Replication English

Molecular Genetics PCB4522 Spring 2004 Lecture 5-Replication-part D Dr. Eva Czarnecka-Verner Course web page: http://PCB4522.IFAS.UFL.EDU --Or go to Microbiology & Cell Science home page and look under course material.

Replication of E. coli chromosome Genes VIII, Chapter 14

Replication requires DNA Polymerase III In E. coli: 1. Single type of catalytic subunit (dnaE) used in replication of both strands

2. Active replicase is a dimer; each half (enzyme unit) contains dnaE subunit & other proteins In B. subtilis: 1. Two different catalytic subunits:

a) Pol C (homolog of E. coli dnaE) synthesizes the leading strand b) dnaEBS synthesizes the lagging strand In eukaryotes: 1. The same overall structure of DNA pol III 2. Different enzyme units synthesize leading and lagging strands 3. Not clear whether the same or different catalytic subunits used

Replication requires DNA Polymerase III • DNA polymerase III holoenzyme: 900 kDa complex a) a catalytic core α subunit (dnaE) b) a 3’-5’ proofreading ε subunit (dnaQ) c) a θ subunit that stimulates exonuclease d) a dimerization component τ that links two cores e) a processivity component β that keeps polymerase on DNA (clamp) f) a clamp loader γ that places the processivity subunit β on DNA (complex of 5 proteins)

Forms of DNA polymerase III from biochemical studies: Core:

α 130 kD catalytic

25 kD ε proofreading

θ 10 kD structuralholds together

Pol III*: ε ε θ θ

α increased processivity

τ

τ

τ maintains dimeric structure

α 71 kD

Forms of DNA polymerase III from biochemical studies: γδ complex makes β clamp bind to primed template Pol III’:

α

Clamp loader γ 52 kD

32 kD δ ε ε θ θ α τ

τ

γδ ATP

Only one γδ complex γδ complex adds a pair of β dimers

Holoenzyme:

δ’ ψ χ

β β

Asymmetric

δ’ ψ χ

ADP + P

α

ε ε θ θ τ

Clamp

τ

δ α

γ

Assembly stages for Pol III holoenzyme: γδ is the clamp loader β dimer & γδ recognize primer-template δ’ ψ χ δ’ ψ χ δ γ Conformation β β δ γ change of β ATP

ADP + P

β dimer clamps on DNA Increased processivity

β ε θ

β

δ’ ψ χ δ

γ

α

Core joins complex

Assembly stages for Pol III holoenzyme: Q:Asymmetric; only one clamp loader- why? Lagging strand β α

β ε θ τ

Leading strand

δ’ ψ χ

β α

β β βδ ε ε θ θ α τ

τ forms dimeric structure

γ

τ

A:Different abilities to holoenzyme dissociate from DNA

Q: Which 2 subunits are encoded by the same DNA? A: tau (τ) and gamma (γ) use different reading frames of the same DNA.

β subunits of DNA pol III (head to tail dimer) 12 α -helices/6-fold symmetry Interactions with DNA via water molecules

The clamp

β dimer makes holoenzyme highly processive β dimer bound to DNA but slides along “Ice-skating”

β dimer is ring shaped; assembly or removal requires energy (γδ )

Fig. 14.18, Genes VIII by B. Lewin

Replication of E. coli chromosome

3’

dnaB helicase

Pol III γ

5’

1

β

Replication of E. coli chromosome

3’ γ

5’

2

Replication of E. coli chromosome

3’ γ

5’

DnaG

3

Replication of E. coli chromosome

3’ DnaG γ

5’

RNA primer

4

Replication of E. coli chromosome

3’

5’

5

γ

The template for a lagging strand is pulled through creating a loop in DNA

DnaG

Replication of E. coli chromosome

3’

5’

γ

6

The template for a lagging strand is pulled through creating a loop in DNA

Replication of E. coli chromosome 2nd Core Pol III 3’ β

5’ γ The loop is released

7

1st Core Pol III

Replication of E. coli chromosome

3’ γ

5’

8

Core Pol III

New β clamp present on DNA

Organization of the oriC Replication Fork ATP→ AMP

Pol III DnaB/DnaC core

DNA is pulled through the primosome

DnaB β β

τ

Note: the loading of DnaB helicase by DnaC only occurs at the origin.

