the idl of e. coli ssb links ssdna and protein binding by ... · 117 or 152 were unable to...

The IDL of E. coli SSB links ssDNAand protein binding by mediatingprotein–protein interactions

Piero R. Bianco,1* Sasheen Pottinger,1 Hui Yin Tan,1 Trong Nguyenduc,1

Kervin Rex,2 and Umesh Varshney2

1Department of Microbiology and Immunology, Center for Single Molecule Biophysics, University at Buffalo, Buffalo,New York 142142Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

Received 22 August 2016; Accepted 17 October 2016

DOI: 10.1002/pro.3072Published online proteinscience.org

Abstract: The E. coli single strand DNA binding protein (SSB) is essential to viability where it func-

tions in two seemingly disparate roles: it binds to single stranded DNA (ssDNA) and to target pro-teins that comprise the SSB interactome. The link between these roles resides in a previously

under-appreciated region of the protein known as the intrinsically disordered linker (IDL). We

present a model wherein the IDL is responsible for mediating protein–protein interactions criticalto each role. When interactions occur between SSB tetramers, cooperative binding to ssDNA

results. When binding occurs between SSB and an interactome partner, storage or loading of that

protein onto the DNA takes place. The properties of the IDL that facilitate these interactionsinclude the presence of repeats, a putative polyproline type II helix and, PXXP motifs that may

facilitate direct binding to the OB-fold in a manner similar to that observed for SH3 domain binding

of PXXP ligands in eukaryotic systems.

Keywords: SSB; RecG; RecO; OB-fold; SH3 domain; PXXP motif

Introduction

The Escherichia coli single stranded DNA binding

protein (SSB) is essential to all aspects of DNA

metabolism. Here it performs two seemingly disparate

roles. First, it functions to stabilize single-stranded

DNA (ssDNA) intermediates generated during DNA

processing.1–5 Second, it interacts with as many as 14

proteins in temporal and spatial fashion, to both store

and target enzymes to the DNA when needed.6,7 How

these roles are linked mechanistically is currently not

known.

The protein, like the majority of eubacterial SSBs,

exists as a stable homo-tetramer with a monomer MW

of 18,843 Da.8 Each 178 amino acid length monomer

can be divided into two general domains defined by

proteolytic cleavage: an N-terminal portion comprising

approximately the first 115 residues and a C-terminal

domain that includes residues 116–178.9 The

N-terminal domain contains elements critical to tetra-

mer formation and the oligonucleotide/oligosaccharide

binding-fold (OB-fold) required for binding to ssDNA.10

DNA binding occurs via the wrapping of ssDNA

around the SSB tetramer using an extensive network

of electrostatic and base-stacking interactions with

the phosphodiester backbone and nucleotide bases,

respectively.10–12

The C-terminal domain can be further subdi-

vided into two additional regions: a sequence of �50

amino acids that has been called the intrinsically

Abbreviations: SSB, single stranded DNA binding protein; ssDNA,single stranded DNA; IDL, intrinsically disordered linker; PPII,polyproline type II helix.

Additional Supporting Information may be found in the onlineversion of this article.

Grant sponsor: National Institutes of Health Grant; Grant number:GM100156.

*Correspondence to: Piero R. Bianco, Center for Single MoleculeBiophysics, Department of Microbiology and Immunology, Universityat Buffalo, Buffalo, NY 14214, USA. E-mail: [email protected]

Published by Wiley-Blackwell. VC 2017 The Protein Society PROTEIN SCIENCE 2017 VOL 26:227—241 227

disordered linker (IDL) and the last 8–10 residues

which are known as the acidic tip or C-peptide and

which is very well conserved among eubacterial pro-

teins.1,13–23 While there is extensive knowledge of

the N-terminal domain, there are only a limited

number of studies of the C-terminus. This is due in

part to the fact that it is highly disordered even

when the protein is bound to ssDNA.24 Recent

studies have focused on the acidic tip only as it is

thought to be critical for mediating interactions with

SSB interactome partner proteins.1

The IDL is the least conserved region among

prokaryotic SSB proteins.1,5 Initially, it was proposed

to be non-essential, as a mutant ssb with residues 116-

167 deleted was able to complement an ssb deletion.9

However, truncated ssb genes terminating at residues

117 or 152 were unable to complement the same ssb

deletion. This led to the proposal that the last 20–50

residues of SSB are essential for protein–protein

interactions.9 Subsequent in vitro studies showed that

deletion of the C-terminal 15–20 residues eliminated

interactions with the psi and chi subunits of the DNA

polymerase III complex.18,25 Further work both in vivo

and in vitro using the ssbDC8 gene and SSBDC8 protein

respectively, have shown that the last eight residues are

critical, but not solely responsible, for target protein

binding.7,22 One interpretation is that the acidic tip resi-

dues contact target proteins directly.23,26–28 An alterna-

tive proposal suggests that these last eight residues play

a regulatory role and that target protein binding utilizes

another region of SSB.29

We propose that this other region is the intrinsi-

cally disordered linker. This proposal makes sense

because the IDL is unique to each bacterium, and

may explain species specificity in interactome func-

tion.6,22 What is known about the IDL is limited. As

alluded to above, early studies suggested that it is a

non-essential part of the protein.9 Recently, the

Lohman group found that when the linker is deleted

or its sequence altered, cooperative ssDNA binding

by SSB is affected.23 They proposed a model wherein

the linkers could self-associate so that the linker of

one tetramer binds to that of an adjacent one and,

this would facilitate interactions between acidic tips

and OB cores. This model has at its core, interac-

tions between the acidic tip and OB-folds of adjacent

proteins. The current data show that this interaction

is at its best, only transient.29,30 Furthermore,

recent crystal structures of part of the acidic tip

bound to interactome partners reveal that the tip

does not bind to OB-folds but instead binds to a

hydrophobic pocket that contains a prominent basic

residue.26–28,31

To begin to understand how the linker region of

SSB may link the seemingly disparate activities of

cooperative ssDNA and protein binding, a combinatori-

al strategy was employed. First a series of linker dele-

tion mutants containing the acidic tip was created,

and then utilized in an in vivo binding assay to

monitor the interaction between small (RecO; 27 kDa)

and large (RecG; 76 kDa) interactome partners.22,32

Results show that a full length linker is required to

bind RecG whereas shorter linker SSBs can still bind

to RecO. We then compared the binding results of our

mutants to that of a naturally occurring short-linker

protein, the Thermotoga maritima SSB and also to

E. coli–Mycobacterium tuberculosis hybrid SSB pro-

teins. Collectively, these results reveal novel insights

into the role of the linker in target protein binding as

well as insight into subdomains within the linker

which are critical to IDL function. These include a

section that could adopt a polyproline type II helix,

repeats that impart flexibility and tensile strength

and also PXXP motifs to facilitate binding specificity.

