Topological locking restrains replication fork reversal

Abstract

Two-dimensional agarose gel electrophoresis, psoralen crosslinking, and electron microscopy were used to study the effects of positive supercoiling on fork reversal in isolated replication intermediates of bacterial DNA plasmids. The results obtained demonstrate that the formation of Holliday-like junctions at both forks of a replication bubble creates a topological constraint that prevents further regression of the forks. We propose that this topological locking of replication intermediates provides a biological safety mechanism that protects DNA molecules against extensive fork reversals.DNA repair ͉ DNA replication ͉ DNA structure ͉ DNA topology ͉ DNA supercoiling G enomic instability is a hallmark of several human diseases (1, 2). During the last decade it was clearly established that a significant portion of this instability arises during the replication of damaged templates. Progression of the replication machinery halts or pauses when the replisome encounters DNA lesions, and this blockage may lead to their collapse (3). All living cells are constantly exposed to a great variety of genotoxic agents. Among these agents, UV light and tobacco smoke, because of their well known carcinogenic potential, are particularly important for humans (4). This correlation explains the increasing interest of the scientific community to understand the mechanisms that cells have developed to repair DNA lesions and reactivate stalled replication forks to ensure that when a cell divides both daughter cells receive a complete and exact copy of the genome of their mother cell (5).In Escherichia coli, current models suggest that once a replication fork meets a DNA lesion RecA (6) and/or RecG (5, 7) promotes regression of the stalled fork, generating a Hollidaylike junction (HLJ) that is subsequently processed by the Ruv-ABC complex to allow replication restart by PriA (8-10). Despite the solid genetic evidence supporting this model, the molecular characterization of HLJs formed in vivo and an estimation of the extent of fork regression are still scarce (11-13). Moreover, replication restart after fork stalling was also suggested to occur by means of mechanisms that do not necessarily need fork reversal (FR) (8,14).One of the possible driving forces for FR at stalled forks in vivo is the positive (ϩ) writhe increase (⌬Wr) generated by other replication or transcription forks progressing in the opposite direction (15-17). FR relaxes this (ϩ) ⌬Wr and lowers the free energy of the constrained DNA. Indeed, in vitro experiments demonstrated that FR compensates the (ϩ) ⌬Wr generated by intercalation of moderate quantities of chloroquine in the unreplicated region of replication intermediates (RIs) (16). In the same study, however, direct scanning force microscopy of RIs exposed to rather high concentrations of ethidium bromide (EthBr) revealed their supercoiled character, suggesting that FR was apparently unable to adsorb all of the (ϩ) ⌬Wr generated by the intercalated drug. In a different study, Olavarrieta et al. (11) also noticed t...

Direct Evidence for the Formation of Precatenanes during DNA Replication

“…All the immunograms were aligned attending to the mobility of OCs. Note that chloroquine's intercalation has no effect on the electrophoretic mobility of nicked unreplicated molecules (29,37,50).…”

“…6). As mentioned previously, it should be noticed that for RIs containing three-way junctions, once all the native (Ϫ)-supercoiling is removed, these molecules cannot acquire (ϩ)-supercoiling because the nascent strands of the sister duplexes form a fourth arm leading to fork reversal with changes in neither mass nor electrophoretic mobility (50,51). Finally, the number and complexity of replication knots were higher for the RIs isolated from DH5␣FЈ cells, DH5αF' (RecA -Topo IV + Gyr Nal ) grown at 37ºC r FIGURE 7.…”

Replication Fork Reversal Occurs Spontaneously after Digestion but Is Constrained in Supercoiled Domains

“…It was recently shown, however, that formation of Holliday-like junctions at both forks of a replication bubble creates a topological constraint that prevents further regression of the forks (33). To confirm this observation for a different plasmid, we used a modification of two-dimensional gels where the agarose lane containing the DNA that came out from the first dimension was heated at 65°C in the presence of 0.1 M NaCl for 4 h before the second dimension took place (33,36). This condition favors branch migration and extrusion of the fourth arm of Holliday junctions in vitro (37,38).…”

“…The RIs of plasmids where the fork stalls at a specific site accumulate, generating a prominent signal in two-dimensional gel autoradiograms that can be readily distinguished on top of the simple-or double-Y patterns (7, 30 -32). Analysis of these plasmids undigested and after restriction enzyme digestion revealed that RFR readily forms in vitro but that fork retreat is severely limited in covalently closed domains (33). Extensive retreat and total extrusion of the nascent-nascent duplex, on the other hand, occurs spontaneously after nicking or restriction enzyme digestion.…”

Comparative Genomics and Molecular Dynamics of DNA Repeats in Eukaryotes

“…It would now be interesting to know if trinucleotide repeats that form strong secondary structures, like (CCG) n and (GAA) n , are also able to induce fork reversal in vivo or if this is restricted to the more labile secondary structures formed by (CAG) n repeats. It is nevertheless interesting that fork reversal cannot occur spontaneously in supercoiled regions and could occur in vivo only if topoisomerases are present to relax the supercoiling induced by the progression of the replication fork (134,135).…”