Accessory Helicase Research Articles

Pfh1 Is an Accessory Replicative Helicase that Interacts with the Replisome to Facilitate Fork Progression and Preserve Genome Integrity.

Replicative DNA helicases expose the two strands of the double helix to the replication apparatus, but accessory helicases are often needed to help forks move past naturally occurring hard-to-replicate sites, such as tightly bound proteins, RNA/DNA hybrids, and DNA secondary structures. Although the Schizosaccharomyces pombe 5’-to-3’ DNA helicase Pfh1 is known to promote fork progression, its genomic targets, dynamics, and mechanisms of action are largely unknown. Here we address these questions by integrating genome-wide identification of Pfh1 binding sites, comprehensive analysis of the effects of Pfh1 depletion on replication and DNA damage, and proteomic analysis of Pfh1 interaction partners by immunoaffinity purification mass spectrometry. Of the 621 high confidence Pfh1-binding sites in wild type cells, about 40% were sites of fork slowing (as marked by high DNA polymerase occupancy) and/or DNA damage (as marked by high levels of phosphorylated H2A). The replication and integrity of tRNA and 5S rRNA genes, highly transcribed RNA polymerase II genes, and nucleosome depleted regions were particularly Pfh1-dependent. The association of Pfh1 with genomic integrity at highly transcribed genes was S phase dependent, and thus unlikely to be an artifact of high transcription rates. Although Pfh1 affected replication and suppressed DNA damage at discrete sites throughout the genome, Pfh1 and the replicative DNA polymerase bound to similar extents to both Pfh1-dependent and independent sites, suggesting that Pfh1 is proximal to the replication machinery during S phase. Consistent with this interpretation, Pfh1 co-purified with many key replisome components, including the hexameric MCM helicase, replicative DNA polymerases, RPA, and the processivity clamp PCNA in an S phase dependent manner. Thus, we conclude that Pfh1 is an accessory DNA helicase that interacts with the replisome and promotes replication and suppresses DNA damage at hard-to-replicate sites. These data provide insight into mechanisms by which this evolutionarily conserved helicase helps preserve genome integrity.

The Balance between Recombination Enzymes and Accessory Replicative Helicases in Facilitating Genome Duplication.

Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair.

Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ.

Using an in vitro reconstituted system in this work we provide direct evidence that the yeast repressor/activator protein 1 (Rap1), tightly bound to its consensus site, forms a strong non-polar barrier for the strand displacement activity of DNA polymerase δ. We propose that relief of inhibition may be mediated by the activity of an accessory helicase. To this end, we show that Pif1, a 5′–3′ helicase, not only stimulates the strand displacement activity of Pol δ but it also allows efficient replication through the block, by removing bound Rap1 in front of the polymerase. This stimulatory activity of Pif1 is not limited to the displacement of a single Rap1 molecule; Pif1 also allows Pol δ to carry out DNA synthesis across an array of bound Rap1 molecules that mimics a telomeric DNA-protein assembly. This activity of Pif1 represents a novel function of this helicase during DNA replication.

CTXϕ: Exploring new alternatives in host factor-mediated filamentous phage replications.

For a long time Ff phages from Escherichia coli provided the majority of the knowledge about the rolling circle replication mechanism of filamentous phages. Host factors involved in coliphages replication have been fully identified. Based on these studies, the function of Rep protein as the accessory helicase directly implicated in filamentous phage replication was considered a paradigm. We recently reported that the replication of some filamentous phages from Vibrio cholerae, including the cholera toxin phage CTXϕ, depended on the accessory helicase UvrD instead of Rep. We also identified HU protein as one of the host factors involved in CTXϕ and VGJϕ replication. The requirement of UvrD and HU for rolling circle replication was previously reported in some family of plasmids but had no precedent in filamentous phages. Here, we enrich the discussion of our results and present new preliminary data highlighting remarkable divergence in the lifestyle of filamentous phages.

Pf Filamentous Phage Requires UvrD for Replication in Pseudomonas aeruginosa.