Consequently, a model is proposed whereby the

linker(s) of an SSB tetramer bind to the OB-folds

present in either a neighboring SSB or a member

of the interactome. The common feature in these

interactions are the PXXP motifs in the intrinsically

disordered linker of SSB and the OB-fold of the

target protein. Here binding occurs in a manner analo-

gous to eukaryotic SH3 domains binding PXXP-

containing protein ligands. When binding of a linker to

the OB-fold of another SSB occurs, this results in rapid

and cooperative ssDNA binding, producing a stable

complex that can resist displacement by a variety of

enzymes that might otherwise damage the exposed

portion of the genome. When the IDLs bind to a protein

such as RecG, the helicase is loaded onto the DNA,

leading to the rescue of stalled replication forks. There-

fore, the linker domain of SSB connects the seemingly

disparate roles of ssDNA and target protein binding by

SSB using a common mechanism.

Results

RecO and SSB bind in vivo

The RecO interaction with SSB in vitro has been dem-

onstrated previously.32,33 To determine whether bind-

ing also occurs in vivo, as shown previously for PriA

and RecG, an identical approach was employed.7 To

test this, expression strains were made by transform-

ing high copy number plasmids expressing either wild

type or N-terminally his-tagged SSB into E. coli

strains. Then, high copy number plasmids expressing

his- or wild type RecO were transformed separately

into wild type and his-SSB cells, respectively. One liter

cultures of cells expressing both proteins were grown,

lysed and the cleared cell lysates subjected to Ni-

sepharose chromatography. After extensive washing,

proteins were eluted using a linear, imidazole gradient

as described in the Materials and Methods. The results

show that both SSB and his-RecO coeluted from the

column [Fig. 1(A)]. When the tag was present on the

C-terminus of RecO (i.e., RecO-his), coelution was not

observed, suggesting that this end of the protein being

228 PROTEINSCIENCE.ORG SSB IDL Links ssDNA and Protein Binding

involved in binding to SSB [Fig. 1(B)32,33]. A vanishing-

ly small amount of SSB could be detected and the

resulting analysis shows that binding decreased 67-

fold compared to his-RecO [Fig. 1(E)]. Coelution of SSB

and RecO was observed when the histidine tag was

present on the N-terminus of either protein [Figs.

1(A,C)]. Analysis of the gels shows that when his-RecO

was used, the ratio of RecO to SSB was 1.7 6 0.5 [Fig.

1(A,E)]. When the tag was on SSB, the ratio of

RecO:SSB in the eluted fractions was 1.4 6 0.4 [Fig.

1(C,E)]. Our previous work with RecG and PriA dem-

onstrates that the SSB-partner complexes exist in vivo

and do not form following cell lysis.7 Therefore, the

data presented in Figure 1 show that RecO binds to

SSB in vivo.

RecO-SSB complex formation requires the

presence of the SSB C-terminus

In vitro studies show that SSBDC8, a mutant which

lacks the last eight residues of SSB, does not form a

complex with RecO.32 Previous work has shown that

binding to the RecG and PriA helicases in vivo

requires the presence of the C-terminal eight resi-

dues of SSB.7 To determine whether these residues

are also required for complex formation between

SSB and RecO in vivo, we made dual-expression

strains, replacing wild type SSB with SSBDC8. One

liter cultures of cells expressing SSBDC8 and his-

RecO were grown, lysed and the cleared cell lysates

subjected to Ni Sepharose chromatography as before.

The results show that his-RecO eluted off the col-

umn as expected, but that these fractions contained

little or no SSBDC8 [Fig. 1(D)]. There is however, a

small amount of SSBDC8 found in the fractions elut-

ed from the column at the front end of the peak.

This indicates that there is a weak interaction

between RecO and SSBDC8, similar to what was

observed with RecG.7 Therefore, and as shown previ-

ously for RecG and PriA, binding of RecO requires

that the last 8 amino acids of the acidic tip of SSB

be present.7

SSB linker length appears to be critical

for binding to interactome partners

As the binding of SSB to interactome partners in

vivo is essential to facilitating many DNA metabolic

processes, we wanted to understand the role of the

linker region in target protein binding.7 To do this,

we constructed a series of SSB linker mutants,

designated SSB125, SSB133, SSB145, and SSB155

[Fig. 2(A,B), Supporting Information Fig. S1]. These

are similar to those of the Lohman group, but differ

in the deletion points and the length of the C-

terminal tip retained.23 For the proteins described

herein, the subscript refers to the number of resi-

dues in each protein, counting the first methionine

as position 1. Each protein contains the N-terminal

115 residues of the protein responsible for tetramer

formation and DNA binding, followed by various

length linker regions and the C-terminus (the last

10 amino acids; 50-PMDFDDDIPF-30). Wild type SSB

has a linker 54 residues in length while that of

mutants is 31 (SSB155; SSBD146–167); 21 (SSB145;

SSBD135–167); 6 (SSB133; SSBD124–167); and 0

(SSB125; SSBD115–167). Next, we coexpressed his-

RecO separately with each of the mutants, and

Figure 1. SSB binds to RecO in vivo. The 1-L cultures of cells expressing RecO and SSB were grown to early log phase, IPTG

added to 500 mM and growth continued until early stationary phase. Cells were harvested by centrifugation, lysed and the

cleared cell lysate applied to a 5-mL nickel column as described in the Materials and Methods. Proteins were then eluted using

an imidazole gradient following extensive washing to remove unbound proteins. Aliquots from various fractions throughout the

purification were subjected to electrophoresis. The resulting Coomassie stained, 15% SDS-PAGE gels of the purifications are

presented. (A), hisRecO binds to SSB; (B), RecO-his does not bind to SSB; (C), RecO binds to hisSSB; (D), formation of the

RecO-SSB complex requires the highly conserved and acidic SSB tip.; (E), Analysis of the gels in panels A–C. For each

analysis, only the five central lanes of the peak were analyzed and the ratios averaged. MW, molecular weight marker; WCL,

whole cell lysate; CCL, cleared cell lysate; FT, flow through; numbers, indicate fraction numbers from the peak elution profile.

