dna replication the basic rules for dna replication dna synthesis at the replication fork ...

DNA Replication

The basic rules for DNA replication

DNA synthesis at the replication fork

Termination of replication

Other modes of DNA replication

DNA Polymerases

Initiation of replication

Regulation of re-initiation

Initiation of replication

Common features of replication origins

Common events of initiation

Priming

(proposed by F. Jacob, S. Brenner and J. Cuzin, 1963)

All the DNA replicated from a particular origin as a replicon.

Binding of the initiator to the replicator stimulates initiation of replication.

The replicon model of replication initiation:

Replicator

Replicator: the entire set of cis-acting DNA sequences that is sufficient to direct the initiation of DNA replication (*the origin of replication is part of replicator)

Initiator

Initiator: the DNA-binding protein that specifically recognizes a DNA element in the replicator and activates the initiation of replication

Initiator binding siteEasily melted region

Replicator

InitiatorDNA binding

DNA unwinding

Protein recruitment

Priming and DNA synthesis

Th

e f

un

cti

on

s o

f in

itia

tor

(Origin)

DNA binding

DNA unwinding

Protein recruitment

Priming and DNA synthesis

oriCDnaA

DnaB/DnaC

E. coli

oriC : the origin of replication in E. coli

Easily melted(AT rich)

Initiator (DnaA) binding site

Figure 14.26

Consensus sequence

GATCTNTTNTTTTCTAGANAANAAAA

Consensus sequence

TTATNCANAAATANGTNT

L, M, R repeats (13 bp) 1~4 repeats (9 bp)

oriC:

The replicator (origin) of S. cerevisiae:

圖引用自： Cooper, G. M. (1997) The cell: a molecular approach. ASM Press. Fig. 5.17

Origin recognition complex (ORC)

(Initiation complex)

ACS: ARS consensus sequence

ARS (Autonomously replicating sequence)

~ 150 bp

Mutation in ARS

Mutations in B elements reduce origin function.

Mutations in core consensus abolish origin function.

Fig

ure

13.2

0

DNA binding

DNA unwinding

Protein recruitment

Priming and DNA synthesis

oriCDnaA

DnaB/DnaC

E. coli

Fig

ure

14.2

7

Watson, J. D. et al. (2004) Molecular Biology of the gene. 5th ed. CSHL Press. Fig. 8-26.

DNA helicase(DnaB)

DNA helicaseLoader (DnaB)

DnaA.ATP

DnaAATP

HU + ATP

(histone like protein)

DNA bending

Super-helical tension

Strand separation

DNA helicase(DnaB)

DNA helicaseLoader (DnaB)

Fig

ure

14.1

0

Two types of function are needed to convert dsDNA to the single-stranded state:

2. Single-strand binding proteins bind to the ssDNA, preventing it from reforming the duplex state

1. Helicases separate the strands of DNA, usually using the hydrolysis of ATP to provide the necessay energy

Single-strand binding proteins

DNA helicase(DnaB)

DNA helicaseLoader (DnaB)

Primase

For DNA replication, a primase is required to catalyze the synthesis of RNA primer.

Primase in E. coli:

An RNA polymerase

Encoded by the dnaG gene

Synthesizing short stretches of RNA

Fig

ure

14.1

4

Protein required to initiate replication at the E. coli origin:

Protein Function

DnaA protein Recognizes origin sequence; open duplex

at specific sites in origin

DnaB protein (helicase) Unwinds DNA

DnaC protein Required for DnaB binding at origin

HU DNA bending protein; stimulates initiation

Primase (DnaG protein) Synthesizes RNA primers

Single-strand DNA-binding protein (SSB)

Binds single-strand DNA

DNA gyrase

(DNA topoisomerase)

Relieves torsional strain generated by DNA

unwinding

Dam methylase Methylates 5’-GATC-3’ sequences at oriC

Summary

DNA Replication

The basic rules for DNA replication

DNA synthesis at the replication fork

Termination of replication

Other modes of DNA replication

DNA Polymerases

Initiation of replication

Regulation of re-initiation

DNA synthesis at the replication fork

Proteins at the replication forks

Coordinating synthesis of the lagging and leading strands

in eukaryotic cells

in E. coli

DNA replication is semidiscontinuous.

Figure 14.9

1000-2000 nt in prokaryotes100-400 nt in eukaryotes

Okazaki fragments

*SSB: Single-strand binding proteins

Topoisomerase

HelicasePrimase

SSB

DNA replicase(DNA polymerase)

Primosome

Proteins required at the replication forks:

Primer removal enzyme

DNA ligase

Different replicase units are required to synthesize the leading and lagging strands.

In E. coli both units contain the same catalytic subunit of DNA Pol III. In other organisms, different catalytic subunits may be required for each strand.

The helicase creating the replication fork is connected to two DNA polymerase catalytic subunits.

Each polymerase catalytic subunit is held on DNA by a sliding clamp.