Pol III core SSB

3’

5’ end of Okazaki fragment 5’

DnaG primase; stimulated by DnaB

What is responsible for recognizing the sites for initiating synthesis of Okazaki fragments? Dual properties of dnaB helicase: 2) Propels the replication fork 3) Interacts with dnaG primase at a correct site

Semidiscontinuous replication: The lagging strand fragments are known as “Okazaki fragments.” Usually 1,000 to 2,000 bases in length. 5’ 3’ lagging strand 1

5’-CTG-3’ GAppp-5’

2 RNA primer (~11-12 bases) (RNA polymerase=dnaG primase)

Schematic of one side of the replication fork

OriC Primosomedirected synthesis DnaB

DnaG primase; stimulated by DnaB

Related fact: At oriC, the primosome consists of DnaB and DnaG.

3’

5’

DnaB Role of DnaB: 1.) propels the replication fork through its helicase activity. 2.) required to activate primase (DnaG).

DnaG primase; stimulated by DnaB

3’

5’

τ binds dnaB to attach pol III core to replication fork.

OriC Primosomedirected synthesis

1.

Role of Pol III: 1.) Synthesis of leading strand. 2.) synthesis of lagging strand by extending the RNA primer. Displaces primase. 3.) pulls the lagging strand template through the holoenzyme.

Speed of DNA synthesis increased 10x

2. Prevent leading strand from falling off (increased processivity)

DnaG (primase)

3’

5’

oriC replication fork

Eukaryotic DNA pol α /primase DNA Pol α (Ι): primase complex- bifunctional Heterotetrameric phosphoprotein 48 kDa PRIMASE- initiates DNA synthesis (makes complementary RNA primer) of new strands 58 kDa protein- tethers primase to 180 kDa subunit 180 kDa polymerase A subunit- extends RNA primer by making a short iDNA (20-30 bases) (only in Drosophila has proofreading activity); not very processive polymerase

70 kDa subunit- no known catalytic function (may recruit polα:primase to the replication fork)

Eukaryotic DNA Replication: 1. DNA pol α (Ι) / primase: a) initiates synthesis of lagging and leading strands. b) RNA (~10 b)-iDNA (~20-30 b) primer. 2. DNA pol δ (ΙΙΙ) : a) elongates leading strand continuously b) highly processive (interacts with RF-C & PCNA) c) can dimerize- may also elongate the lagging strand 3. DNA pol ε : a) may be involved in lagging strand synthesis b) other functions 4. Replication factor C (RF-C): a) clamp loader; binds to 3’ end of iDNA & loads PCNA b) ATPase activity used to open PCNA ring

Eukaryotic DNA Replication: 5. PCNA (proliferating cell nuclear antigen) : a) tethers DNA pol δ to the template b) acts as processivity factor for strand (like β clamp) elongation d) trimer forms a ring that surrounds DNA 6. Replication factor RF-A a) single strand binding protein 7. Topoisomerases I & II: maintains DNA winding 8. Exonuclease MF1 a) removes RNA primers 9. T antigen helicase; T antigen loading helicase 11. DNA ligase I a) seals the nicks

Eukaryotic DNA Replication: Notes:

Eukaryotic replication fork contains one complex of DNA polα/primase & two other pol complexes; either 2x δ’s or one δ & one δ/ε In mammalian systems (DNA pol has no 5’-3’ exo activity) Okazaki fragments removed by: c) RNAse HI (specific for RNA-DNA hybrid) endonuclease cuts b) FEN1 exonuclease removes the RNA (5’-3’)

Similar functions at bacterial and mammalian replication forks: Function

E. coli

HeLa/SV40

•helicase

DnaB

T antigen

•loading helicase • single strand • priming

DnaC SSB DnaG

T antigen RF-A Pol α(Ι)/primase

• sliding clamp

β

PCNA

• clamp loading

γδ

RF-C

• catalysis

Pol III core

Pol δ(ΙΙΙ)

• holoenzyme dimerization

τ

???