Proteins were eluted using a linear imidazole gradient from 30 to 500 mM.

Bianco et al. PROTEIN SCIENCE VOL 26:227—241 229

subjected the cleared cell lysates to nickel column

chromatography.

The results show co-elution of his-RecO with

each SSB protein consistent with in vivo complex

formation [Fig. 2(C)]. In the gel shown, we have

normalized each lane to the amount of his-RecO

eluted in the peak fractions from each purification.

When presented in this manner, it is clear that the

average ratio of RecO to SSB is 2.2 for wild type.

This ratio decreases in almost linear fashion as

length of the linker decreases to zero, reaching a

minimum of 1.3 6 0.4 for SSB125. Thus complete

binding to RecO requires a fully intact linker.

Full length SSB binds to RecG whereas linker

mutants do not

Recent work demonstrates that RecG (MW 5 76,430

Da) and SSB, bind to each other in vivo in the

absence of DNA.7 The mass of the RecO protein is

27,391 Da, approximately one third that of RecG. It

is conceivable that the truncated SSB proteins bind

to RecO due to its small size but perhaps binding to

the larger RecG requires a longer linker.

To test this, we co-expressed the histidine

tagged version of each of the SSB mutants with

RecG. As a positive control for the linker mutants,

we repeated the experiment with full length SSB

and RecG. As expected, both his-SSB and RecG

eluted from the column [Fig. 3(A,D)]. In contrast,

when the linker mutants replaced full length SSB,

co-elution to the level of wild type was not observed

[Fig. 3(B,C)]. In fact, binding to RecG was

diminished 10-fold for SSB155 and 26-fold for the

SSB125 mutant. A simple interpretation of these

data is that longer linkers are required to bind

larger interactome partner proteins. However, and

as explained below, this turns out not to be the case.

Key components of the linker are

required for optimal function

To understand why the linker mutants bind to RecO

but not to RecG, two separate sequence analyses

Figure 2. SSB linker length affects binding to RecO. (A), Schematic of the wild type and linker domain mutants. The black line

demarcates the region deleted in each mutant. (B), Sequence alignment of full length and linker domain mutants of E. coli SSB with

T. maritima SSB. The alignment was done using PRALINE multiple sequence alignment and the result shows residues 112–178 (E.

coli numbering)66. The green boxed region indicates the C-terminal tail with invariant residues in red. (C) SSB linker domain mutants

co-elute with his-RecO. The 100 mL cultures were grown to early log phase and expression induced with 500 mM IPTG. Cells were

harvested in early stationary phase by centrifugation and the resulting pellets lysed. The cleared cell lysates were applied to 1mL

nickel columns, extensively washed and eluted with elution buffer as described in the Materials and Methods. A Coomassie stained,

15% SDS-PAGE gel with the apex fraction of each coelution is shown. To allow direct comparison, each lane was normalized to the

amount of RecO loaded. (D), Quantification of SDS-PAGE gels such as those shown in (C).


were done. First, we examined the primary amino

acid sequence of SSB focusing on the C-terminal 69

residues. This analysis shows that this region is

over-represented for Gly 24.6%, Gln 17.4%, Pro

14.5%, and to a lesser extent, Ala and Ser [8.7 and

5.8%, respectively; Fig. 4(A)]. The presence and

spacing of these residues in the N-terminal half of

this region, ending at residues 148 and 149 (Phe

and Ser, respectively), is consistent with the forma-

tion of polyproline II helices (PPII) found in proteins

such as collagen.34 However, the spacing of the pro-

line residues in collagen occurs at every fourth posi-

tion which is not the case for SSB. Instead, we

observed that glycine, glutamine and arginine occur

in those positions in SSB. A recent study has shown

that these amino acids can substitute for proline

without disrupting a model PPII helix.35 As one

strand of a collagen fiber is essentially a PPII helix,

we wanted to determine whether this region of SSB

could be modeled on a collagen-like peptide.36,37

Indeed, the SSB sequence can adopt a PPII helix

that superimposes well with the peptide, with an

RMSD 5 0.8 Angstroms for the backbone atoms

(Supporting Information Fig. S3). In contrast, the

C-terminal 30 residues of SSB downstream of F148/

S149 could not be modeled on the collagen peptide

using the same approach (data not shown).

Second, and as the gly, pro and gln residues

appear in clusters in the SSB C-terminal domain

(Fig. 4, lines a and b), we analyzed the entire 69 res-

idue region for repeats using REPRO, which identi-

fies protein sequence repeats using a graph-based

iterative clustering procedure.38 Eleven of the identi-

fied repeats are shown in Figure 4 (lines 1–11; yel-

low box). Some are these are identical (highlighted

in red), while the remaining identified repeats

possess a high degree of similarity with one another

(quasi-repeats), for example, repeats 1 and 10. We

also noticed the presence of seven hydrophilic GGX

repeats (line 11). In addition, of the first ten repeats,

nine cluster in the N-terminal half of the IDL

sequence upstream of F148. Likewise, six of the

seven GGX repeats also occur in this region of the

protein.

Similar repeats containing G, P, and Q have

been identified in several eukaryotic proteins such

as the X-type HMW subunit of wheat gluten, spider

silk protein and x-protein where they have been

shown to confer important structural features and

special functions in addition to conferring elastomer-

ic properties to that region of the protein.39 The

over-representation of gly, gln, pro, and ser residues

and the presence of the repeats, may impart similar

elastomeric properties to the C-terminus of SSB,

Figure 3. Alterations in the SSB linker eliminate binding to RecG. The 1-L cultures of cells expressing RecG and SSB or linker

mutants were grown to early log phase, IPTG added to 500 mM and growth continued until early stationary phase. Cells were

harvested by centrifugation, lysed and the cleared cell lysate applied to a 5-mL nickel column as described in the Materials and

Methods. Proteins were then eluted using an imidazole gradient following extensive washing to remove unbound proteins.

