Major Research Areas
Molecular Biology
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Figure 5
Figure 5 Replication Fork Regression in Early Replication Intermediates
Positively supercoiled ERIs can be cut by the Holliday junction resolvase RuvC, however, regression is suppressed in negatively supercoiled ERIs, requiring the addition of the branch migration protein RecG to catalyze regression.

We modeled nascent strand regression (NSR) by exploiting a system that we had developed previously in which replication fork progression stalled because of DNA topology. Initiation of replication of an oriC plasmid in vitro will occur on negatively supercoiled templates in the absence of any topoisomerase. As the replication fork progresses, the negative supercoils are converted to positive ones. Without any relief of the positive supercoiling, the fork stalls, generating an early replication intermediate (ERI). These ERIs could be cut by both RuvC and RusA, the 2 Holliday junction resolvases in E. coli (Figure 5), demonstrating that they contained regressed forks that formed HJs.


Figure 6
Figure 6 The influence of superhelicity on nascent strand regression at stalled replication forks.

A central issue with respect to NSR is identification of the driving force, because the reaction is favored by positive supercoiling and suppressed by negative supercoiling (Figure 6). Given the presence of DNA gyrase in the cell, it is likely that the domains of the chromosome containing the replication forks are negatively supercoiled. Thus, if NSR plays a role in replication fork repair in the cell, there is likely to be an enzyme(s) that catalyzes the reaction.

We showed as scored by cleavage of the ERI by RuvC, that the branch migration protein RecG was able to catalyze NSR in negatively supercoiled ERIs (Figure 5). We have also compared the action of RecG and the other branch migration protein in E. coli, RuvAB, in this reaction (Figure 5). We find that the 2 proteins act differently: RuvAB unwinds the nascent DNA in the ERI (UW in Figure 5), irrespective of its supercoiled state (+, positive; -, negative). In addition, unlike RecG action, which clearly generates HJs that can be cleaved by RuvC (generating a linear form, FIII in Figure 5), no evidence of coupling between RuvAB and RuvC was manifest. Nevertheless, the product generated by RuvAB is double stranded, indicating that unwinding is via a branch migration reaction that is mediated by a HJ.

Furthermore, we find that RuvAB can unwind the nascent DNA even when the ERI has been linearized. Because positive supercoiling is required to maintain the HJs formed by NSR, the linearized ERI will not contain them, indicating that RuvAB will recognize replication forks directly and regress them. This observation represents an important new finding about RuvAB action. Because we know that the presence of the replisome inhibits NSR, we are currently developing assays for replisome remodeling at stalled forks. Using a new template that we have developed (see Multiple Pathways of Replication Restart), we will also be assessing the influence of the disposition of the nascent strands at stalled forks on NSR and modeling the coupling of NSR to replication restart.
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