lox substrates and Cre have shown

similar results (Lee et al., 1997, Guo et al., 1997) indicating that bending of the DNA

- 1-3 1 -

site influences protein-mediated cleavage. While some bending defective venions of

Cre and Flp are also defective in cleavage, some bending defective Flp variants, such

as Flp TA232 and the P32 peptide, are still able to cleave (Amin and Sadowski, 1990,

MSc. Thesis, A.C. Shaikh-1997).

The Cre-induced bend in the target site occurs without cleavage of the site. as

co-crystal structures of catalytically inactive Cre variants also show a similar bending

of the DNA to that present in structures generated with wild type Cre (Guo et al.,

1999). However, a number of Cre variants that are defective in cleavage activity and

synaptic complex formation (Cre A36V, T4 1 F, G3 14R) can be complemented in

tram by a cleavage-defective, bending-competent Cre variant (Y324C) to restore

cleavage activity and synaptic complex formation (Wieabicki et al., 1987, A.C.

Shaikh. unpublished results, Guo et ab , 1999). A possible explanation is that the Cre

Y324C provides the bend in a mixed protein-DNA complex that allows the putative

bending-deficient Cre variant to cleave.

C) Strond Cleavage

The main catalytic activity of Cre and Flp is the breakage and rejoining of

DNA strands. As integrases, both proteins establish a covalent linkage between the

conserved tyrosine residue (Cre Y324, Flp Y343) of their respective COOH-terminal

- 1-32 -

domains and the 3'-phosphate at the cleavage site (Sadowski, 1995) through a

nucleophilic attack of the aromatic hydroxyl of the tyrosine residue on the DNA

backbone (Evans et al., 1990, Gronostajski and Sadowski, 1985). The covalent

intermediate is analogous to those found in topoisornerase systems as the energy of

the scissile bond is conserved for a later protein-mediated rejoining of DNA strands

(Landy, 1993, Sadowskil 1995).

A major question in integmse-mediated recombination is the location of the

cleaving monomer within a protein-DNA complex. In cis cleavage, the cleaving

monomer is bound adjacent to the scissile bond (Figure 1-1 1) and exarnples of

proteins employing this type of mechanism are the AInt and XerC proteins (Nunes-

Düby et ai.. 1994, Arciszewska and Sherratt, 1 995). In contrast, Flp-mediated

cleavage has been shown to occur in tram (Chen et ai., 1992). In this type of

cleavage, a Flp monomer bound adjacent to the cleavage site activates the scissile

bond but the catalytic tyrosine is donated by another Flp monomer bound elsewhere

in a protein-DNA complex. In principle, there are three type of trans cieavages. If

Flp donates its tyrosine to another Flp monomer bound across the core on the same

DNA site, frans-horizontal cleavage occurs. Tram-vertical or frans-diagonal

cleavage occurs when the donor Flp is bound to one partner DNA site but cleaves

another site in the synapse (Figure 1-1 1). In al1 cases, the characteristic feature is the

- 1-33 -

shared active site fonned between two Flp monornets. Some studies have suggested

that Flp uses solely a nans-horizontal mode of cleavage (Lee et al., 1994, Zhu and

Sadowski, 1995). However, it is still possible that Flp employs both cross-core and

synaptic cleavages dunng the recombination process (Qian and Cox, 1995).

There is conflicting data on the mechanism of Cre-mediated cleavage. My

data in Chapter II (Shaikh and Sadowski, 1997) support a pans mechanism of

cleavage. In direct contrat, the crystal structure of a Cre synapse shows cis cleavage

(Guo et al., 1997). To resolve these differences, 1 designed the experiments in

Chapter III. The following description refers to the cis mode of cleavage observed

for Cre as detailed in the first Cre crystd structure (Guo et al., 1997).