Aliquots from various fractions throughout the purification were subjected to electrophoresis. The resulting Coomassie stained,

12% SDS-PAGE gels of the purifications are presented. (A) RecG binds to full-length SSB as shown previously.7 (B) RecG does

not bind appreciably to SSB125. (C) RecG does not bind appreciably to SSB155. MW, molecular weight marker; WCL, whole cell

lysate; CCL, cleared cell lysate; FT, flow through; numbers, indicate fraction numbers from the peak elution profile.


enabling it to interact with a large range of partners

of different sizes.

Finally, we also identified three PXXP motifs:

PQQP, PAAP and the similar, PQQS (Fig. 4, motif).

Two of these are deleted in SSB155 and all three are

absent in SSB145. In summary, these analyses sug-

gest that in addition to length, the sequence compo-

sition of the SSB linker is critical to protein–protein

interactions.

TmSSB binds to EcRecO in vivoTo understand whether linker length or sequence

composition is more critical to binding, we decided

to test these independently. First, to evaluate the

role of linker length in protein binding, we selected

an SSB that had a naturally occurring short linker

region relative to the E. coli protein. For this pur-

pose, we selected the Thermotoga maritima SSB

(TmSSB) which has a linker 22 residues in length,

approximately the same as that of SSB145 [Fig. 2(B),

Supporting Information Fig. S1]. In addition, the

last six residues of TmSSB are 50% identical to

that of EcSSB. Correspondingly, we cloned the

Thermotoga RecG protein, which is similar to

EcRecG, but has additional N-terminal residues of

unknown function.40

To test binding, the TmRecG and his-TmSSB

proteins were expressed in E. coli and the resulting

cleared cell lysate subjected to nickel column chro-

matography. The results show that as for the E. coli

proteins, Thermotoga RecG and SSB coelute

although the amount of RecG eluted is significantly

lower [Fig. 5(A)]. The ratio of TmSSB:TmRecG is on

average, 25 whereas for Ec proteins, the average

was 7 [Fig. 5(D)]. Next, we tested binding of TmSSB

to EcRecG. As before, cleared cell lysates were sub-

jected to nickel column chromatography and pro-

teins eluted with a linear imidazole gradient. The

resulting SDS-PAGE gel shows a significant level of

TmSSB eluted and only a small, but detectable level

of EcRecG [Fig. 5(B)]. The ratio of TmSSB: EcRecG

eluted is 39 6 3 and this is comparable to what we

observed for SSB155 where the ratio was 70 6 10

(Fig. 3). This suggests that binding of TmSSB to

EcRecG is impaired.

Next, to test binding to the smaller RecO pro-

tein and as the T. maritima genome does not con-

tain recO, EcRecO had to be used instead. The

results from the nickel column show that the

EcRecO coeluted with TmSSB and the ratio of

SSB:RecO was 3.9 6 0.6 [Fig. 5(C)]. However, the

amount of SSB was in excess of RecO indicating

that binding was not stoichiometric as we observed

for full length E. coli proteins [Fig. 1(E)]. This ratio

is twofold higher than what was observed for the

SSB145 linker mutant [Fig. 2(D)]. Because the

sequences of the linkers in the TmSSB and SSB145

proteins are different while the lengths are similar

[Fig. 2(B)], these data suggest that the sequence of

the linker is more important than length for target

Figure 4. The linker of EcSSB has a specialized sequence composition. The sequence of the C-terminal 69 residues of EcSSB

(top line) contains repetitive elements consisting of glycine, proline and glutamine. Sequence analysis of the protein was done

using REPRO at http://www.ibi.vu.nl/programs/.38 The position of beta sheet 6 is indicated.34,43 Lines a and b illuminate the

over-represented residues in the SSB C-terminus (explained in the next). Lines 1–11 (yellow box) show a subset of the repeated

sequence elements identified using REPRO.35,38 The PXXP motifs were identified by eye. The amino acids spanning the puta-

tive PPII-helix are indicated by the black line and the positions of the deletions in the linker mutants are also shown.


http://www.ibi.vu.nl/programs/

protein binding, as suggested by the sequence

analyses.

Binding of SSB to EcRecO requires

more than the acidic tip

To further understand the role of sequence composi-

tion of the linker region in target protein binding,

we utilized a series of five hybrid SSB proteins and

determined their ability to bind EcRecO in vivo.

These hybrid SSBs are combinations of the E. coli

and Mycobacterium tuberculosis proteins and are

shown schematically in Figure 6(A).41,42 The sequen-

ces of each hybrid aligned to full length E. coli SSB

are shown in Supporting Information Figure S2. In

both instances, MtSSB is shown for comparison only

and was not studied here. We selected MtSSB as the

protein has been well studied in vivo and in vitro,

its N-terminal core is similar to that of EcSSB and

the last six residues of the acidic tip are 67% identi-

cal. The hybrid proteins are designated EM followed

by a number. EM-1 and 2 are control proteins with

altered core domains and otherwise E. coli linker

and C-termini, while the test proteins EM-3, 4, and

5 have chimeric linker regions as explained below.

Hybrid proteins EM-1 and EM-2 each have an

E. coli C-terminus and split core domains [Fig.

6(A)]. For EM-1, the N-terminal 73 residues are

Mycobacterial in origin with the remainder of the

protein from E. coli. In contrast, for EM-2, the

C-terminal part of the core, corresponding to resi-

dues 73–131, is from MtSSB. EM-3 is an otherwise

E. coli protein with only part of beta sheet 6 and the

downstream sequence from the Mycobacterium

protein replacing the corresponding region. EM-4

has part of beta sheet 6 as well as the C-terminus

from M. tuberculosis. Finally, EM-5 is an E. coli

protein with only its C-terminal 33 amino acids

replaced with the Mycobacterial sequence. Each of

the hybrids selected forms homotetramers, retains

binding to ssDNA and with the exception of EM-2,

are functional when expressed in E. coli.41,42

To test whether these hybrid proteins could bind

to EcRecO, separate strains expressing his-RecO

and the five hybrid proteins were made, grown,

lysed, and subjected to nickel column chromatogra-

phy as before. Analysis of the resulting gels reveals

some surprising results [Fig. 6(B)]. First, binding of

the EM-1 and EM-2 proteins to RecO is reduced 2.5-

fold. This was surprising given that EM-2 is inactive

in vivo.41,42 Second, and perhaps the most surprising

result is that the efficiency of binding of EM-4 is

reduced 10-fold, while EM-3 and EM-5 bind as well

as wild type E. coli SSB. The failure of EM-4 to bind

is not simply due to a length change because the

EM-5 hybrid is three residues shorter and it retains

wild type levels of binding. Nor is it the presence of

Figure 5. TmSSB forms stable complexes with TmRecG and EcRecO but not EcRecG. The 1-L cultures of cells expressing RecG

(Ec or Tm) with his-TmSSB were grown to early log phase, IPTG added to 500 mM and growth continued until early stationary phase.

