Lecturr 4 DNA Replication 2 Duplication

Question 1

Single origin of replication Ori C

Answer 1

E.coli has a single origin of replication - where chromosome replication is initiated- called Ori C ( C for chromosome) DNA synthesis starts here at short RNA primer synthesised by DnaG primase. 2 bidirectional replication forks formed with leading & lagging strand. DnaB helicase unwinds parental duplex to allow assembly of DNA replication proteins.

Question 2

Ori C

Answer 2

Consists of 245bp sequence for replication
5 repeats of 9bp sequence are binding sites (R1-5) for key initiator protein
DnaA protein associates with these sequences at ori c 3 additional repeats (I1-3) are bound to DnaA when complexed with ATP.

IHF and FIS are DNA bending proteins aiding initiation reaction

DNA unwinding element (DUE) is AT rich sequence and sit where DNA opened for replication

As DUE melts (base pairs pulled apart) DnaA becomes more compact facilitating unwinding

DnaA DNA melting protein not helicase

Question 3

DnaB loadedat fork by DnaC

Answer 3

Loading DnaB ring protein onto DNA without ends (most bacterial chromosomes are circular) is difficult. DnaC loads DnaB at Ori C opened by DnaA.

DnaC ATP ring binds to DnaB ring and opens it for loading. Binding of DnaBC complex to DnaA promotes assembly of two DnaB rings one for each replication fork (DnaB encircles lagging strand template) ATP hydrolysis releases DnaC leaving DnaB bound to DNA

Question 4

Accessory proteins: SSB

Answer 4

1)Stops unwound parent strands from reannealing
2) prevents intramolecular strands annealing (hairpins can lead to deletions)
3) protects DNA from degradation/damage
4) acts as assembly point for multiple proteins involved in DNA metabolism (via flexible SSB C-terminus)

However SSB can block access of proteins involved in DNA replication recombination and repair

Question 5

Accessory proteins DNA gyrase

Answer 5

Prevents supercoiling Infront of replication fork which could block progression.

By cutting 2 strands of DNA molecule and passing another through the break before resealing the cut strands.

Gyrase removes pos supercoils so DNA relaxes ahead of fork facilitating uncoiling

Question 6

3 DNA polymerases in E. Coli

Answer 6

DNa pol 1) removes RNA primer and replaced with DNA

DNA pol 2) fills gaps following repair of DNA damage

DNA pol 3) chromosome replication - adds nucleotides 250-1000 nt/second) highly processive. Initiates synthesis by extending an RNA primer. Lagging strand loops resulting in directional alignment of leading/lagging strand polymerases.

Question 7

Polymerase proofreading

Answer 7

All 3 can proofread ( check and correct errors of nucleotide insertion)
Without 3’5’ exonuclease activity of proofreading subunit of DNA Poly 3 one nucleotide every 7x10^6 (7million) would be incorrect

E. Coli has a genome of 4.6x10^6 genome so every second replication could have a mutation.

After 30 gen (overnight growth of culture) every cell would have approx 15 mutations.

Proofreading of DNA Poly 3 reduces error to 1 in 10^9 i.e. 1 mutation per 1000 replications

Question 8

DNA polymerase 3

Answer 8

Consists of 2 alpha subunits.
A separate epsilon subunit specifies 3’5’ exonuclease for proof reading.

Two tau units attach to the alpha polymerase subunits and hold them together.

Gamma complex or clamp loader consists of gamma, δ (Delta), δ’ and τ (tau). It loads the beta sliding clamp.

Beta clamp is a ring (homodimer) that encircles parental strand on each daughter template. It ensures that polymerase complex does not fall off during DNA synthesis (enhancing processivity.) Because it’s a ring it must be opened to load newly primed lagging strand. Gamma complex bound to ATP allows opening of the clamp while ATP hydrolysis allows clamp release and ring closure.

Leading strand primed at both replication origins (bidirectional fork) lagging strand primed at each Okazaki fragment by DnaG. New beta clamp loaded on each RNA primer by clamp loader (gamma complex)

As lagging strand polymerase synthesises Okazaki fragment clamp loader opens a new clamp and helicase (DnaB) recruits primase (DnaG) to replication fork to initiate next fragment. Completion of fragment triggers recycling of lagging strand polymerase to newly loaded clamp leaving old clamp behind. Lagging strand synthesises new Okazaki fragment completing full cycle. Fork unwinding and leading strand synthesis continue throughout the cycle.

Question 9

Summary

Answer 9

DnaA opens parental duplex at Ori C (single origin of replication)

DnaC loads DnaB helicase

DnaB (replicative helicase) unwinds duplex at fork

SSB keeps unwound strands apart

DnaG synthesises RNA primer on lagging strand (and one on leading strand)

Alpha subunits synthesise DNA on leading strand and lagging strands ( as part of larger DNA Poly 3 complex)

Tau subunits ensure dimerization of core polymerase subunits

Gamma complex loads beta clamp at each Okazaki fragment

Beta clamp encircles DNA and binds complex to fork

RNA primers are removed by DNA poly 1

DNA ligase seals nicks

Question 10

How long does it take E. Coli to replicate?

Answer 10

E. Coli has 4.6 x 10^6 BP in its single circular chromosome. DNA pol 3 works at 900nt per second

Divide length of genome by 900 giving 511 seconds (÷60) is 85 mins. However there are 2 replication forks so answer is 42 mins. It takes around 40 mins for E. Coli to replicate it’s chromosome and 20 mins to form septum and complete cell division.

Although it can divide at faster rates as quick as 20-25 mins ( next round of replication initiated before first completed)