dna repair 6 ( 2 0 0 7 ) 945–952 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/dnarepair Non-replicative helicases at the replication fork Ryan C. Heller, Kenneth J. Marians ∗ Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA article info Article history: Published on line 26 March 2007 Keywords: Non-replicative helicases Replication fork DNA replication abstract Reactivation of stalled or collapsed replication forks is an essential process in bacteria. Restart systems operate to restore the 5 ′ → 3 ′ replicative helicase, DnaB, to the lagging- strand template. However, other non-replicative 3 ′ → 5 ′ helicases play an important role in the restart process as well. Here we examine the DNA-binding specificity of three of the latter group, PriA, Rep, and UvrD. Only PriA and Rep display structure-specific fork bind- ing. Interestingly, their specificity is opposite: PriA binds a leading-strand fork, presumably reflecting its restart activity in directing loading of DnaB to the lagging-strand template. Rep binds a lagging-strand fork, presumably reflecting its role in partially displacing Okazaki fragments that originate near the fork junction. This activity is necessary for generating a single-stranded landing pad for DnaB. While UvrD shows little structure-specificity, there is a slight preference for lagging-strand forks, suggesting that there might be some redun- dancy between Rep and UvrD and possibly explaining the observed synthetic lethality that occurs when mutations in the genes encoding these two proteins are combined. © 2007 Elsevier B.V. All rights reserved. 1. Introduction The Escherichia coli replisome is a highly efficient machine capable of duplicating the genome with high rates of speed and high levels of processivity. Much of this ability is owed to the replicative helicase, DnaB, a hexameric-type helicase that encircles the lagging-strand template at the replication fork and translocates in the 5 ′ → 3 ′ direction [1] using the energy of ATP to separate the strands of the parental duplex. Though DnaB is sufficient to maintain DNA replication in vitro [2],a class of non-replicative, non-hexameric, 3 ′ → 5 ′ helicases is thought to aid in the maintenance of DNA replication through roadblocks in the form of bound proteins and DNA template lesions in vivo. The helicase activities of three of these pro- teins, PriA, Rep, and UvrD, are not essential alone, but evidence is accumulating that these proteins have important and over- lapping functions in DNA replication. Along with several other components, PriA was purified based on its requirement in the reconstituted bacteriophage ∗ Corresponding author. Tel.: +1 212 639 5890; fax: +1 212 717 3627. E-mail address: kmarians@sloankettering.edu (K.J. Marians). X174 replication reaction [3]. Lacking a direct role in cellular replication, PriA was later identified as a key factor in repli- some assembly outside the origin of replication, a process that enables replication restart after fork stalling or breakage. After processing of the stalled fork or the broken chromosome, PriA utilizes its ability to recognize DNA in a structure-specific manner, binding to specific stalled forks and recombination- generated D-loop structures [4,5]. PriA then catalyzes the assembly of a multiprotein complex including PriB and DnaT, which together allow the loading of the replicative helicase DnaB from a DnaB–DnaC complex to single-stranded DNA- binding protein (SSB)-coated single-stranded DNA [6]. The loading of DnaB nucleates the assembly of a new replisome through protein-protein interactions with a component of the DNA polymerase III holoenzyme (Pol III HE) [7,8] and with the primase, DnaG [9]. Although PriA is a SF2 family member DNA helicase with 3 ′ → 5 ′ polarity [10,11], its unwinding activity is distinct from its ability to load DnaB for the replication restart process. Cells lacking PriA have a number of defects including 1568-7864/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2007.02.014