Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate.

PriA and other primosome assembly proteins of Escherichia coli recruit the major replicative helicase DnaB for replisome assembly during bacteriophage Mu transposition and replication. MuA transposase catalyzes the transfer of Mu ends to target DNA, forming a potential replication fork that provides the assembly site for the replisome. However, this fork lacks the single-stranded DNA needed to load DnaB. Although no pre-existing primosome assembly sites that bind PriA were found within the Mu end sequences, PriA was able to bind to the forked DNA structure created by MuA. The helicase activity of PriA could then open the duplex to create the DnaB binding site. In a tightly coupled reaction on synthetic forked substrates, PriA promoted both the unwinding of the lagging strand arm and preprimosome assembly to load DnaB onto the lagging strand template. PriA apparently translocated 3' to 5' along the lagging strand template until sufficient single-stranded DNA was exposed for binding of DnaB, which then translocated 5' to 3' in the opposite direction. Mutant PriA lacking helicase activity was unable to promote this process, and loss of PriA helicase impaired Mu DNA replication in vivo and in vitro. This suggests that the opening of the duplex by PriA helicase is a critical step in the initiation of Mu DNA replication. Concerted helicase and primosome assembly functions would allow PriA to act as initiator on recombination intermediates and stalled replication forks. As part of the replisome, PriA may act as a mobile initiator that minimizes interruptions in chromosomal replication.     

Escherichia coli PriA helicase: fork binding orients the helicase to unwind the lagging strand side of arrested replication forks

Escherichia coli PriA is a primosome assembly protein with 3' to 5' helicase activity whose apparent function is to promote resumption of DNA synthesis following replication-fork arrest. Here, we describe how initiation of helicase activity on DNA forks is influenced by both fork structure and by single-strand DNA-binding protein. PriA could recognize and unwind forked substrates where one or both arms were primarily duplex, and PriA required a small (two bases or larger) single-stranded gap at the fork in order to initiate unwinding. The helicase was most active on substrates with a duplex lagging-strand arm and a single-stranded leading-strand arm. On this substrate, PriA was capable of translocating on either the leading or lagging strands to unwind the duplex ahead of the fork or the lagging-strand duplex, respectively. Fork-specific binding apparently orients the helicase domain to unwind the lagging-strand duplex. Binding of single-strand-binding protein to forked templates could inhibit unwinding of the duplex ahead of the fork but not unwinding of the lagging-strand duplex or translocation on the lagging-strand template. While single-strand-binding protein could inhibit binding of PriA to the minimal, unforked DNA substrates, it could not inhibit PriA binding to forked substrates. In the cell, single-strand-binding protein and fork structure may direct PriA helicase to translocate along the lagging-strand template of forked structures such that the primosome is specifically assembled on that DNA strand.     

The Escherichia coli primosome can translocate actively in either direction along a DNA strand

The primosome is a mobile multiprotein DNA replication-priming apparatus that requires seven Escherichia coli proteins (replication factor Y (protein n'), proteins n and n", and the products of the dnaB, dnaC, dnaT, and dnaG genes) for assembly at a specific site (termed a primosome assembly site) on single-stranded DNA binding protein-coated single-stranded DNA. Two of the protein components of the primosome have intrinsic DNA helicase activity. The DNA B protein acts in the 5'----3' direction, whereas factor Y acts in the 3'----5' direction. The primosome complex has DNA helicase activity when present at a replication fork in conjunction with the DNA polymerase III holoenzyme. In this report, evidence is presented that the multiprotein primosome per se can act as a DNA helicase in the absence of the DNA polymerase III holoenzyme. The primosome DNA helicase activity can be manifested in either direction along the DNA strand. The directionality of the primosome DNA helicase activity is modulated by the concentration and type of nucleoside triphosphate present in the reaction mixture. This DNA helicase activity requires all the preprimosomal proteins (the primosomal proteins minus the dnaG-encoded primase). Preprimosome complexes must assemble at a primosome assembly site in order to be loaded onto the single-stranded DNA and act subsequently as a DNA helicase. The 5'----3' primosome DNA helicase activity requires a 3' single-stranded tail on the fragment to be displaced, while the 3'----5' activity does not require a 5' single-stranded tail on the fragment to be displaced. Multienzyme preprimosomes moving in either direction are capable of associating with the primase to form complete primosomes that can synthesize RNA primers.     

