Template-switching during replication fork repair in bacteria

Abstract

Replication forks frequently are challenged by lesions on the DNA template, replication-impeding DNA secondary structures, tightly bound proteins or nucleotide pool imbalance. Studies in bacteria have suggested that under these circumstances the fork may leave behind single-strand DNA gaps that are subsequently filled by homologous recombination, translesion DNA synthesis or template-switching repair synthesis. This review focuses on the template-switching pathways and how the mechanisms of these processes have been deduced from biochemical and genetic studies. I discuss how template-switching can contribute significantly to genetic instability, including mutational hotspots and frequent genetic rearrangements, and how template-switching may be elicited by replication fork damage.

Keywords: Copy number variation; DNA replication; Genetic recombination; Mutagenesis; Postreplication repair; Quasipalindrome.

Figure 1

Post-replication repair pathways that operate in gap-filling. A. Homologous recombination is initiated by RecFOR promoted RecA binding to ssDNA gaps. The RecA filament on ssDNA signals induction of the SOS DNA damage response and initiates strand invasion of the gap with the duplex DNA of the sister chromosome. Resolution of a double Holliday junction by RuvABC restores an intact chromosome. B. Translesion synthesis involving the exchange of Pol II, IV or V for the replicative Pol III polymerase can fill gaps, especially those caused by template lesions. C. A cross-fork template switch pathway can provide an alternative template for nascent strands. Due to mispairing of nascent strands in the annealing step, this pathway can lead to RecA-independent rearrangements between tandem direct repeats. This mechanism can also lead to crossovers between sister chromosome (see Fig. 3).

Figure 2

Fork reversal and template-switching. Step 1: A replication fork is blocked by a leading strand lesion (blue triangle). Branch migration factors drive reversal of the fork, allowing the nascent strands to pair. This reversal generates a Holliday junction, with one arm as a double-strand ended “nub”. Step 2: If the 3′ nascent strand is recessed relative to the 5′ nascent strand, synthesis can occur from this alternative template. Step 3: Reversed branch migration regenerates a fork structure, with the newly synthesized DNA bypassing the blocking lesion. Step 4: Alternatively, cleavage of the Holliday junction can break the fork, generating a double-strand end that can be repair by subsequent RecABCD-mediated DSB repair. Step 5: From the 4-stranded reversed fork, the nub may be degraded by RecBCD exonuclease. The fork has backed away from the blocking lesion and, because it is now in a dsDNA, can be repaired by excision repair (or other repair processes).

Fig. 3

SCE-associated template-switching mechanism for deletion formation between repeated sequences. In a replication fork blocked on the lagging strand, nascent strands are unwound. Mispairing of the nascent strands at direct repeats (highlighted in blue) and Holliday junction resolution (indicated by scissors) generates: A. a reciprocal crossover product between sister chromosomes, with one deletion and a triplication; or B: a non-reciprocal crossover product with a deletion and the original duplication. Similar structures with different mispairing at the repeats can generate a triplication associated with SCE (see [61]).

Fig. 4

Simple replication “slippage” (“slipped mispairing”) mechanism for RecA-independent deletion between direct repeats. Replication stalls during replication of a tandem direct repeat. The nascent strand is unwound, which allows it to mispair with the downstream repeat. This mispaired structure will generate a deletion. Similar events with synthesis of both repeats, unwinding and mispairing to the template at the first repeat can generate a triplication (see [61]).

Fig. 5

Comparison of template-switching mediated by: A. Cross-fork pairing; and B. Fork reversal. A. Unwinding is initiated from the 3′ ends of the leading nascent strand and 3′ end of a blocked lagging strand. RecA-independent pairing of nascent strands generates two Holliday junctions that can be resolved to yield RecA-independent crossover products. B. Unwinding is initiated from the 3′ end of the nascent leading strand and 5′ end of the nascent lagging strand. This produces a Holliday junction whose cleavage generates a double-strand break. Subsequent repair of this break could yield crossovers, although unlike cross-fork template switching, these crossovers would be RecA-dependent.

Fig. 6

Quasipalindromes (imperfect palindromes). These are inverted repeats with “imperfections” shown in pink and blue. Such sequences can form hairpin and cruciform structures. An intramolecular template switch from the nascent strand to itself (A) can template a mutational change, that upon a second, restoration template switch (B) generates the sequence change (boxed). Note that depending on the direction of replication, different sides of the quasipalindrome are mutated. A leading strand template switch generates a mutation in the rightward repeat, as shown; a lagging strand template switch generates a mutation in the leftward repeat.

Fig. 7

Quasipalindrome-associated mutation (QPM) hotspots. A. The first recognized QPM site, in Saccharomyces cerevisiae CYC1, original sequence at left and mutated product at right. B. QPM site in bacteriophage T4 rII gene identified by Ripley et al. [106] C. The E. coli thyA QPM hotspot that accounts for 60% of mutations that inactivate the gene. Shown are the three types of mutational products recovered. The most common product mutates thyA131 alone (the middle imperfect site). In ExoI⁻ ExoVII⁻ strains, a complex, co-mutation at thyA124-127 (the bottom imperfect site) occurs frequently. More rarely, a single mutation at thyA124-127 is recovered.

Fig. 8

Intramolecular and intermolecular template-switching mechanisms for QPM. Diagrammed is a model QP containing imperfections in the inverted repeats colored pink or gold in a replication fork, moving rightward. The mutational outcome for each illustrated event is indicated at right. A. An intramolecular template-switch on the lagging strand generates a mutation in the left repeat. B. An intramolecular template-switch on the leading strand generated a mutation in the right repeat. C. A cross-fork intermolecular template-switch, involving a leading nascent strand pairing with the lagging strand template at the leftward repeat, generates a mutation in right inverted repeat. D. A leading strand, cross-fork intermolecular template-switch from the left to the right repeat primes synthesis across the repeat center. Its mutational outcome is a mutation in the right repeat plus an inversion of the repeat center (as illustrated here, TTC to AAG).