DNA Repair II

what happens when a lesion is encountered by the DNA replication machinery?

depends on where it is encountered and how severe it is

can be repaired or bypassed in various ways

lesion repair methods based on nature of lesion

1. translesion synthesis (error prone repair)

2. stalled replication fork followed by replication fork regression

3. gap repair of a lesion bypass

4. double strand break repair

translesion synthesis

error prone repair

involves replicating the lesion without correcting the error, requires DNA polymerases that do not possess proof-reading activities

fork stalls and regression (recombination)

if lesion cannot be bypassed (replicated or immediately repaired), replication fork can stall and backtrack

occurs when DNA pol III and other bypass pols cannot replicate DNA in vicinity of lesion- presents a bigger problem than the pol stalling and being replaced

two outcomes

1. regressed fork permits recruitment of nucleotide excision repair (NER) machinery, NER occurs, daughter strands are degraded by the exonuclease activity, then replication continues

2. the daughter DNA lacking the lesion can serve as a template for replication without correcting the lesion in the parent strand. lagging strand is temporarily used as a template for leading strand which are then reunited with their parents following branch migration

double-strand break repair (recombination)

if the lesion in the parent DNA is large the replication fork can collapse and reestablish

this involves recombination machinery that generates diversity of chromosomes

gap repair of a lesion bypass (recombination)

if the lesion in the parent DNA can be bypassed the replication fork continues, i.e. reinitiates replication downstream. this leaves the parent template single stranded in the region of the DNA lesion

if this occurs, recombination proteins (RecA) can trigger a strand invasion across the replication fork

the daughter strand of the opposite template can be used as a template

Note: DNA lesion still present, it was only shifted onto the other parental DNA template. However, now it can be repaired by NER or BER

translesion synthesis steps

1. DNA pol III (responsible for chromosomal replication) is stalled at a DNA lesion because the mismatched base pair or DNA damage distorts DNA too heavily

2. a bypass polymerase can temporarily take over DNA replication

3. the bypass polymerase continues to replicate DNA selecting the least bad dNTP(s) for the lesion. eventually it is replaced by DNA pol III again

how does a bypass polymerase take over replication?

have larger active sites that may accommodate bulky lesions

main one in E. coli is Pol V although Pol II and Pol IV could also be recruited

why is translesion synthesis so error prone?

both pol IV and pol V lack 3’ to 5’ proof reading (exonuclease activity)

can give rise to mutation including indels

fork stalls and regression (recombination) steps

1. no DNA pol can bypass DNA lesion

2. replication fork stalls with the daughter strand of the opposite DNA template having been duplicated beyond the DNA lesion in the problem template

3. recruitment of RecA, bacterial recombination protein, can trigger replication fork regression (reversal) reuniting the parental template DNA before the site of the DNA lesion → this permits formation of Holliday intermediate comprising the junction of 4 DNA strands, Holliday intermediate is formed when the longer daughter strand serves as a template for the stalled daughter strand

4. either lesion repaired by NER and daughter strands degraded by exonuclease activity, or lagging strand temporarily used as a template for the leading strand which then reunite with parents following branch migration

Holliday junctions

branched structures that contain 4 double stranded arms, can exist in different conformations

branch migration

occurs when DNA strands relocate (originating from Holliday junctions) to form alternative base-pairing without changing the number of paired nucleotides in comparison to previous structure

gas repair of a lesion bypass (recombination) steps

1. DNA pol dissociates from lesion DNA and reinitiates replication downstream

2. strand invasion, facilitated by RecA, results in the lesion baring parental template being swapped for the other daughter strand (lagging strand)

3. branch migration facilitates DNA replication across the gap using the daughter strand from the undamaged parent DNA as a template

4. recombination ensues then resolve the two Holliday junction intermediates and DNA ligase closes the remaining nicks