Cells were harvested by centrifugation, lysed and the cleared cell lysate applied to a 5-mL nickel column as described in the

Materials and methods. Proteins were then eluted using an imidazole gradient following extensive washing to remove unbound

proteins. Aliquots from various fractions throughout the purification were subjected to electrophoresis. Coomassie stained, 15%

SDS-PAGE gels showing various stages during the purification are presented. (A) TmRecG binds to TmSSB. (B) EcRecG does not

bind appreciably to TmSSB. (C) TmSSB binds to EcRecO. (D), Analysis of the gels in panels A–C. The data for EcSSB/EcRecG are

from column 1, Figure 3(D) and are presented here for comparison. MW, molecular weight marker; WCL, whole cell lysate; CCL,

cleared cell lysate; numbers indicate fraction numbers from the peak elution profile.


the Mycobacterial terminal eight residues replacing

the E. coli acidic tip, as EM-5 binds well. Instead,

residues 111–131 from MtSSB in combination with

the Mycobacterial C-terminus provide insight into

how the linker may be involved in protein-protein

interactions.

Perturbation of the putative PPII helix-like

region eliminates binding

To gain further insight into the contributing factors

affecting binding in the hybrids, sequence analysis for

composition and repetitive elements was done. Analysis

of the MtSSB sequence reveals it to be over-represented

for Ser (19%), Gly (18%), Ala (14%), and Pro (12%).

Furthermore, and not surprisingly, the REPRO

analysis identified repeats that were different from that

of EcSSB and, in contrast to E. coli, these repeats are

clustered in the C-terminal half of the sequence

(Supporting Information Fig. S4). The clustering within

this region of the C-terminus, prompted us to ascertain

whether a model could be built using collagen as a tem-

plate. Surprisingly, we were able to build a model of the

C-terminal 33 residues of MtSSB using the collagen-

like peptide (Supporting Information Fig. S5). In

contrast, a model of the core-proximal region of the

linker of this SSB could not be built (data not shown).

Next we analyzed hybrid proteins 3, 4, and 5.

Replacement of residues 114–130 as in the EM-3

protein does not significantly alter the over-

representation of amino acids but eliminates five of

the ten repeats identified in EcSSB and only repeats

similar to 2 and 4–7 are retained. In addition, and

Figure 6. The sequence composition of the EcSSB linker influences binding to RecO. (A) Schematic of EcSSB, five Mt-Ec-SSB

hybrid proteins and MtSSB is shown. Residue numbering is from the E. coli sequence with methionine as 1. Numbers

immediately above the hybrid proteins demarcates where the sequence changes from E. coli to M. tuberculosis. (B) Some

hybrid proteins bind to EcRecO while others do not. Quantification of the Coomassie-stained gels of apex fractions from each

coelution of his-RecO and the five hybrid SSB proteins is shown. The 100 mL cultures of his-RecO and each of the five hybrid

were grown separately to early log phase and expression induced with 500 mM IPTG. Cells were harvested in early stationary

phase by centrifugation and the resulting pellets lysed. The cleared cell lysates were applied to 1-mL nickel columns,

extensively washed and eluted with elution buffer as described in the Materials and methods. Eluted fractions were subjected

to electrophoresis in 15% SDS-PAGE gels.


in contrast to the wild type protein, the clusters

identified occur in the C-terminal half of the

sequence (data not shown). In addition, while the

region corresponding to the proposed PPII-like helix

is perturbed, the PXXP motifs are retained. Analysis

of EM-4, shows a reduction in Gln to only 2 resi-

dues, Gly and Pro are reduced while Alanine and

Serine are increased. In addition, all of the repeats

present in the wild type E. coli SSB C-terminal 69

residues are eliminated (not shown). Finally, EM-5

retains the PPII-like region but the sequences of the

PXXP motifs are all different. In summary, these

data suggest that when the sequence and/or position

of the proposed PP-II helix are altered it has profound

effects on SSB-protein interactions.

To understand how the changes in sequence in

each of the hybrids relative to wild type E. coli SSB

could lead to the elimination of binding to RecO, we

performed homology modeling of the intact hybrid

proteins using Swiss-model.36,37 In each case, models

of each hybrid were built using subunit 1 of 1QVC

as a template as in this structure, residues 2–145

are visible.43 The resulting Connolly surface repre-

sentations for residues 112 to the C-terminal residue

of each model are shown in Figure 7 with the same

region from subunit 1 from the SSB structure 1QVC

as a reference. When viewed in this way, it is evi-

dent that the N-terminal part of this region presents

as a PPII-like helix with minimal additional second-

ary structure for the EcSSB and EM-3 proteins

(Fig. 7, yellow shaded region). Even though this

region of SSB can adopt different conformations, res-

idues 112–130 still present as a PPII-like helix with

minimal additional secondary structure. In contrast,

the same region in the EM-4protein does not present

as a PPII-like helix but has increased secondary

structure. This results in this region being more

compact and possibly more rigid, stabilized by

hydrogen binding (not shown), resulting in a protein

that cannot bind to RecO. Even though these are

models, they suggest that the changes in primary

amino acid sequence of residues 112–130 leading to

disruption of the putative PPII helix, has profound

effects on the ability of EcSSB to interact with

target proteins.

Discussion

The primary conclusion of this article is that the

intrinsically disordered linker (IDL) connects ssDNA

binding and protein–protein interactions of SSB

using a common mechanism. This mechanism

involves binding of the IDL of an SSB tetramer to

the OB-fold of a nearby protein as explained below.