• RNA removal

Pol I

MF 1 exonuclease

• ligation

ligase

ligase 1

Origins: 1. ColE1 (RNA II acts as primer) 2. ΦΧ174 replicative form 3. oriC hairpin ColE1 plasmid

ΦΧ174 (+)

RNA II

primosome

Creating replication forks at oriC 1. the strands melt at the origin over a short distance. 2. DNA is unwound. 3. first nucleotides synthesized into RNA primer. Occurs only once for leading strand-many times for lagging strand.

Creating replication forks at oriC 1. Initiation at oriC starts with complex formation of 6 proteins: a) DnaA, DnaB, DnaC, HU, Gyrase and SSB 2. DnaA uniquely involved in initiation 3. DnaB/C “engine”of initiation at origin

Minimal oriC DnaA binding Region of melting L M

R

1

2

13-mers

3

4

9-mers 245 bp

GATCTNTTNTTTT

TTATNCANA Note: GATC is Dam methylation site; 11 copies of GATC in oriC

Creating replication forks at oriC 1. Binding of DnaA to four 9 bp sites at on right side of the origin. 2. 2-4 DnaA monomers form a tetramer and DNA melts at the three 13 bp sites on the left side. 3. DnaB/DnaC joins the complex to form bidirectional replication forks.

2-4 monomers bind cooperatively

Minimal oriC DnaA protein

(4) 9-mer sites ATP→ AMP DNA strands melted DnaB/DnaC DnaB hexamers at (3) 13-mer sites

Central core

DnaB binds & displaces DnaA from 13 bp repeats

Minimal oriC Q: Does DnaA act as the titrator that measures number of origins vs. cell mass? A: Mutations DnaA-replication asynchronous; overproduction of DnaA-initiation starts at reduced cell mass ATP→ AMP

DnaB/DnaC

DnaB hexamers

Creating replication forks at an origin Other proteins are involved in replication: 1. Gyrase: acts as a swivel allowing one strand to rotate around the other. 2. SSB stabilizes single stranded DNA as it forms 3. HU: (HU1/HU2) general DNA (double stranded & single stranded) binding protein. Bends DNA; structural role? Similar to histones. No cooperativity in binding. Causes DNA to bend and fold into structure that leads to open complex formation & resembles beaded chromatin

Creating replication forks at an origin ATP required in replication: 1. 2. 3. 4.

For helicase to unwind the strands For gyrase to swivel strands For primase to initiate For DNA pol III to be activated

Methylation state of DNA may regulate replication Active origin (N6) Me

Me Single round replication

GATC CTAG

(13 min. delay) Dam methylase

GATC CTAG

methylated DNA

Me

methylated DNA

Active origin

GATC CTAG

Inactive origin

Me hemimethylated DNA accumulates

Methylation state of DNA may regulate replication Me

Membrane-bound inhibitor- competes with DnaA for oriC

GATCnnnnnnnnnnn CTAGnnnnnnnnnnn

hemimethylated DNA

Inactive origin Dam methylase (delayed)

~13 min. at oriC vs <1.5 min for GATC elsewhere in the genome Me

DnaA protein

GATCnnnnnnnnnnn CTAGnnnnnnnnnnn Me Active origin

Inhibitor released; DnaA can initiate

dnaA promoter repressed; also has delayed methylation; reduced level of DnaA protein

Methylation state of DNA may regulate replication 1. SeqA inhibitor binds to hemimethylated DNA what delays re-replication 2. SeqA may interact with DnaA 3. Hemimethylated origins bind to cell membraneinaccessible to methylases 4. Methylated origins do not bind to membranes 5. No clear connection between the origin and membrane

The end of lecture 5