The crystal structure of a Cre synaptic complex demonstrated two primary

asyrnmetries between the cleaving and non-cleaving monomers that comprise the

catalytically active dimer within the synapse (Guo et aL, 1997). Firstly, the active site

of the Cre monomer that provides the catalytic tyrosine diffen fiom the active site of

the opposing non-cleaving Cre monomer. As expected, the active site consists of the

conserved integrase residues Arg173, His289, and Arg292, the cleaving nucleophile

Tyr324, and an additional residue Trp3 15. Within the cleaving monomer, the active

site is organized around the scissile phosphate with four hydrogen bonds between the

- 1-34 -

Arg 1 73. His289 and Ag292 residues and the first and second phosphate oxygen

atorns of the scissile bond. A fifth hydrogen bond interaction is also present between

the Trp3 15 amino acid and the second phosphate oxygen (Guo et al., 1997). Trp3 15

is not conserved amongst the integrases. most of which contain a histidine at this

position (Nunes-Düby et al., 1998). The histidine residue could participate in similar

hydrogen bond interactions suggesting that the CO-ordination of this interaction

between the scissile bond and the protein may be a conserved feature that ensures a

functional active site (Guo et al., 1997, Nunes-Düby et al., 1 998). These interactions

position the scissile phosphate for nucleophilic attack by the catalytic tyrosine

residue. The active site of the non-cleaving Cre monomer shows a similar

arrangement between the Arg173, k g 2 9 2 and Trp3 15 residues and the scissile

phosphate. In contrast, both His289 and the M helix that contains the catalytic

tyrosine residue are shified away fiom the scissile bond. Furthemore, the catalytic

tyrosine is hydrogen-bonded to the oxygen atom of the upstrearn phosphate group and

thus prevented from attacking the scissile phosphate (Guo et al., 1997).

The asymmetry exhibited in the active sites of the cleaving and non-cleaving

monomers insures that only one scissile bond in the lox site is cleaved. While the

Cre-induced asymrnetric bend in the fox site hints at a possible method by which Cre

selects one cleavage site over the other for activation of the scissile bond, the exact

- 1-35 -

mechanism by which the bend asymrnetry directs the asymmetry in the active sites of

two Cre monomee is unclear (Guo et al., 1997, 1999).

The most dramatic difference between the cleaving and non-cleaving

monomers of Cre is the arrangement of the L, M and N helices of the COOH-terminal

domains of the Cre monomers in the active dimer. The cleaving Cre monomer

donaies its N helix in tram to the non-cleaving monomer that is bound across the core

in a (psuedo) full-lox site. This monomer accepts the incoming helix in a

hydrophobic pocket created by the L and M helices (Figure 1-12)(Guo et al., 1997).

This represents the major cross-core protein-protein interaction between the COOH-

terminal domains of Cre monomers. As previously mentioned, the M helix of the

non-cleaving monomer shifts away firom the scissile phosphate and allows the capture

of the N helix in tram. The region between the M and N helices in the cleaving

monomer of Cre protein is in an extended conformation while the same region in the

non-cleaving unit is compacted. As a result, the N helix of the non-cleaving

monomer is directed away from the cleaving monomer bound on the same lox site and

towards the cleaving monomer bound to the partnered site in the synapse, where it

interacts using a dockinp site that differs from the one formed by the L and M helices

(Guo et ai., 1997) and thus forms a COOH-terminal based synaptic interface between

paired sites.

- 1-36 -

Cis-cleavage by one Cre monomer is accompanied with the burial of the N

helix tram in the non-cleaving monomer of the active dimer (Guo et al., 1997). This

conformation is also observed even in the absence of Cre-mediated cleavage (Gopaul

el al., 1 998, Guo et a/. , 1 999). Gopaul and Van Duyne (1 999) have proposed a

mechanism whereby tram-cleavage by Cre could occur. In this scheme, the N helix

is buned in cis in order to allow for the extension of the M helix toward the active site

of the monomer bound across the core that acts as the tyrosine acceptor.

d) Synapsis

The association of the two recombining sites into a higher order complex is

accomplished through protein-protein and protein-DNA interactions in a process

tenned synapsis. It is in within this framework of aligned protein-DNA complexes

that the actual exchange of strands in the recombination reaction occurs. The various

crystal structures of Cre-DNA complexes illustrate that both the NH,- and COOH-

terminal regions of the Cre protein participate in the assembly of the synaptic

complex (Guo et al., 1997). The interactions between the NH2-terminal A and E

helices of the non-cleaving and cleaving Cre monomers, respectively, bound to the

same fox site form a cross-core interface. In addition, the E helix of the non-cleaving

rnonomer is positioned to interact with the A helix of the cleaving monomer bound to

the partnered lox site in the synapse, thereby creating synaptic interface. Similarly,

- 1-37 -

the donation of the N helk of the cleaving monomer to the acceptor non-cleaving

rnonomer constitutes a cross-core interface that originates in the COOH-terminal

region of Cre. Additionally, a synaptic interaction is generated between the synapsed

DNAs by the contact of the N helix of the non-cleaving monomer fkom one dimer

with the COOH-terminal domain of the cleaving monomer of the other Cre dimer

found at the partner site. Combined, the NH,- and COOH-terminai interfaces

network al1 four Cre monomers into a highly stable cyclic complex where the actual

exchange of strands will occur (Figure 1- l3)(Guo et al., 1997).