The importance of the IDL became clear when

sequence analysis revealed several previously unidenti-

fied features that impart specificity, tensile strength,

and flexibility. These properties are conferred on SSB in

the following ways. First, for the linker region to func-

tion correctly, either all, or a subset, of the amino acids

present must be able to adopt a PPII-like helix confor-

mation. This may impart tensile strength when SSB

tetramers associate with one another along a stretch of

ssDNA or when bound to a target protein. Second, the

primary amino acid sequence of the C-terminal 69 resi-

dues arranged in various repeats, imparts flexibility

possibly in a similar fashion to that observed in proteins

such as wheat gluten. This enables SSB to bind to all

interactome partners, regardless of their size or the posi-

tion within multi-subunit complexes and also between

SSB tetramers. Finally, the presence of PXXP motifs

suggests that this region of SSB is directly involved in

protein-protein interactions: either between SSB tet-

ramers during cooperative binding to ssDNA or between

SSB and interactome partners. These interactions

Figure 7. The proposed PPII helix region is important to EcSSB function. Homology modeling of the EM-3 and 4 proteins was

done at Swiss-model in automated mode using PDB file 1QVC as a template.37,39,43 Models were built on the first subunit of

1QVC. As residues 1–111 in each model are identical to 1QVC they are not displayed. Instead, the Connolly surfaces of resi-

dues 110 to the C-terminus of each model are shown with the N-termini at the top left of each image.44,67 For EcSSB this is to

residue 145; for EM-3 it extends to position 149, and for EM-4 to amino acid 161. Colouring is done according to hydrophobici-

ty as indicated and the orientation of each region is approximately the same. A sequence alignment of the hybrids to EcSSB is

shown in Supporting Information Figure S2.


involve binding between the PXXP regions of SSB and

the OB-fold in the target protein, either another SSB or

an interactome partner.

The in vivo binding studies revealed that SSB

binds to RecO in vivo, similar to what was observed

previously for PriA and RecG.7 Binding was not

observed when SSBDC8 replaced wild type and when

the histidine tag was present on the C-terminus of

RecO, consistent with previous work showing that

this region was involved in SSB binding.32 The in vivo

binding assay was subsequently used to determine

the effects of deletions of residues within the linker on

binding to RecO and separately, to RecG. Results

show that the deletion of 21 residues eliminated bind-

ing to RecG, whereas binding to RecO was only affect-

ed when 42 or more residues were removed. To

determine whether linker length is critical or whether

sequence may be important, we used the Thermotoga

maritima SSB. The IDL in this protein is 16–18 resi-

dues in length, comparable to that in the SSB145

mutant. In vivo binding to EcRecO was observed but

this was reduced compared to full length EcSSB, and

more importantly, to SSB145 as well. Surprisingly,

TmSSB bound to TmRecG but not to EcRecG. Tm/Tm

binding was reduced compared to Ec/Ec binding, but

this was not surprising given these are thermostable

proteins. Collectively, these results suggested that

while the length of the linker is important for protein–

protein interactions, the sequence is more critical, as

illuminated below.

The analysis of the sequence of the C-terminal 69

residues of EcSSB revealed an over-representation of

glycine, glutamine, proline, serine, and alanine. Fur-

ther analysis with REPRO showed the presence of

repeats of the over-represented residues in various com-

binations [Fig. 4(A)]. Similar sequence composition and

repeats have been found in a large number of eukaryot-

ic proteins where they have been shown to confer elas-

tomeric properties to that region of each protein.39

These include the flagelliform silk proteins (repeats of

GPGGx), spider silk proteins (GGX), the X-type HMW

subunit of wheat gluten and x-protein (repeats of

PGQGQQ and GQQ) and dragline protein (repeats of

GPGQQ).44 Each of these repeats occurs in the linker

region of E. coli SSB with most being repeated once and

GGX seven times [Fig. 4(A)]. In the eukaryotic proteins,

the same sequence is typically repeated many times.

The presence of this collection of repeats confers

elastomeric properties such as those observed in the

eukaryotic proteins enabling the C-terminal 69 resi-

dues to adopt different conformations thereby facilitat-

ing a larger range of partner proteins with which SSB

can bind. This sequence composition likely contributes

to the movements of the C-terminal domain of the pro-

tein associated with ssDNA binding.45

Our analysis of the linker region of EcSSB sug-

gests that the amino acids present in the region from

Q117 to Q147 have a propensity to form a PPII-like

helix.46–48 This is supported by modeling which

showed that this region could adopt a structure

resembling a collagen monomer. While this analysis

does not prove the existence of a PPII-helix (experi-

ments are underway to determine this possibility

experimentally), it provides some insight into what

this region of SSB could be doing. First, PP-II helices

are known to bind nucleic acids and to be involved in

protein–protein interactions.34,48,49 Our results

showing that perturbations in the sequence of this

region, as well as in changes in its position relative

to the core of SSB, severely impaired target protein

binding is consistent with a role in protein–protein

interactions. Second, the accessibility of PPII helices

is greatly enhanced by the fact that they are

frequently found either at the amino- or carboxyl ter-

mini of proteins where they form extended structures

that have been described as “sticky arms.”46–48 Third,

PPII-helices are flexible, and this has been suggested

to be the major feature underlying its function in pro-

tein interactions.50

However, this is not the only important compo-

nent of the linker region. The identification of PXXP

motifs in the C-terminus of SSB suggests a possible

mechanism for binding specificity. PXXP motifs are

most well known for their ability to bind structurally

conserved Src homology 3 (SH3) domains. These

domains are �50 residue modules that are ubiquitous

in biological systems and which often occur in signal-

ing and cytoskeletal proteins in eukaryotes.48,51–53

The SH3 domain has a characteristic fold which con-

sists of five or six beta-strands arranged as two tightly

packed anti-parallel beta sheets and is similar in

structure to the OB-fold.54 SH3-like domains have

been identified in several E. coli proteins, including

Exonuclease I, an SSB-interactome partner.14,55 This

prompted us to look at the crystal structures available

of SSB interactome partners to determine whether

they contained an OB-fold. Indeed, each structure

examined contains an OB-fold (not shown and

P.Bianco, manuscript in preparation).