Pf is a lysogenic filamentous phage that promotes biofilm development in Pseudomonas aeruginosa. Pf replicates by a rolling circle replication system which depends on a phage-encoded initiator protein and host factors usually involved in chromosome replication. Rep, an accessory replicative DNA helicase, is crucial for replication of filamentous phages in Escherichia coli. In contrast, here we show that, instead of depending on Rep, Pf replication depends on UvrD, an accessory helicase implicated in DNA repair. In this study, we also identified the initiator protein of Pf and found that it shares similarities with that of Vibrio phages CTXφ and VGJφ, which also depend on UvrD for replication. A structural comparative analysis of the initiator proteins of most known filamentous phages described thus far suggested that UvrD, known as a nonreplicative helicase, is involved in rolling circle replication of filamentous phages in diverse bacteria genera. This report consolidates knowledge on the new role of UvrD in filamentous phage replication, a function previously thought to be exclusive of Rep helicase. IMPORTANCE Biofilm development is a key component of the ability of Pseudomonas aeruginosa to evade host immune defenses and resist multiple drugs. Induction of the filamentous phage Pf, which usually is lysogenized in clinical and environmental isolates of P.aeruginosa, plays an important role in biofilm assembly, maturation, and dispersal. Despite the clinical relevance of Pf, the molecular biology of this phage is largely unknown. In this study, we found that rolling circle replication of Pf depends on UvrD, a DNA helicase normally involved in DNA repair. We also identified the initiator protein of Pf and found that it shares structural similarity with that of Vibrio cholerae phages CTXφ and VGJφ, which also use UvrD for replication. Our results reveal that, in addition to DNA repair, UvrD plays an essential role in rolling circle replication of filamentous phages among diverse bacteria genera, adding a new, previously unrecognized function of this accessory helicase.

Overexpression of the Replicative Helicase in Escherichia coli Inhibits Replication Initiation and Replication Fork Reloading

Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6–DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6–DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6–DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6–DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096–22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825–831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication.

Mitochondrial functions of RECQL4 are required for the prevention of aerobic glycolysis-dependent cell invasion

Germline mutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome, which is characterized by a predisposition to cancer. RECQL4 localizes to the mitochondria, where it acts as an accessory factor during mitochondrial DNA replication. To understand the specific mitochondrial functions of RECQL4, we created isogenic cell lines, in which the mitochondrial localization of the helicase was either retained or abolished. The mitochondrial integrity was affected due to the absence of RECQL4 in mitochondria, leading to a decrease in F1F0-ATP synthase activity. In cells where RECQL4 does not localize to mitochondria, the membrane potential was decreased, whereas ROS levels increased due to the presence of high levels of catalytically inactive SOD2. Inactive SOD2 accumulated owing to diminished SIRT3 activity. Lack of the mitochondrial functions of RECQL4 led to aerobic glycolysis that, in turn, led to an increased invasive capability within these cells. Together, this study demonstrates for the first time that, owing to its mitochondrial functions, the accessory mitochondrial replication helicase RECQL4 prevents the invasive step in the neoplastic transformation process.

Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage.

Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

The B. subtilis Accessory Helicase PcrA Facilitates DNA Replication through Transcription Units.

In bacteria the concurrence of DNA replication and transcription leads to potentially deleterious encounters between the two machineries, which can occur in either the head-on (lagging strand genes) or co-directional (leading strand genes) orientations. These conflicts lead to replication fork stalling and can destabilize the genome. Both eukaryotic and prokaryotic cells possess resolution factors that reduce the severity of these encounters. Though Escherichia coli accessory helicases have been implicated in the mitigation of head-on conflicts, direct evidence of these proteins mitigating co-directional conflicts is lacking. Furthermore, the endogenous chromosomal regions where these helicases act, and the mechanism of recruitment, have not been identified. We show that the essential Bacillus subtilis accessory helicase PcrA aids replication progression through protein coding genes of both head-on and co-directional orientations, as well as rRNA and tRNA genes. ChIP-Seq experiments show that co-directional conflicts at highly transcribed rRNA, tRNA, and head-on protein coding genes are major targets of PcrA activity on the chromosome. Partial depletion of PcrA renders cells extremely sensitive to head-on conflicts, linking the essential function of PcrA to conflict resolution. Furthermore, ablating PcrA’s ATPase/helicase activity simultaneously increases its association with conflict regions, while incapacitating its ability to mitigate conflicts, and leads to cell death. In contrast, disruption of PcrA’s C-terminal RNA polymerase interaction domain does not impact its ability to mitigate conflicts between replication and transcription, its association with conflict regions, or cell survival. Altogether, this work establishes PcrA as an essential factor involved in mitigating transcription-replication conflicts and identifies chromosomal regions where it routinely acts. As both conflicts and accessory helicases are found in all domains of life, these results are broadly relevant.

Phylogenetic relationship of some "accessory" helicases of plant positive-stranded RNA viruses: toward understanding the evolution of triple gene block.