Formation of a synaptic complex by Cre and Flp cm be monitored by non-

denaturing PAGE (Wierzbicki et ai., 1987, Amin et al., 199 1 ). While synapsis is not

a requirement for Cre- or Flp-mediated cleavage (Voziyanov et al., 1996, see Chapter

III). Cre variants that are defective for cleavage also fail to generate synaptic

complexes in vitro (Wierzbicki et ai., 1987, A.C. Shaikh, unpublished results).

Restoration of cleavage activity for these altered Cre proteins via complementation

with the cleavage defective Cre Y324C protein also results in a formation of a

synaptic complex (A.C. Shaikh, unpublished results). Hence, there is a strong

association between the cleavage activity of Cre and the ability to form higher order

complexes. Furthemore, the combined data obtained fiom the Cre crystal structures

and the biochemical assays suggest that protein-induced bending of the lox site directs

- 1-38 -

cleavage of the DNA, which in tum arranges the NH,- and COOH-terminal interfaces

between the paired recombining sites for assembly of the synaptic complex (Guo et

al., 1997, 1999. Gopaul et al., 1998, A.C. Shaikh, unpublished results).

In both the Cre and Flp systems, interaction of the bent protein-bound target

sites occurs by random collision (Beatty et al., 1986). Both parallel and anti-parallel

alignments of recombining FRT sites have been observed in the Flp reaction (Amin el

al., 199 1 ). However, recent evidence suggests that the asymrnetry of the FRT core

region favors parallel alignments of the recombining sites within the synaptic

complex to ensure the formation of a Holliday junction isomer that readily proceeds

to the resolution step (Hufhan and Levene, 1999). The Cre crystal structures show

that an anti-parallel alignment of the recombining lox sites is preferred. However,

since the Cre-induced bend direction in the lox site can occur in either direction, a

synapse resulting fkom a parallel alignment of lox sites should be possible as well

(Guo et al., 1997, 1999).

The inability of Cre25 and P32 to f o m synaptic complexes suggested a role

for the NH,-terminal domains of Cre and Flp in synapsis (Hoess et al., 1990, Pan and

Sadowski, 1 993, M.Sc. Thesis, AC. Shaikh- 1997). Furthemore, mutations in the

NHz-terminal regions of both Cre and Flp result in proteins that fail to f o m higher

- 1-39 -

order complexes (Wierzbicki et al., 1997. Lee et al., 1997). Given the importance of

the NHflerrninal domain of Cre in establishing the synapse seen in the crystal

structures and despite the sequence heterology that exists between the Cre L 3 and P 13

domains, it is likely that the crystal structure of the Flp synapse will also reveal a role

for P 13 in synapsis.

e) Ligation and Sttartd Exchange

The 5'-OH DNA end and the 3'-covalent phosphotyrosine end can be rejoined

in a protein-dependent manner called ligation. If the event occurs at the 5'- and 3'-

ends of the sarne nick, the cleavage reaction is simply reversed. Altematively, a

strand exchange occun when the nucleophilic attack of the S'-OH end of one DNA

site is directed to the 3'-phosphotyrosine Linkage of the partnered site in the synapse.

While Flp-mediated ligation has been shown to occur in cis, the mode of

ligation for the Cre recombinase has not been determined (Pan et al., 1993b).

However, the Cre crystal structures do suggest the chemistry involved in Cre-

mediated ligation. The active site of the cleaving monomer of Cre arranges Argl73,

His289, Arg292 and Trp3 15 around the scissile phosphate in a proton cradle (Guo et

al., 1997). In this configuration, the negative charges of the phosphate groups are

balanced and the His289 residue acts as a proton donor. As a result, the nucleophilic

- 1 4 0 -

attack of the 5'-OH DNA end is attracted to the tyrosine covalent linkage at the

scissile phosphate (Guo et al., 1997). This view of the protein-mediated ligation

mechanism is especially appealing as mutations in the equivalent conserved Flp

residues result in defects in ligation (Zhu and Sadowski. 1995, 1998, Pan and

Sadowski, 1992, Friesen and Sadowski, 1992).