Therefore, since SSB has PXXP motifs and each

partner as well as SSB has an OB-fold, we propose the

following model, shown in schematic fashion in Figure

8. The linker(s) from one SSB tetramer bind to the

OB-fold of the interaction partner. When the partner

is another SSB tetramer, cooperative binding to

ssDNA results with multiple interactions forming a

stable complex wherein the ssDNA is extremely well

protected. In similar fashion, when the nearby protein

is a member of the SSB interactome, storage or load-

ing of that protein on to the DNA results, as shown for

the fork rescue DNA helicase RecG.22,56 While the

OB-fold is most well-known for its ability to bind oligo-

saccharides and single stranded nucleic acids, peptide

binding by an OB-fold is not without precedent. A

scan of the PDB reveals more than fifteen instances


including matrix metalloproteinases, Staphylococcal

enterotoxin B, and yeast RNA polymerase II.15,57–59

If the model suggested above is correct, then

there are some obvious predictions. First, loss of

these motifs should eliminate binding to interactome

partners. This was clearly the case for RecG but not

for RecO, indicating that other parts of the IDL may

be important for binding as well. Second, removal of

these motifs should negatively impact cooperative

binding to ssDNA. This is exactly what was

observed by the Lohman group.23 Third, mutation of

the PXXP motifs should negatively impact. Prelimi-

nary data where the motifs were interrupted or

replaced eliminated binding (P. Bianco, manuscript

in preparation). Finally, elimination of the OB-fold

in an interacting partner should eliminate binding.

Consistent, removal of the corresponding regions

in both RecO and RecG eliminate binding entirely

(P. Bianco, unpublished).60

In summary, we propose that the linker region

of E. coli SSB is essential for optimal functioning of

the protein. While previously thought of as being

unimportant, our results show that this is no longer

the case. SSB has two seemingly disparate roles:

it binds rapidly and cooperatively to ssDNA and it

binds to partner proteins that constitute the SSB

interactome (Fig. 8). These two roles are not dispa-

rate but are instead, intimately linked. They are

linked by the properties identified in the IDL that

facilitate protein-protein interactions. When these

interactions occur between an SSB tetramer and an

interactome partner, loading of that protein onto

DNA takes place. When the interactions take place

between SSB tetramers, cooperative ssDNA binding

occurs. When these key elements—the putative

PPII-helix, the repeats and the PXXP motifs are

perturbed, SSB no longer functions correctly.23

Materials and Methods

Chemicals and reagents

All chemicals were reagent grade and were made up

in Nanopure water and passed through 0.2-mm pore

size filters. Ampicillin, IPTG, NaCl, Na2HPO4, tryp-

tone, and yeast extract were from Fisher Scientific

(NJ). Coomassie brilliant blue R-250, kanamycin,

lysozyme, sucrose, tris-base, triton x-100, sodium

dodecyl sulfate were from AMRESCO (OH). EDTA,

NaH2PO4, and nickel sulfate were from J.T. Baker

(NJ). Sodium deoxycholic acid and imidazole were

from Acros Organics (NJ). The infusion kit was from

Clontech (CA). Nonidet P40 was from USB (OH).

Protease and phosphatase inhibitor mini tablets

were from Thermo Scientific (IL). PMSF was from

Omnipur (NJ). The 1 and 5 mL HisTrap FF crude

columns, Ni Sepharose 6 Fast Flow were from GE

Healthcare Life Sciences (NJ). Oligonucleotides were

purchased from IDT (Coralville, IA) and are listed in

Supporting Information Table SI.

Cloning

A pET21a1 plasmid containing the E. coli ssb gene

was obtained from Dr. James Keck (UW-Madison).

The ssb gene was amplified by PCR and subcloned

into pET15b to produce his-SSB. Positive clones

were identified by restriction enzyme mapping and

verified by DNA sequencing. All oligonucleotides

used are listed in Supporting Information Table SI.

To create a series of truncated C-terminal linker

domain SSBs, the region comprising amino acids

113–168 was deleted in separate reactions by infusion

cloning. In these reactions, a single forward primer

(PB513) was used in combination with four reverse

primers (PB514-517) in separate reactions. PCR was

done using the various primer combinations and Infu-

sion to create the final construct. The mutants contain

the SSB N-terminal domain (residues 1–112), various

length linker regions (residues 113–168) and the

C-terminal tail (residues 169–178). The linker domain

mutants constructed were: SSB125 (53 residues delet-

ed; final length: 125aa), SSB133 (48 residues deleted;

final length: 130aa), SSB145 (33 residues deleted; final

length: 145aa) and SSB155 (23 residues deleted; final

length: 155aa). Positive clones were identified by

Figure 8. SSB linkers mediate all protein–protein interac-

tions. Schematics of wild type SSB proteins binding to

ssDNA and to partners. The colouring of SSB monomers is

as follows the core domain in green, linker in blue and acidic

tip in red. Here tetramers have their functional C-terminal

domains exposed in solution. Upon binding to ssDNA the

IDLs of monomers 1 and 2, bind to the OB-folds of mono-

mers 10 and 20, respectively. Concurrently, the linkers of

monomers 10 and 20 bind to the OB-folds of monomers 3 and

4, and their IDLs bind to monomers 30and 40, respectively.

The C-termini of subunits 30 and 40 are available to bind to an

incoming tetramer as shown on the right. On the opposite

side of each tetramer, C-termini are available for binding to

interactome partners such as RecG or RecO (purple and blue

ovals). For simplicity, SSB–SSB interactions are shown in the

top subunits only, and SSB interactome partners binding in

the lower subunits.


restriction enzyme mapping and verified by DNA

sequencing. The resulting plasmids created were:

pET21a1-SSB125, pET21a1-SSB133, pET21a1-SSB145,

and pET21a1-SSB155. Next, each mutant was cloned

using Infusion technology into pET15b to create

histidine-tagged fusion proteins. Positive clones were

identified by restriction enzyme mapping and verified

by DNA sequencing.