Figure 14.19

The polymerase that synthesizes the lagging strand dissociates at the end of Okazaki fragment and then reassociates with a primer in the single- stranded template loop to synthesize the next fragment.

The polymerase that synthesizes the leading strand moves continuously.

Figure 14.19

In E. coli: DnaB

DnaGDNA Pol III

holoenzyme

’

E. coli DNA Polymerase III holoenzyme

Based on Figure 14.17

Core enzyme

proofreading

polymerizationpolymerization

Sliding

clamp

’

Clamp loader

Core enzyme dimerization

Sliding

clamp

’

Clamp loader

ATP

’ATP

’ADP

’ATP

Sliding clamp

ATP

hydrolysis

Pi

Core enzyme: 10 ~ 15

Holoenzyme: >500000

converts Pol III from a distributive enzyme to a highly processive

enzyme.

Processivity

Figure 14.18

’

subunits maintain dimeric structure of Pol III and interact with DnaB

DnaB(helicase)

Lagging strand synthesis

Leading strand

synthesis

Each catalytic core of polymerase III synthesizes a daughter strand.

DnaB (helicase) is responsible for forward movement at the replication fork.

Figure 14.20

What happens to the loop when the Okazaki fragment is completed?

Figure 14.21

Initiation of

Okazaki fragment

Termination of

Okazaki fragment

Dissociation of core

and clamp

Reassociation of clamp

1

2 3

4

Figure 14.20

5. Reassociation of core

Each Okazaki fragment is synthesized as a discrete unit.

Primase synthesizes RNA primer.

DNA Pol III extends primer into Okazaki fragment.

Next Okazaki fragment is synthesized.

Leading strand

Lagging strand

Okazaki fragments are linked together.

DNA Pol I uses nick translation to replace RNA primer with DNA.

Ligase seals the nick.

Figure 14.22

5’3’3’ 5’

RNA primer

5’3’ Exonuclease activity of DNA Pol I:

Figure 14.5

Ligase NH3+

Ligase NH2

+P

O

O-

O Ribose Adenine

+

Adenylylation of DNA ligase

P

O

O-

O Ribose AdenineOR

AMP

NAD+ (R = NMN)or ATP (R = PPi)

11

Mechanism of the DNA ligase reaction

+ NMN (or PPi)

Ligase NH2

+P

O

O-

O Ribose Adenine

Ligase NH3+

Activation of 5’ phosphate in

nick

22

33

Fig

ure

14.2

3

*

PO O-

O

O

PO O-

O

RiboseAdenine

HO●●

The 3’-hydroxyl group attacks the phosphate and displaces AMP, producing a phosphodiester bond.

3’

33

Pol/primase

Eukaryotic cellEukaryotic cell

(RFC)

PCNA

Pol

Pol

Eukaryotes have many DNA polymerases.

Fig

ure

14.2

4

Eukaryotic DNA polymerases for replication in nucleus:

DNApolymera

se

Primaseactivity

Processivity

Proof-reading

Function

+ moderate - Primer synthesis

- High +

Leading/lagging? strand

synthesis

- High +laggingstrand

synthesis

DNA polymerase Pol/primase):

2 subunits: Pol

2 subunits: primase

DNA synthesis

RNA synthesis

3’ 5’

RNA DNA (iDNA)

5’ 3’OH

~ 10 bp 20-30 bp

DNA polymerase switching during eukaryotic DNA replication:

DNA Pol / primase RNA primer synthesis

by primase

DNA synthesis by Pol

P

RNA

iDNA Wats

on,

J. D

. et

al. (

20

04

) M

ole

cula

r B

iolo

gy o

f th

e g

ene.

5th

ed.

CSH

L Pre

ss.

Fig.

8-1

6.

Slidingclamp Pol/primase

DNA Pol (or

iDNA

*

R-FC binds to the 3’ end of iDNA and displaces pol /primase

PCNA binds pol or

RF-C: Clamp loader

PCNA: Sliding clamp

*

R-FC attracts PCNA

PCNA- Proliferating cell nuclear antigen

：

圖引用自： Voet, D., Voet, J. G. and Pratt, C.W. (1999) Fundamentals of Biochemistry. John Wiley & Sons, Inc. Fig. 24-1

(trimer)

Proteins required at the replication forks:

Summary

DNA topoisomerases are also required!

Fig

ure

14.2

5

There are two ways to think of the relative motion of the DNA and replication machinery:

1. The replication machinery moves along

the DNA. (similar to a train moving along its

track)2. The DNA moves while the replication machinery is static. (similar to film moving into a movie projector)

The two replisomes of E. coli are linked together and tethered to one point on the bacterial inner membrane.

Helicases(double-hexamers)

Pol III holoenzyme

Pol III holoenzyme

圖引用自： Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-18a

Origin

TerminatorChromosome

Replisomes

Replication begins

Origins separate

Cell elongates as replication continues

Chromosomes

separate

Cells divid

e

圖引用自： Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-18a