RuvAB Acts at Arrested Replication Forks

Abstract

Replication arrest leads to the occurrence of DNA double-stranded breaks (DSB). We studied the mechanism of DSB formation by direct measure of the amount of in vivo linear DNA in Escherichia coli cells that lack the RecBCD recombination complex and by genetic means. The RuvABC proteins, which catalyze migration and cleavage of Holliday junctions, are responsible for the occurrence of DSBs at arrested replication forks. In cells proficient for RecBC, RuvAB is uncoupled from RuvC and DSBs may be prevented. This may be explained if a Holliday junction forms upon replication fork arrest, by annealing of the two nascent strands. RecBCD may act on the double-stranded tail prior to the cleavage of the RuvAB-bound junction by RuvC to rescue the blocked replication fork without breakage.

Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells

“…Such intermediates (Fig. 6e) include hairpins 42 or slipped-strand structures [36][37][38] that form between nascent and template strands; between the two nascent strands, which may occur through replication fork reversal [43][44][45] ; or between the two template strands, which may block or pause fork progression 20 . The sequence (CTG or CAG) and the number of repeats at the terminus might affect both the type and the propensity of structure formation.…”

“…Longer rather than shorter nascent repeat tracts at Okazaki termini might form these structures more readily. The metabolism of the structural intermediates may lead to efficient or error-prone processing by replication 22,24,[32][33][34] , repair 37,46,47 or recombination proteins [43][44][45]48 , all of which could lead to instability. Although other explanations are possible, it is clear that striking differences in (CTG)•(CAG) stability result from the location of replication initiation relative to the repeat tract.…”

Links between replication, recombination and genome instability in eukaryotes

“…In addition, studies in bacteria have indicated that stalled replication forks are actively converted to DSBs as part of the replication fork restart process 2,3,22 and, although not yet demonstrated, it seems likely that a similar mechanism of replication fork restart might occur in eukaryotes. These types of damage/ processing events are likely candidates for activation of the G1/G2-phase damage and Sphase checkpoints 48 .…”

“…If tandem repeats (indicated as arrows) are present at the breakpoint and repair is initiated out of register, it can lead to deletions (1) of the sequences between the repeats. When the distal part of the chromosome is lost, the centromerecontaining fragment (with the centromere depicted as a solid black circle) can be repaired using another chromosome as the template leading to non-reciprocal translocations (2) where the broken chromosome acquires sequences from another chromosome (shown as gray shaded boxes) and in some cases it can be repaired using intrachromosomal regions (3). In all cases, the broken chromosome can capture telomere sequences (depicted as colored regions at the end of each chromosome) by BIR copying to the end of the template.…”

“…In addition, some cellular processes, like DNA replication, can generate DSBs. These could arise by the action of nucleases at unprotected sites where replication forks stall, as a consequence of DNA polymerases (Pols) replicating across nicked chromosomes, as well as by the active processing of stalled replication forks by specific enzyme systems [2][3][4] . DSBs can also occur naturally and play important roles in processes such as meiosis 5 , mating-type switching 6 and mammalian V(D)J recombination 7 .…”