SummaryNucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.

PriA helicase is the major DNA replication restart initiator in Escherichia coli and acts to reload the replicative helicase DnaB back onto the chromosome at repaired replication forks and D-loops formed by recombination. We have discovered that PriA-catalysed unwinding of branched DNA substrates is stimulated specifically by contact with the single-strand DNA binding protein of E.coli, SSB. This stimulation requires binding of SSB to the initial DNA substrate and is effected via a physical interaction between PriA and the C-terminus of SSB. Stimulation of PriA by the SSB C-terminus may act to ensure that efficient PriA-catalysed reloading of DnaB occurs only onto the lagging strand template of repaired forks and D-loops. Correlation between the DNA repair and recombination defects of strains harbouring an SSB C-terminal mutation with inhibition of this SSB-PriA interaction in vitro suggests that SSB plays a critical role in facilitating PriA-directed replication restart. Taken together with previous data, these findings indicate that protein-protein interactions involving SSB may coordinate replication fork reloading from start to finish.

The frequency with which replication forks break down in all organisms requires that specific mechanisms ensure completion of genome duplication. In Escherichia coli a major pathway for reloading of the replicative apparatus at sites of fork breakdown is dependent on PriA helicase. PriA acts in conjunction with PriB and DnaT to effect loading of the replicative helicase DnaB back onto the lagging strand template, either at stalled fork structures or at recombination intermediates. Here we showed that PriB stimulates PriA helicase, acting to increase the apparent processivity of PriA. This stimulation correlates with the ability of PriB to form a ternary complex with PriA and DNA structures containing single-stranded DNA, suggesting that the known single-stranded DNA binding function of PriB facilitates unwinding by PriA helicase. This enhanced apparent processivity of PriA might play an important role in generating single-stranded DNA at stalled replication forks upon which to load DnaB. However, stimulation of PriA by PriB is not DNA structure-specific, demonstrating that targeting of stalled forks and recombination intermediates during replication restart likely resides with PriA alone.

Helicases play critical roles in all aspects of nucleic acid metabolism by catalyzing the remodeling of DNA and RNA structures. UvrD is an abundant helicase in Escherichia coli with well characterized functions in mismatch and nucleotide excision repair and a possible role in displacement of proteins such as RecA from single-stranded DNA. Helicases and translocases use the energy derived from NTP hydrolysis to translocate along single-stranded or doublestranded nucleic acids to remodel nucleic acid structures. Many of these enzymes have RecA-like motor domains, reflecting close similarities in translocation mechanisms (1-3). Specificity is often, therefore, conferred by additional domains within these motor enzymes (4). It is also becoming apparent that helicases and translocases often function as part of larger multisubunit complexes rather than in isolation and that such interactions impact on motor function and specificity. The complex interactions between replicative helicases and other proteins acting at the replication fork have long been known to be critical for replisome function (5-7). Many helicases also interact with, and their activities modulated by, ssDNA 2 -binding proteins (8 -13), while helicase/translocase interactions with RNA polymerases are emerging (14, 15).The Escherichia coli 3Ј-5Ј helicase UvrD (16) has roles in mismatch (17) and nucleotide excision repair (18) and may also act to displace proteins such as RecA at replication forks or ssDNA gaps in duplex DNA (19 -21). UvrD is likely the most abundant helicase in E. coli (22). There is also a second helicase in E. coli, Rep, that shares 40% identity with UvrD but has no known role in mismatch or nucleotide excision repair or in protein displacement in vivo. Employment of UvrD, but not Rep, in a diversity of roles in vivo might, therefore, demand specific physical or functional interactions between UvrD and other proteins in each system. However, specific interaction of UvrD has only been documented with a component of the mismatch repair system, MutL (23). This interaction appears to be essential in allowing a motor with limited dsDNA processivity to unwind the large tracts of DNA necessary during mismatch repair (23)(24)(25)(26).Little is known concerning the protein displacement function of UvrD in vivo. Lack of UvrD leads to a hyperrecombination phenotype (27), whereas UvrD can both promote (20,21) and inhibit (20) RecA-catalyzed strand exchange in vitro, depending on reaction conditions. UvrD might, therefore, promote turnover of RecA-ssDNA complexes at blocked forks and gaps in duplex DNA. Inhibition of RecA function at blocked forks might facilitate other pathways of fork repair that do not rely on strand exchange, possibly minimizing the risks to genome stability that blocked forks present (28). However, whether abortion of strand exchange by UvrD in vivo requires other proteins is unknown. In contrast, the role of UvrD in nucleotide excision repair is well characterized. Nucleotide excision repair is needed to remove and replace ...

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