Replication fork reversal and the maintenance of genome stability

Abstract

The progress of replication forks is often threatened in vivo, both by DNA damage and by proteins bound to the template. Blocked forks must somehow be restarted, and the original blockage cleared, in order to complete genome duplication, implying that blocked fork processing may be critical for genome stability. One possible pathway that might allow processing and restart of blocked forks, replication fork reversal, involves the unwinding of blocked forks to form four-stranded structures resembling Holliday junctions. This concept has gained increasing popularity recently based on the ability of such processing to explain many genetic observations, the detection of unwound fork structures in vivo and the identification of enzymes that have the capacity to catalyse fork regression in vitro. Here, we discuss the contexts in which fork regression might occur, the factors that may promote such a reaction and the possible roles of replication fork unwinding in normal DNA metabolism.

Figure 1.

Blockage of replication fork movement and the possible unwinding of leading and lagging daughter strands to form a four-stranded DNA structure. 3′ ends of DNA strands are indicated by arrowheads.

Figure 2.

Possible structures of forks halted by single-strand-specific blocks such as UV-light-induced pyrimidine dimers located on either the lagging strand template (A) or the leading strand template (B).

Figure 3.

(A) X-ray crystal structure of Thermatoga maritima RecG bound to a forked DNA having a lagging strand but no leading strand (100). Helicase domains 1 and 2 are shown in blue whilst domain 3 is in grey. (B) Model of RecG catalysis at forked DNA structures. Dashed arrows indicate relative movements of duplex arms.

Figure 4.

Action of RuvAB on Holliday junctions and forks. (A) Action of RuvAB on Holliday junction structures. Translocation of the two RuvB hexamers along opposing duplex arms results in movement of duplexes as indicated by dashed arrows. Strand separation results from spooling of the DNA strands across the ‘acidic pins’ found on the surface of the RuvA tetramer (176). (B) Catalysis of fork regression by RuvAB would necessitate loading of a single RuvB hexamer onto the parental duplex. (C) Binding of two RuvB hexamers onto opposing duplex arms of a fork would result in unwinding of the junction in the direction opposite to that required for regression.

Figure 5.

Fork regression followed by reversal. (A) A lesion in the leading strand template could result in the formation of a blocked fork with a gap on the leading strand. (B) Fork regression would reposition the 3′ end of the blocked leading strand so that it would be paired with the nascent lagging strand, whilst the DNA lesion would be relocated back into the reformed parental duplex. (C and D) Bypass of the lesion could be effected by extension of the leading strand using the lagging strand as a template followed by reversal of fork regression. (E and F) Repositioning of the lesion back into the parental duplex could also facilitate repair rather than bypass. Extension of the leading strand using the nascent lagging strand and reversal of regression would reconstitute a fork structure on to which the replication apparatus could be reloaded.

Figure 6.

Restoration of a fork structure after regression by degradation of the extruded duplex arm. Blockage of a replisome (A) followed by fork regression and exonuclease-mediated degradation of the dsDNA end (B) would restore a fork structure onto which the replisome could be reassembled (C). Concomitantly, repositioning of the blocking lesion away from the fork may facilitate access of repair enzymes to the lesion. Note that, in this model, a block is depicted in which both leading and lagging strand synthesis is inhibited resulting in formation of a blunt dsDNA end by regression. A leading strand template-specific lesion might result in an extruded duplex arm with an extended ssDNA overhang (Figure 5B) rather than a blunt dsDNA end. Given the DNA structure specificities exhibited by exonucleases, different extruded DNA ends would require exonucleases with appropriate specificities.

Figure 7.

Regression and Holliday junction cleavage. Blockage of a fork followed by regression and cleavage of the four-stranded structure (A–C) would generate a dsDNA end (D). Processing of this end to promote loading of strand exchange proteins would result in D-loop formation with the intact sister duplex (E). Loading of the replication machinery onto this D-loop and resolution of the connected Holliday junction would restore an intact replication fork (F). Assuming the original block could be cleared, possibly aided by repositioning of the block away from the fork to promote access by repair enzymes (C), then replication fork progression could resume.

Figure 8.

Regression followed by recombination of the extruded dsDNA end. Any exonucleolytic processing of the dsDNA end generated by fork regression might result in generation of a 3′ ssDNA tail rather than complete degradation (A–C). Recombination between this ssDNA and the homologous sequence in the reformed parental duplex would result in formation of a D-loop linked to two Holliday junctions (D). Cleavage of the two linked Holliday junctions by resolvases (as shown in E) or dissolution by a RecQ-type helicase in conjunction with a topoisomerase, coupled with reassembly of the replisome at the D-loop would restore a replication fork (F).