Flp-mediated ligation can be either coupled to or independent of strand

cleavage (Zhu and Sadowksi, 1998). However, a Cre variant with an alteration at

Ar@ 73 is defective for both cleavage and ligation (Gopaul et aL, 1998, Guo et al..

1999), while the equivalent alteration in the Flp protein results only in a ligation

defect (Freisen and Sadowski, 1992). This suggests that Cre-mediated cleavage and

ligation may be coupled activities.

Cre initiates the first strand exchange event at the bottom strand of the iox site

(Hoess and Abremski, 1985, Hoess et al., 1987). While such a bias has not been

defined for the Flp system, a number of studies have suggested that the FRT b

element is the site of the first strand exchange in Flp-mediated recombination (Zhu

and Sadowski, 1998, Azam et al., 1997, H u h a n and Levene, 1999). In both the Cre

and Flp systems, strand exchange followed by protein-mediated ligation results in the

formation of a Holliday intemediate (Holliday, 1964, Jayaram et al., 1988, Hoess et

al., 1987).

The actual mechanism of strand exchange can be inferred from the first

Cre/lox crystallographic structure. Fhtly, the association of the bent protein-DNA

complexes mimics the four way junction of a Holliday isomer, the end product of the

first strand exchange reaction (Guo et al., 1997). Secondly, as a result of the bend in

the cleaved recombining sites, the DNA strands in the core regions are separated

resulting in an un-stacking of about three base pairs at the 5'-OH end of the nick,

while simultaneously maintaining the pairing of the 3'-phosphotyrosine DNA end

(Guo et ai., 1997, 1999). Through this limited separation of the crossing (cleaved)

and continuous (un-cleaved) DNA strands in the core regions of the two sites in the

synapse. strand exchange between the recombining sites is favored over the re-

ligation of the nicked site (Guo et al., 1997).

As the Cre synaptic complex adopts a Holliday-like structure, the S'-OH DNA

ends of the nicked strands are in close proximity to the 3'-phosphotyrosine ends of the

partnered sites. In addition, the reaction is driven by the base pairing between the

incoming 5'-OH DNA end and the complementary unbroken strand region opposite

the 3'-covalent Iinkage of the receiving site. Coupled with the environment created

by the active site residues around the covalent linkage at the scissile phosphate, the

- 1-42 -

nucleophilic attack of the S'-OH end of one cleaved site is strongly directed toward

the 3'-end of a nick at the partnered site in the synapse (Guo et al., 1997).

The crystal structure of the Cre synapse illustrates how both the cleavage and

strand exchange events can be CO-ordinated and encouraged within the same protein-

DNA complex, with very little change to the overall structure of both the DNA and

proteins in the synapse. This efficient mode1 of strand exchange was previously

suggested by biochemical studies of the AInt recombination system where the

swapping of DNA strands was driven by homology and not branch migration through

the core region of the recombining sites (Nunes-Duby et al., 1995, Dixon and

Sadowski. 1 994). Similarly, the XerClXerD and Flp recombinases are believed to

also un-stack bases at the branch point upon target site binding in their respective

recombination systems (Arciszewska et al., 1997, Zhu and Sadowski, 1998). Hence,

the formation of a protein-DNA complex that mimics the transition state of the

reaction may be a general feature of integrme-mediated recombination reactions that

favors strand exchange between nicked sites in a synapse.

The Holliday interrnediate, or X-structure, generated after the first strand

exchange event is the platform for a second round of protein-mediated cleavage,

- 1-53 -

strand exchange and ligation reactions that resolve this transition intennediate into

recombinant products (Dixon and Sadowski, 1993, 1994). Both Flp and Cre resolve

synthetic X-structures with out bias (ie. in the fonvard direction favoring recombinant

products. or the reverse direction favoring parental products; Dixon and Sadowski.

1 993, Hoess et al., 1987, A.C. Shaikh, unpublished results). Howevet, in vivo, both

recombinases show a definite bias toward resolution that generates recombinant

products and the way that this is achieved remains unclear (Hoess et al., 1987, Dixon

et al., 1995, Azam et al., 1997). The second cleavage reaction in the tesolution step

of the Flp system has been shown to occur in pans (Dixon et al., 1995). While Cre-

mediated cleavage of pstnictures in cis or trons has not been determined in vitro

(AC. Shaikh. unpublished results), the crystal structures of Cre complexed with

synthetic X-structures are consistent with cis-cleavage (Gopaul et al, 1 998).