Differential ATP requirements distinguish the DNA translocation and DNA unwinding activities of the Escherichia coli PRI A protein

The Escherichia coli primosome is a mobile multiprotein DNA replication-priming apparatus that assembles at a specific site (termed a primosome assembly site (PAS] on single-stranded DNA-binding protein-coated single-stranded DNA. The PRI A protein (factor Y, protein n') is a PAS sequence-specific (d)ATPase as well as a DNA helicase and is believed to direct the assembly of the primosome at a PAS. In this report, the PRI A DNA helicase reaction is dissected in vitro, by use of a strand displacement assay, into three steps with distinct ATP requirements. First, the PRI A protein gains entry to the DNA via an ATP-independent, PAS sequence-specific binding event. Second, the PRI A protein translocates along the single-stranded DNA in the 3'----5' direction at a maximal rate of 90 nucleotides/s. DNA translocation requires ATP hydrolysis. The ATP concentration required to support half of the maximal translocation rate is 100 microM, which is identical to the Km for ATP of the PRI A protein DNA-dependent ATPase activity. Finally, the PRI A protein unwinds duplex DNA. The ATP concentration required for duplex DNA unwinding depends upon the length of the duplex region to be unwound. Displacement of a 24-nucleotide long oligomer required no more ATP than that required for the translocation of PRI A protein along single-stranded DNA, whereas displacement of a 390-nucleotide long DNA fragment required a 10-fold higher concentration of ATP than that required for oligomer displacement.     

ATPase-deficient mutants of the Escherichia coli DNA replication protein PriA are capable of catalyzing the assembly of active primosomes.

The PriA replication protein of Escherichia coli (formerly replication factor Y or protein n') is multifunctional. It is a site-specific, single-stranded DNA-dependent ATPase (dATPase), a 3'----5' DNA helicase, and guides the ordered assembly of the primosome, a mobile, multiprotein DNA replication priming/helicase complex. Although PriA is not absolutely required for viability, priA null mutant cells grow very slowly, have poor viability, and form extensive filaments. In order to assess which of the multiple activities of PriA are required for normal replication and growth, site-directed mutagenesis was employed to introduce single amino acid substitutions for the invariant lysine within the consensus nucleotide-binding motif found in PriA. Biochemical characterization of the representative purified mutant PriA proteins revealed them to be completely deficient in nucleotide hydrolysis, incapable of translocation along a single-stranded DNA binding protein-coated single-stranded DNA template, and unable to manifest the 3'----5' DNA helicase activity of wild-type PriA. These mutant proteins were, however, capable of catalyzing the assembly of active primosomes in vitro. Furthermore, when supplied in trans to insertionally inactivated priA cells, plasmids containing a copy of these mutant priA genes restored the wild-type growth rate, viability, and cell morphology. Based on these results, a model for PriA function in vivo is discussed.     

Early steps of Bacillus subtilis primosome assembly

Primosomes are nucleoprotein assemblies designed for the activation of DNA replication forks. Their primary role is to recruit the replicative helicase onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. PriA is conserved in bacteria, but its primosomal partners are not. In Bacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB and DnaD proteins unravels their role in primosome assembly. They are both multimeric and bind individually to DNA. Furthermore, DnaD stimulates DnaB binding activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork.     