Recently, we hypothesized that silencing suppression activity gained by a viral replicative helicase led to the emergence of the second helicase possessing activity of the viral silencing suppressor and/or movement protein (MP). Our hypothesis accounted for the evolutionary origin of the specialized ‘triple gene block’ (TGB) in plant virus genomes encoding the MPs TGB1, TGB2, and TGB3 required for viral cell-to-cell transport through plasmodesmata. Here, we used public transcriptome databases to identify previously unrecognized viruses. The analysis of novel viral genomes further supported the previously proposed scenario of TGB origin and evolution, which included the following steps. First, the accessory helicase gene could have been acquired by horizontal gene transfer (HGT) presumably occured independently in different virus groups. Second, the TGB2 gene evolved by HGT or autonomization of the C-terminal transmembrane domain found in at least one TGB1 helicase. Third, the TGB3 gene has most likely emerged in the genomic block consisting of the TGB1 and TGB2 genes.

The B. subtilis accessory helicase PcrA facilitates replication through transcription units genome‐wide (LB126)

In bacteria, transcription and DNA replication occur concurrently, inevitably causing replication‐transcription conflicts. Conflicts can occur both co‐directionally (leading strand genes) and head‐on (lagging strand genes), leading to replisome stalling and restart. Because the majority of genes are co‐oriented with replication in most bacteria, co‐directional conflicts are expected to be highly prevalent, impacting replication speed and genomic stability. Consequently, mechanisms that deal with these conflicts should be critical for the timely and accurate duplication of genomes. Here we report that the essential Bacillus subtilis accessory helicase, PcrA, mitigates both co‐directional and head‐on conflicts between the replication and transcription machineries. Using ChIP‐seq, we have identified genomic regions where PcrA associates and facilitates replication progression. We find that PcrA predominantly associates with co‐directional rDNA and tRNA genes, in a transcription‐dependent manner. When PcrA is depleted, and only when transcription is active, replisome stalling and restart increases in the same areas. PcrA also reduces transcription‐induced replication stalling and restart at other regions, including some head‐on genes. Differential levels of PcrA at engineered head‐on and co‐directional conflicts indicate that its association and activity correlate with conflict severity, rather than transcription levels. These results show that PcrA reduces replisomes stalling, and restart, caused by head‐on and co‐directional replication‐transcription conflicts. Furthermore, these data indicate that during vegetative growth, and in the absence of exogenous stress, the most difficult regions to replicate are co‐directionally transcribed genes. Both the genome co‐orientation bias and these accessory helicases are widely conserved, suggesting that our results are relevant for many other bacterial species.Grant Funding Source: NIH

The Conserved C-Terminus of the PcrA/UvrD Helicase Interacts Directly with RNA Polymerase

UvrD-like helicases play diverse roles in DNA replication, repair and recombination pathways. An emerging body of evidence suggests that their different cellular functions are directed by interactions with partner proteins that target unwinding activity to appropriate substrates. Recent studies in E. coli have shown that UvrD can act as an accessory replicative helicase that resolves conflicts between the replisome and transcription complexes, but the mechanism is not understood. Here we show that the UvrD homologue PcrA interacts physically with B. subtilis RNA polymerase, and that an equivalent interaction is conserved in E. coli where UvrD, but not the closely related helicase Rep, also interacts with RNA polymerase. The PcrA-RNAP interaction is direct and independent of nucleic acids or additional mediator proteins. A disordered but highly conserved C-terminal region of PcrA, which distinguishes PcrA/UvrD from otherwise related enzymes such as Rep, is both necessary and sufficient for interaction with RNA polymerase.

Protein–DNA complexes are the primary sources of replication fork pausing in Escherichia coli

Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks. Using RecD2 to probe replisome pausing in vivo, we demonstrate that most pausing events do not lead to replisome inactivation, that transcription complexes are the primary sources of this pausing, and that an accessory replicative helicase is critical for minimizing the frequency and/or duration of replisome pauses. These findings reveal the hidden potential for replisome inactivation, and hence genome instability, inside cells. They also demonstrate that efficient chromosome duplication requires mechanisms that aid resumption of replication by paused replisomes, especially those halted by protein-DNA barriers such as transcription complexes.

Replication Fork Reversal after Replication–Transcription Collision

Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle.

Interaction of Rep and DnaB on DNA

Genome duplication requires not only unwinding of the template but also the displacement of proteins bound to the template, a function performed by replicative helicases located at the fork. However, accessory helicases are also needed since the replicative helicase stalls occasionally at nucleoprotein complexes. In Escherichia coli, the primary and accessory helicases DnaB and Rep translocate along the lagging and leading strand templates, respectively, interact physically and also display cooperativity in the unwinding of model forked DNA substrates. We demonstrate here that this cooperativity is displayed only by Rep and not by other tested helicases. ssDNA must be exposed on the leading strand template to elicit this cooperativity, indicating that forks blocked at protein–DNA complexes contain ssDNA ahead of the leading strand polymerase. However, stable Rep–DnaB complexes can form on linear as well as branched DNA, indicating that Rep has the capacity to interact with ssDNA on either the leading or the lagging strand template at forks. Inhibition of Rep binding to the lagging strand template by competition with SSB might therefore be critical in targeting accessory helicases to the leading strand template, indicating an important role for replisome architecture in promoting accessory helicase function at blocked replisomes.

Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication

Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required for Rep to maintain rapid genome duplication. Furthermore, this Rep–DnaB interaction is critical in minimizing the need for both recombinational processing of blocked replication forks and replisome reassembly, indicating that colocalization of Rep and DnaB minimizes stalling and subsequent inactivation of replication forks. These data indicate that E. coli contains only one helicase that acts as an accessory motor at the fork in wild-type cells, that such an activity is critical for the maintenance of rapid genome duplication and that colocalization with the replisome is crucial for this function. Given that the only other characterized accessory motor, Saccharomyces cerevisiae Rrm3p, associates physically with the replisome, our demonstration of the functional importance of such an association indicates that colocalization may be a conserved feature of accessory replicative motors.

RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases

We observed that cells lacking Rep and UvrD, two replication accessory helicases, and the recombination protein RecF are cryo-sensitive on rich medium. We isolated five mutations that suppress this Luria–Bertani (LB)-cryo-sensitivity and show that they map in the genes encoding the RNA polymerase subunits RpoB and RpoC. These rpoB (D444G, H447R and N518D) and rpoC mutants (H113R and P451L) were characterized. rpoBH447R and rpoBD444G prevent activation of the Prrn core promoter in rich medium, but only rpoBH447R also suppresses the auxotrophy of a relA spoT mutant (stringent-like phenotype). rpoCH113R suppresses the thermo-sensitivity of a greA greB mutant, suggesting that it destabilizes stalled elongation complexes. All mutations but rpoCP451L prevent R-loop formation. We propose that these rpo mutations allow replication in the absence of Rep and UvrD by destabilizing RNA Pol upon replication–transcription collisions. In a RecF+ context, they improve growth of rep uvrD cells only if DinG is present, supporting the hypothesis that Rep, UvrD and DinG facilitate progression of the replication fork across transcribed sequences. They rescue rep uvrD dinG recF cells, indicating that in a recF mutant replication forks arrested by unstable transcription complexes can restart without any of the three known replication accessory helicases Rep, UvrD and DinG.

Origin Activation Requires both Replicative and Accessory Helicases during T4 Infection

The bacteriophage T4 has served as an in vitro model for the study of DNA replication for several decades, yet less is known about this process during infection. Recent work has shown that viral DNA synthesis is initiated from at least five origins of replication distributed across the 172 kb chromosome, but continued synthesis is dependent on recombination. Two proteins are predicted to facilitate loading of the hexameric 41 helicase at the origins, the Dda accessory helicase and the 59 loading protein. Using a real time, genome-wide assay to monitor replication during infections, it is shown here that dda mutant viruses no longer preferentially initiate synthesis near the origins, implying that the Dda accessory helicase has a fundamental role in origin selection and activation. In contrast, at least two origins function efficiently without the 59 loading protein, indicating that other factors load the 41 helicase at these loci. Hence, normal T4 replication includes two mechanistically distinct classes of origins, one requiring the 59 helicase loader, and a second that does not. Since both mechanisms require an additional factor, repEB, for sustained activation, normal T4 origin function appears to include at least three common elements, origin selection and initial activation, replisome loading, and persistence.

Roles of Pif1-like helicases in the maintenance of genomic stability.

The Pif1p family of DNA helicases is conserved from yeast to humans. To date, four members of this family have been analyzed in some detail by in vitro and in vivo assays: the two baker's yeast helicases, ScPif1p and Rrm3p, the fission yeast Pfh1p and the human enzyme hPif1p. In vitro, these enzymes are 5′ to 3′ DNA helicase and show little processivity. In vivo, ScPif1p, Rrm3p and probably Pfh1p, function in both the nucleus at specific genomic loci and in mitochondria, where they are needed for the stable maintenance of the genome as accessory helicases to the replication machinery. Interestingly, they act on common DNA substrates but appear to have largely non-overlapping cellular functions, ranging from Okazaki fragment processing, telomerase inhibition, to helping the replication fork progress through non-nucleosomal protein–DNA complexes. For example, both ScPif1p and Rrm3p affect the replication of telomeres, but in a different way: Pif1p inhibits telomerase-mediated telomere elongation by directly removing telomerase from a DNA end, whereas Rrm3p facilitates replication through telomeric DNA. Here we review the current knowledge on the Pif1-like helicases, as a first step towards understanding the basis of their functional specialization and mechanism of action.