The crystal structure of the Cre-bound Holliday junction suggests how

resolution might occur. Afier the first set of strand exchanges, a short 1-2 bp branch

migration in the lox core occurs. This shifis the bend center in the sites to the

opposite end of the core (Gopaul et al., 1998). As the bend asymmetry in the lox site

defines the directionality in the cleaving monomer, this limited isomenzation imposes

an inactive conformation upon the Cre proteins that were previously active in the first

strand exchange event and configures the two Cre monomers that were inactive in the

-1-44-

fmt set of reactions to an active state (Figure Id)(Gopaul et al., 1998, Dixon et al.,

1995). These Cre monomers are now responsible for the cleavage, strand exchange

and ligation reactions for the resolution of the Holliday intermediate.

As with the fint strand exchange reaction, the Cre-mediated resolution step

requires no extreme branch migration or helical re-stacking of the core region,

consistent with previous observations fiom the AInt and Flp systems that showed that

branch migration was not required for recornbination @ixon and Sadowski, 1994,

Nunes-Düby el al., 1995). The recombination reaction progresses from the first

strand exchange event to the second, resolution step without major reorganization of

the multi-protein-DNA complex fmt assembled in the synapse.

10. Thesis Outline

The major question addressed in this thesis is the mode of cleavage by Cre

recombinase. 1 performed complementation experiments described in Chapter II

(Shaikh and Sadowski, 1997) before the publication of the first Cre crystal structure.

1 determined that the Cre recombinase used a tram mode of cleavage. However, the

publication of the crystal structure of the Cre/lox synapse (Guo er ai., 1997) clearly

showed Cre cleaving in cis, and caused me to re-examine the mechanism of cleavage

by Cre. It was possible that my use of the sites and proteins in the complementations

- 145 - in Chapter II predisposed Cre to act in tram. In the data shown in Chapter III, 1

constructed Cre and Flp chimeras with aitered DNA binding specificity through

swaps of the NH,-terminal regions of both proteins. I characterized the distinct

binding activities of these novel chimeras and developed specific target sites for

them. Using hybrid target DNA sites for reactions of the chimeras with either Cre or

Flp. 1 determined that the Cre and Flp proteins cleave in cis and trons, respectively. 1

also found that both Cre and Flp had flexible catalytic domains that allowed for a

transition in the protein conformation to accommodate both cis- and tram-cleavage

modes.

Figure 1-1. The effect of general and site specific recombination on Pl plasmid maintenance.

a. The PI plasmid (inset circle) is present as one DNA copy per bacterial cell, (outer oval)(l). Replication of Pl DNA (2) results in two copies of the P l DNA which can be partitioned between the resulting daughter cells at ce11 division (3-5). General recombination gives dirneric Pl DNA (6), which cannot be partitioned between two daughter cells at ce11 division (7). Only one daughter ce11 receives P1 DNA, while the other is cured of the plasmid (empty oval)(8).

b. The lox site (black box) is present in each copy of Pl DNA (1). If generalized recombination dimerizes the P l DNAs (2 and 6), the dimeric DNA can then be resolved into monomenc P 1 plasmids by Cre-mediated recombination between the two Zox sites (7), allowing for partitioning of one P l DNA into each daughter ce11 at ce11 division (8).

Figure 1-2. Peptide maps of Cre and Flp recombinase.

a. Cre peptide map. The NH2-terminal domain, Cre 13 represents the first 1 1 8 amino acids. Cre25, the COOH-terminal peptide, includes the remaining amino acids. 1 19 to 343 of Cre. The four conserved cataiytic residues are shown in green.

b. Flp peptide map. The NH,-terminal domain, P l 3 contains the fint 123 amino acids of Flp. The COOH-terminal domain, P32, stretches fiom residue 124 to amino acid 423. The four catalytic residues of Flp are shown in green.

Figure 1-3. The Cre recombinase.

a. Secondary structure assignment for Cre. The p h a r y amino acid sequence of Cre is rnatched with the secondary structures observed fiom the crystal structure of a Cre synaptic complex (Guo et aL, 1997). The helices (lettered) and P strands (numbered) are denoted as barrels and arrows, respectively, while the intervening regions are depicted as the continuous line. The conserved catalytic residues, Arg 1 73, His289, kg292 and Tyr323, are shown in red. The original demarcation between residues 1 18-1 19 that divided Cre into NH,- and COOW-terminal domains (Hoess ei al., 1990) is shown in purple, while the seiaration of the domains from the crystal structure at amino acids 130-1 3 1 is shown in green. (Adapted fiom Guo et ai.. 1997)

b. Ribbon mode1 of the tertiary structure of Cre. The five helices of the NH2- terminal region (A-E) and the nine helices of the COOH-terminal region (F-N) of Cre are labeled. For clarity, the five P strands (yellow) are not labeled. The demarcation behveen Cre 13 and Cre25 is s h o w as a purple region in the E helix (purple arrow). The separation of the domains fiom the crystal structure at amino acids 1 30- 1 3 1 is shown as a green segment in the intervening region between the E and F helices (green arrow).

(The figure was generated using the Swiss-PDB Viewer and the ICRX set of CO-ordinates fiom Guo et al., 1997)

Figure 1-4. The lox and FRT target sites.

a. The lox site contains two inverted, identical 13 bp symmetry elements, (horizontal arrows. green sequence), that surround an asymmetric 8 bp core region, (open box, magenta sequence). Each element is divided into a 4 bp core-proximal region (underlined) and a 9 bp core-distal region. Cre binds to these symrnetry elements, cleaves at the phosphodiester bonds sites indicated by the two vertical arrows, and attaches covalently to the 3'-phosphate group of the dA nucleotide (top strand) or dG nucleotide (bottom strand), respectively.

b. The FRT site contains three 13 bp syrnmeay elements, a, b and c, (horizontal arrows. red sequences), that surround a 8 bp core region, (open box, blue sequence). Symmetry elements a, and b are in inverted orientation with respect to each other and differ from each other at a single base pair. Syrnmetry elements b and c are directly onented copies of one another. Each symmetry element is divided into a 4 bp core- proximal region (underlined) and a 9 bp core-distal region. Flp binds to these symmetry elements and cleavage by Flp occurs at the core-symmetry element junction. (vertical arrows). Pyrimidine rich tracts (solid green bars) radiate fiom the core towards each adjoining symmetry element.

5' ATAACTTCGTATAATGTATGCTATACGAAGTTAT

TATTGAAGCATATTACATACGATATGCTTCAATA

5' GAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGCTTC 3'

3' CTTCAAGGATAAGGCTTCAAGGATAAGAGATCTTTCATATCCTTGAAG 5u

Figure 1-5. The 2 p M plasmid of Saccharomyces cerevisiue.

The plasmid is divided into two unique regions, large (L). and small (S), by two 599 bp inverted repeats (green bars), each containing an FRT site. The relative locations of the four open reading h e s with known functional products are shown, (REP 1 , RAF, REP2 and FLP). Two cis acting sequences, Stb, and the origin of replication, (Ori), are shown. Flp recombinase mediates a site-specific recombination event between the two FRT sites converthg the A isoform (top), into the B isofonn (bottom).

Figure 1-6. Steps in Cre-mediated recombination.

a. DNA Bioding. Two Cre monomers (ovals) bind specifically to the lox site at the symmetry elements @lue and red arms) that flank the core region (central black region). For simplicity in this figure, the extensive network of protein-protein interactions is not shown.

b. DNA Bending. Upon binding the lox site, Cre induces a bend in the center of the site and sets up an asymmetry in the Cre dimer where one monomer assumes an active state for cleavage (blue oval) while the opposing Cre monomer is in an inactive state (green ovai) (Guo er ul., 1999).

c. Cleavage and Covalent Attachment. The active monomer nicks one strand of the fox site in cis and f o m s a covalent Iinkage with the 3'-phosphoryl end of the nick (red dot and arm) and a free 5'-OH DNA end (open circle). The bottom strand nicked first is the bottom strand of the lox site (See Figure 1-4a).

d. Synapsis. Reactions a-c occur on a second lox site (green and purple arms, yellow core region). Protein-protein interactions between two dimeric Cre-lox complexes aiigns the sites in an anti-parallel manner to generate a synaptic cornplex. As a result of the bends in the synapse the crossing strands in the cross-over regions of both the fox sites are separated in the synapse (squiggly strands with yellow and black open circled ends).