Identification of Subunit Binding Positions on a Model Fork and Displacements That Occur during Sequential Assembly of the Escherichia coli Primosome

When replication stalls and forks disassemble, the restart primosome is required to reload the replicative helicase so that chromosomal replication can be reinitiated. We have taken a photo-cross-linking approach, using model replication forks containing a phenyl diazirine placed at single locations, to determine the positions of primosomal protein binding and changes in interactions that occur during the assembly reaction. This approach revealed a novel mode for single-stranded DNA-binding protein (SSB)-DNA binding, in which SSB interacts with both the leading and lagging single-strand segments and the parental duplex of the fork. Cross-linking to a novel region within SSB is observed only when it is bound to forked structures. This binding mode is also followed by PriB. PriA binds to the fork, excluding SSB and PriB, interacting with the primer terminus, single-stranded leading and lagging strands and duplex in immediate proximity of the fork. SSB binds to flanking single-stranded segments distal to the fork in the presence of PriA. The addition of PriB or DnaT to a PriA-SSB-fork complex does not lead to cross-linking or displacement, suggesting that their association is through protein-protein interactions at early stages of the reaction. Upon addition of DnaC and the DnaB helicase in the presence of ATPγS, helicase is assembled, leading to contacts within the duplex region on the tracking (lagging) strand and strong contacts with the displaced leading single strand near the fork. PriA is displaced from DNA upon helicase assembly.     

Properties of the PriA helicase domain and its role in binding PriA to specific DNA structures

Primosome assembly protein PriA functions in the assembly of the replisome at forked DNA structures. Whereas its N-terminal DNA binding domain (DBD) binds independently to DNA, the affinity of DBD protein for forked structures is relatively weak. Although the PriA helicase domain (HD) is required for high affinity fork binding, HD protein had very low affinity for DNA. It had only low levels of ATPase activity, and it hydrolyzed ATP when DNA was absent whereas PriA did not. HD catalyzed unwinding of a minimal substrate composed of a duplex with a 3' single-stranded tail. Single-strand binding protein (SSB) bound to the tail of this substrate inhibited this reaction by full-length PriA but enhanced the reaction by HD. SSB stabilized binding of PriA but not of DBD or HD to duplexes with a 5' or 3' single-stranded tail. On forked substrates SSB enhanced helicase action on the lagging-strand arm by PriA but not by HD. The results indicate that synergy of the DBD and HD allows stable binding at the interface between duplex and single-stranded DNA bound by SSB. This mode of binding may be analogous to fork binding, which orients the helicase to act on the lagging-strand side of the fork.     

PriB stimulates PriA helicase via an interaction with single-stranded DNA

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.     

The 3'-tail of a forked-duplex sterically determines whether one or two DNA strands pass through the central channel of a replication-fork helicase

DnaB helicase is a ring-shaped hexamer that unwinds DNA at a replication fork. To understand how this protein interacts with DNA during unwinding, DnaB from Thermus aquaticus was incubated with chemically modified forked-duplex DNA substrates and the unwinding rates were measured. Unwinding was inhibited by modifications made to the 5'-tail, but not the 3'-tail, suggesting that the helicase interacts with the 5'-tail but not the 3'-tail during unwinding. Using oligonucleotides of mixed polarity, it was confirmed that DnaB translocates in the 5' to 3' direction as it unwinds DNA. A substrate was synthesized that contained two duplexes in tandem. Experiments involving various modifications of this tandem duplex demonstrated that when the 3'-tail is short, two stands of DNA pass through the central channel of DnaB with no resultant unwinding. Thus, the role of the 3'-tail in stimulating unwinding has been elucidated. The 3'-tail does not bind to DnaB during unwinding, but sterically determines whether one or two DNA strands pass through the central channel of DnaB. Furthermore, a new substrate for DnaB locomotion has been discovered. DnaB may actively translocate in the 5' to 3' direction along single-stranded DNA, even when a complementary strand is also present within the protein's central channel. This new mode of action may regulate DnaB activity by inhibiting unwinding at regions of DNA that are not forked. Furthermore, this new function for DnaB may coordinate abortion of leading and lagging strand replication if a nick is encountered on the leading strand.     

Non-replicative helicases at the replication fork

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 binding. 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 redundancy between Rep and UvrD and possibly explaining the observed synthetic lethality that occurs when mutations in the genes encoding these two proteins are combined.     