To construct N-terminal his6 tagged truncated

SSBs, primers (PB549 and 550) were used to amplify

truncated ssb genes from the vectors described above

followed by Infusion cloning of the PCR fragments

into pET15b at NdeI and BamHI sites.pET15b-wt-

recO was made by amplifying genomic E. coli recO

from MG1655 using primers (PB458 and 459) and

inserted into pET15b at NcoI and EcoRI sites via Infu-

sion Cloning. To create C-terminal histidine tagged

recO, the recO gene was amplified using primers

(PB462 and 463) and then inserted into pET28a1 at

NcoI and HindIII sites using Infusion Cloning. Posi-

tive clones were identified by restriction enzyme map-

ping and verified by DNA sequencing. The resulting

plasmid was pET28a1-his-rec0.pUC57 plasmids con-

taining the Thermotoga maritima (Tm) ssb and recG

genes were purchased from Genescript. The original

Thermotoga sequences were codon optimized for

expression in E. coli to produce SSB (Q9WZ73); and

RecG (Q9WY48). To create N-terminal his-TmSSB,

the pUC57 Tmssb gene was amplified by PCR using

primers (PB523 and 525) and inserted into NdeI and

BamHI sites of the pET15b vector (Novagen) using

Infusion cloning (Clontech). To create a pET28a1 plas-

mid expressing TmRecG, pUC57 containing TmrecG

gene was amplified by PCR using (PB526 and 528)

and inserted into NcoI and EcoRI sites of the pET28a1

vector using Infusion cloning. Positive clones were

identified by restriction enzyme mapping and

DNA sequencing. The resulting clones were: pET15b-

his-Tmssb, and pET28a1-TmrecG.

Construction of the E. coli–M. tuberculosis hybrid

genes was described previously.41–43 These genes were

amplified by PCR and cloned by Infusion into pET15b.

Positive clones were identified by restriction enzyme

mapping and DNA sequencing. The resulting plas-

mids and strains are listed in Supporting Information

Tables SII and SIII, respectively.

Dual plasmid expression

To facilitate coexpression of his-RecO with potential

binding partners, pET28a1-his-recO was transformed

into Tuner (kDE3) cells. A colony in which high levels

of expression of his-RecO was observed was selected

and made competent. Next, these cells were trans-

formed separately with test plasmids encoding wtSSB,

SSB125, SSB133, SSB145, and SSB155. Next, expression

of his-RecO and the SSB proteins were determined

using 5-mL cultures. Fresh overnights were used to

inoculate LB1 antibiotics (ampicillin and kanamycin,

250 and 25 lg mL21, respectively) and cells grown at

378C for 3 h. IPTG was added to either 100 lM, 500

lM, or 1 mM final, followed by an additional 2 h of

growth. Protein expression levels were assessed using

SDS-PAGE. A colony in which high levels of both his-

RecO and the SSBs were observed was selected and

grown overnight in LB1 antibiotics and 0.2% glucose.

The overnights were used to inoculate 0.1 and 2 L

cultures the following day at a 1:100 ratio.

To facilitate coexpression of TmhisSSB with poten-

tial binding partners, Tuner (kDE3) cells that

expressed his-Tmssb from the pET15b plasmid were

made competent and transformed separately with

pET28a1-TmrecG, pET28a1-EcrecG, or pET28a1-

EcrecO plasmids. Next, expression was determined as

described above except IPTG was used at 500lM. Pro-

tein expression levels were assessed using SDS-PAGE.

A colony in which overexpression of both his-TmSSB

and the target binding proteins was observed was

selected and grown overnight as described above. The

overnights were used to inoculate 1- and 2-L cultures

the following day at a 1:100 ratio.

0.1, 1, and 2 liters cell growth

The 1, 10, and 20 mL of fresh overnight cultures

were added to 100 mL, 1 L, and 2 L of LB, respec-

tively. LB contained ampicillin (250 lg mL21) and

kanamycin (25 lg mL21). Cells were grown at 378C

with vigorous shaking. When the OD600 was between

0.4 and 1, IPTG was added to 500 lM final, and

growth continued for an additional 3 h. Cells were

harvested by centrifugation at 48C and cell pellets

were resuspended in lysis buffer (50 mM Tris-HCl (pH

8.0), 20% sucrose) using 2.05 mL g21 of wet cell

weight. Resuspended cells were frozen at 2208C or

stirred at 48C overnight for lysis the following day.

Elution of proteins using an imidazole gradientThe 100 mL, 1 L, and 2 L cultures were lysed and

the cleared cell lysate was subjected to affinity chro-

matography using a 1 mL or 5 mL HisTrap FF crude

column. To lyse the cells, lysozyme (1 mg mL21

final), benzonase 5 lL, Mg(OAc)2 (4 mM final),

CaCl2 (2 mM final), PMSF (1 mM final), protease

inhibitor mini tablets (50 mL cells/tablet) were

added and mixture stirred for 30 min at 48C. Deoxy-

cholate (0.5% final) and triton x-100 (0.1% final)

were added slowly followed by stirring for 40 min.

Imidazole and NaCl were then added to 30 and

600 mM final, respectively, and stirred for 5 min.

The whole cell lysate of each was centrifuged at

35,000g at 48C for 30 min. The cleared cell lysate

was loaded onto a nickel column equilibrated in

Binding Buffer (20 mM sodium phosphate; 600 mM

NaCl; 30 mM Imidazole; pH 7.4). The column was

sequentially washed with 400 mL binding buffer,

350 mL binding buffer with 0.2% NP40 and 250 mL

binding buffer only. A linear gradient of 30–500 mM


of Imidazole was used to elute bound proteins from

the column. Proteins were assessed by SDS-PAGE.

Following electrophoresis, gels were stained with

Coomassie brilliant blue, destained, and photo-

graphed. Protein fractions were pooled and then pre-

cipitated in 70% ammonium sulfate for short term

storage or further processing.

Small scale, step-elution of proteinsCell lysis, centrifugation and buffers were identical

to that of larger cultures with volumes adjusted

accordingly. The cleared cell lysate was loaded onto

the 1 mL HisTrap FF crude column. The column

was washed with 45 mL binding buffer. Proteins

were eluted using 6 mL of Elution buffer containing

500 mM Imidazole. The 1 mL fractions were collect-

ed and aliquots subjected to SDS-PAGE. The majori-

ty of protein(s) eluted in the third fraction.

Analysis of coomassie stained gels

Photographs of stained, SDS-PAGE gels were ana-

lyzed using the Gels Analysis function in Fiji.61,62

Here, the area under each peak was determined from

the densitometric trace of each lane. This value was

then divided by the molecular weight of each protein

to correct for the different amounts of Coomassie dye

being present in bands, assuming each protein stained

equally well with Coomassie. The ratio of protein A:

protein B was then determined as is displayed in vari-

ous graphs throughout the article. Each coelution

experiment was repeated two to five times, with

a minimum of three lanes per gel analyzed and

combined to yield the resulting graphs.

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