e.f. Strand Exchange and the formation of the Holliday Intermediate. The 5'-OH ends of the crossing strands (incoming squiggly strands terminating in yellow and black open circles) are swapped. The 5'-OH terrnini attack the Cre covalent linkage and reform continuous strands of DNA (filled yellow and black circles in f.) As a result, a Holliday interrnediate is generated.

g. Resolution. i. An isomerization in the core region shifis the bends in the sites to the opposite side of the core region and inactivates the Cre monomers that catalyzed the first set of reactions (Cre monomers bound to the blue and purple arms) and activates the previously inactive Cre monomers (Cre monomers bound to the green and red arms). ii. A second set of Cre-mediated cleavage reactions initiate the next round of strand exchange events. üi. The second set of crossing strands (squiggly strands terminating in yellow and black open circles) are exchanged and ligated. iv. Resolution of the Holliday intennediate yields recombinant products. Note the swapping of the arms in the products and the heteroduplex nature of the core regions.

a. Binding b. Bending C, Cleavage

d. Synapsis e, Strand Exchange f. Holliday Intemediate

g. Resolution

i. Isomerization ii. Second Cleavages iii. Second Strand Exchange

iv. Recombinant Products

Figure 1-7. Modelo of binding of Cre to ibe lox site.

In al1 cases, the lox site is shown with the symmetry elements depicted in green sequences and horizontal arrows. The red sequence and the open box represent the core. Vertical arrows denote cleavage sites and are omitted in subsequent cartoons. Two Cre monomers are shown bound to each lox site on opposite DNA strands.

a. The initial binding of Cre to a single lox symmetry elernent occurs with the binding of the NH,-terminal domain of Cre, Crel3, to the fmt 4 bp of the symmetry element at the core-proximal region (underlined). The COOH-terminal domain. Cre25, contacts the remaining 9 bp of the core-distal region of the symmetry element. Hence. the entire symmetry element is contacted by the full-length Cre protein (M.Sc. Thesis, A.C. Shaikh-1997).

b. When two Cre monomers bind to a full-fox site, protein-protein interactions extend the contact region of the Cre domains so that Cre25 is positioned over both the core-proximal and -distal regions of the symmetry element, while Cre 13 is situated solely in the core. Thus, Cre binding to a full-lox site results in two Cre molecules occupying the core region at the sarne time, (shown here, on opposite strands).(Hoess et al., 1990)

c. Composite view of the above models derived from the Cre/lox crystallographic structure (Guo et al., 1997). The COOH-terminal domain of Cre contacts the entire syrnmetry element and the fvst 2 bp of the core region. The NH,-terminal domain interacts with the core-proximal region of the symmetry elemen&d the first 3 bp of the core region. Two Cre monomers cover the entire fox site except for the central 2 bp of the core region.

ATAACTTCGTATMTCTATGCTATWWGTTAT 3'

3' TATTGIUCCATATTACATACGATATGCTTCAATA 5'

5' ATMCTTCGTAT~TGTATGCTATACGAAGTTAT 3'

3' TATTGAAGCATATTACATACGATATGCTTCAATA 5'

3 ATAACTTCGTATAATGTATGCTATACGAAGTTAT 3'

3' TATTGAAGCATATTACATACGATATGCTTCAATA 5'

Figure 1-8. Tertiary structure mode1 of binding of Cre to the fox site.

For clarity, only some helices of the Cre protein are labeled and one Cre monomer, a cleaving subunit, is shown binding to the two-half lox sites.

The Cre protein binds the lox site in a clamp-like fashion, with the DNA sandwiched between the two domains of the protein. Extensive sugar-phosphate contacts occur between the core region (nucleotides of the core region of the continuous strand of the bound half-lox site shown in blue) and the E helix. The NH,-terminal domain forms direct base contacts primarily through the B and D helices. The COOH- terminal domain forrns several contacts with the DNA. Direct base contacts occur between the DNA and the J helix, the 4-5 P sheet and a Lys201 residue (green residue) contained in between the p strands 2 and 3. The catalytic tyrosine (red residue) is shown covalently attached (black oval) to the 3'-phosphoryl end of the cleavage site (dG nucleotide of the cleaved strand shown in purple). Accompanied with Cre binding, a bend is introduced in the lox site in the direction of the green arrow and would serve to separate the crossing (cleaved) and continuo