A DNA helicase induced by herpes simplex virus type 1.

We have identified and partially purified a DNA-dependent ATPase that is present specifically in herpes simplex virus type 1-infected Vero cells. The enzyme which has a molecular weight of approximately 440,000 differs from the comparable host enzyme in its elution from phosphocellulose columns and in its nucleoside triphosphate specificity. The partially purified DNA-dependent ATPase is also a DNA helicase that couples ATP or GTP hydrolysis to the displacement of an oligonucleotide annealed to M13 single-stranded DNA. The enzyme requires a 3' single-stranded tail on the duplex substrate, suggesting that the polarity of unwinding is 5'----3' relative to the M13 DNA. The herpes specific DNA helicase may therefore translocate on the lagging strand in the semidiscontinuous replication of the herpes virus 1 genome.     

Two modes of PriA binding to DNA.

The role of PriA, required for the assembly of the phiX174-type primosome on DNA, in cellular DNA replication has been unclear since its discovery. Recent evidence, based on the phenotypes of strains carrying priA null mutations, has led to proposals that the primosome assembly activity of PriA was required to load replication forks at intermediates such as D loops during homologous recombination. McGlynn et al. (McGlynn, P., Al-Deib, A. A., Liu, J., Marians, K. J., and Lloyd, R. G. (1997) J. Mol. Biol. 270, 212-221) demonstrated that PriA could, in fact, bind D loops. We show here that there are two modes of stable binding of PriA to DNA. One mode, in which the enzyme binds 3'-single-stranded extensions from duplex DNAs, presumably reflects the 3' --> 5' DNA helicase activity of PriA. The D loop DNA binding activity of PriA can be accounted for by the second mode, where the enzyme binds bent DNA at three strand junctions.     

Polar arrest of the simian virus 40 tumor antigen-mediated replication fork movement in vitro by the tus protein-terB complex of Escherichia coli.

The effect of the tus protein-terB sequence complex of Escherichia coli on the movement of the SV40 large tumor antigen (T antigen)-mediated replication fork during SV40 DNA replication in vitro has been examined. In the monopolymerase and dipolymerase systems, the tus protein-terB complex efficiently blocked the replication fork movement in a polar fashion, as observed in prokaryotic replication systems. With crude cytosolic extracts of HeLa cells, the same polarity of fork arrest was observed, but the block of replication fork movement was inefficient. These results indicate that the structure of the prokaryotic tus protein-terB complex allows it to block replication fork movement in an orientation-dependent manner. We also show that the tus protein-terB complex blocks the 3'----5' helicase action of T antigen in a polar fashion, using substrates comprised of single-stranded M13 DNA with either a 52-base pair (bp) or 29-bp duplex containing the terB sequence. The tus protein-terB complex formed on the 52-bp duplex was less effective than the complex formed on the 29-bp duplex in blocking the helicase action of T antigen. With the 52-bp duplex substrate, T antigen movement was only partially (30%) blocked by the tus protein-terB sequence complex in the active orientation, whereas the E. coli dnaB helicase moving 5'----3' was blocked more than 90% by the complex in the active orientation. However, with the shorter 29-bp duplex substrate, the complex blocked the T antigen helicase activity about 75%, whereas the dnaB helicase activity was completely blocked. Altogether, these results suggest that the T antigen helicase activity, when coupled to DNA replication, is more susceptible to arrest by the tus protein-terB complex than the T antigen functioning as a helicase alone.     

Isolation of a DNA helicase from HeLa cells requiring the multisubunit human single-stranded DNA-binding protein for activity.

A DNA helicase, dependent on the multisubunit human single-stranded DNA binding protein (HSSB), was isolated from HeLa cells. At low levels of helicase, only the multisubunit SSBs, HSSB and yeast SSB, stimulated DNA helicase activity. At high levels of the helicase Escherichia coli SSB partially substituted for HSSB whereas other SSBs such as T4 gene 32 and adenovirus DNA binding protein did not stimulate the enzyme activity. Maximal activation of helicase activity occurred in the presence of one molecule of HSSB for every 20 nucleotides of single-stranded DNA. The addition of E. coli SSB significantly lowered the amount of HSSB required for strand displacement, suggesting that the HSSB plays at least two roles in the activation of the helicase. One is to bind single-stranded DNA, thereby preventing sequestration of the helicase, the other involves the interaction of the HSSB with the helicase. Monoclonal antibodies that interact with the 70- and 34-kDa subunits of HSSB inhibited its stimulation of the helicase activity. The DNA helicase acted catalytically in displacing duplex DNA and translocated in the 3' to 5' direction. The helicase displaced fragments from both ends of a DNA substrate that contained duplex region at both termini, but the 3' to 5' fragment was displaced 20 times faster than the 5' to 3' fragment. Since this helicase also displaced fully duplex DNA, the release of the 5' to 3' fragment may have occurred by entry of the helicase through the duplex end in a 3' to 5' direction.     

The Escherichia coli dnaB replication protein is a DNA helicase

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.     

Unwinding of the nascent lagging strand by Rep and PriA enables the direct restart of stalled replication forks

During origin-independent replisome assembly, the replication restart protein PriC prefers to load the replication fork helicase, DnaB, to stalled replication forks where there is a gap in the nascent leading strand. However, this activity can be obstructed if the 5'-end of the nascent lagging strand is near the template branch point. Here we provide biochemical evidence that the helicase activities of Rep and PriA function to unwind the nascent lagging strand DNA at such stalled replication forks. PriC then loads the replicative helicase, DnaB, onto the newly generated, single-stranded template for the purposes of replisome assembly and duplex unwinding ahead of the replication fork. Direct rescue of replication forks by the Rep-PriC and PriA-PriC pathways in this manner may contribute to genomic stability by avoiding the potential dangers of fork breakage inherent to recombination-dependent restart pathways.     

PriA supports two distinct pathways for replication restart in UV-irradiated Escherichia coli cells

The Escherichia coli PriA protein loads the DnaB replicative helicase at branched DNA structures independently of the replication initiator protein, DnaA, and thereby facilitates assembly of functional replisomes at sites removed from oriC. It is therefore a critical factor in the rescue of replication forks stalled at DNA lesions. It is also a DNA helicase. We describe insertions near the 3' end of priA that interfere with PriA activity. These insertions and the previously described priA300 encoding helicase-defective PriA K230R are shown to be effective suppressors of the DNA repair defect in recG strains, but substantially reduce the ability of ruv mutants to survive DNA damage. The data presented suggest that PriA helicase in conjunction with RecG can promote direct rescue of stalled forks independently of the recombinational pathway promoted by the combined activities of the RuvABC, RecBCD and RecA proteins, which requires only the primosome assembly activity of PriA to load DnaB at D loops. In cells lacking the helicase activity of PriA, we propose that stalled forks can be redirected to the recombination pathway via a Holliday junction intermediate common to both pathways, thus explaining the resistance of these cells to DNA damage.     

DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome.

Phenotypes of Bacillus subtilis priA mutants suggest that they are deficient in the restart of stalled chromosomal replication forks. The presumed activity of PriA in the restart process is to promote the assembly of a multiprotein complex, the primosome, which functions to recruit the replication fork helicase onto the DNA. We have proposed previously that three proteins involved in the initiation of replication at oriC in B. subtilis, DnaB, DnaD and DnaI, are components of the PriA primosome in this bacterium. However, the involvement of these proteins in replication restart has not yet been studied. Here, we describe dnaB mutations that suppress the phenotypes of B. subtilis priA mutants. In a representative mutant, the DnaC helicase is loaded onto single-stranded DNA in a PriA-independent, DnaD- and DnaI-dependent manner. These observations confirm that DnaB, DnaD and DnaI are primosomal proteins in B. subtilis. Moreover, their involvement in the suppression of priA phenotypes shows that they participate in replication fork restart in B. subtilis.     

DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion

DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases (MCM and DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions.