phiX174-type Primosome Research Articles

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.

Recombinational DNA Repair in Bacteria and the RecA Protein

In bacteria, the major function of homologous genetic recombination is recombinational DNA repair. This is not a process reserved only for rare double-strand breaks caused by ionizing radiation, nor is it limited to situations in which the SOS response has been induced. Recombinational DNA repair in bacteria is closely tied to the cellular replication systems, and it functions to repair damage at stalled replication forks, Studies with a variety of rec mutants, carried out under normal aerobic growth conditions, consistently suggest that at least 10-30% of all replication forks originating at the bacterial origin of replication are halted by DNA damage and must undergo recombinational DNA repair. The actual frequency may be much higher. Recombinational DNA repair is both the most complex and the least understood of bacterial DNA repair processes. When replication forks encounter a DNA lesion or strand break, repair is mediated by an adaptable set of pathways encompassing most of the enzymes involved in DNA metabolism. There are five separate enzymatic processes involved in these repair events: (1) The replication fork assembled at OriC stalls and/or collapses when encountering DNA damage. (2) Recombination enzymes provide a complementary strand for a lesion isolated in a single-strand gap, or reconstruct a branched DNA at the site of a double-strand break. (3) The phi X174-type primosome (or repair primosome) functions in the origin-independent reassembly of the replication fork. (4) The XerCD site-specific recombination system resolves the dimeric chromosomes that are the inevitable by-product of frequent recombination associated with recombinational DNA repair. (5) DNA excision repair and other repair systems eliminate lesions left behind in double-stranded DNA. The RecA protein plays a central role in the recombination phase of the process. Among its many activities, RecA protein is a motor protein, coupling the hydrolysis of ATP to the movement of DNA branches.

The phiX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition.

Initiation of Escherichia coli DNA synthesis primed by homologous recombination is believed to require the phiX174-type primosome, a mobile priming apparatus assembled without the initiator protein DnaA. We show that this primosome plays an essential role in bacteriophage Mu DNA replication by transposition. Upon promoting transfer of Mu ends to target DNA, the Mu transpososome undergoes transition to a pre-replisome that permits initiation of DNA synthesis only in the presence of primosome assembly proteins PriA, DnaT, DnaB and DnaC. These assembly proteins promote the engagement of primase and DNA polymerase III holoenzyme, initiating semi-discontinuous replication preferentially at the Mu left end. The results indicate that these proteins play a crucial role in promoting replisome assembly on a recombination intermediate.

The Ordered Assembly of the φX174-type Primosome: III. PriB FACILITATES COMPLEX FORMATION BETWEEN PriA AND DnaT

The properties of two mutant PriA proteins, PriA C439Y and PriA C445Y have been used to reveal the role of PriB during assembly of the phiX174-type primosome. The replication defects of both mutant PriA proteins could be rescued by high concentrations of DnaT. Analysis of the formation of intermediate complexes in primosome assembly and the effect of PriB on PriA binding to DNA demonstrated that the mutant PriA proteins could not form a PriA-PriB complex on DNA carrying a primosome assembly site. Consequently, the mutant proteins also could not form PriA-PriB-DnaT complexes at concentrations of DnaT sufficient to form such a complex with wild-type PriA. In addition, PriB was found to stabilize wild-type but not mutant PriA proteins on DNA. At high concentrations of DnaT, both mutant and wild-type PriA proteins could form a PriA-DnaT complex and support PriB-independent phiX174 complementary strand DNA replication. Thus, during primosome assembly, PriB facilitates complex formation between PriA and DnaT.

The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair

The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein capable of catalyzing the assembly of an active primosome but which is missing the n'-pas-dependent ATPase, helicase, and translocase activities associated with PriA, alleviates the defects of priA::kan mutants in homologous recombination, double-strand break repair, and inducible stable DNA replication. Furthermore, spa-47, which was isolated as a suppressor of the broth sensitivity of priA::kan mutants, suppresses the Rec- and mitomycin C sensitivity phenotypes of priA::kan mutants. The spa-47 suppressor mutation maps within or very near dnaC. These results suggest that PriA-dependent primosome assembly is crucial for both homologous recombination and double-strand break repair and support the proposal that these processes in E. coli involve extensive DNA replication.

Escherichia coli PriA protein is essential for inducible and constitutive stable DNA replication.

Under certain conditions, Escherichia coli cells exhibit either of two altered modes of chromosomal DNA replication. These are inducible stable DNA replication (iSDR), seen in SOS-induced cells, and constitutive stable DNA replication (cSDR), seen in rnhA mutants. Both iSDR and cSDR can continue to occur in the absence of protein synthesis. They are dependent on RecA protein, but do not require DnaA protein or the oriC site. Here we report the requirement for PriA, a protein essential for assembly of the phi X174-type primosome, for both iSDR and cSDR. In priA1(Null)::kan mutant cells, iSDR is not observed after induction by thymine starvation. Replication from one of the origins (oriM1) specific to iSDR is greatly reduced by the priA1::kan mutation. cSDR in rnhA224 mutant cells deficient in RNase HI is also completely abolished by the same priA mutation. In both cases, SDR is restored by introduction of a plasmid carrying a wild-type priA gene. Furthermore, the viability of an rnhA::cat dnaA46 strain is lost at 42 degrees C upon inactivation of the priA gene, indicating the lethal effect of priA inactivation on those cells whose viability depends on cSDR. These results demonstrate that a function of PriA protein is essential for iSDR and cSDR and suggest the involvement of the PriA-dependent phi X174-type primosome in these DnaA/oriC-independent pathways of chromosome replication. Whereas ColE1-type plasmids, known to be independent of DnaA, absolutely require PriA function for replication, DnaA-dependent plasmid replicons such as pSC101, F, R6K, Rts1 and RK2 are able to transform and to be maintained in the priA1::kan strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of phi X174 type primosome- and G4 type primase-dependent primings in initiation of lagging and leading strand syntheses of DNA replication.

Eleven single strand initiation sequences (ssi) were isolated from various plasmid genomes using a plaque-morphology assay. Out of seven ssi that require dnaB and dnaC functions for replication in a crude in vitro system, six use a phi X174 type priming mechanism, and a phi X174 type primosome is assembled at these sequences from the purified proteins, n'(priA), n(priB), n"(priC), dnaT, dnaB, dnaC, and primase. The same ssi potentiate dATPase activity of n' protein, and thus represent new n' protein recognition sequences (n'-pas). Based on sequence homology, two structural groups are evident. Two sequences show a strong homology with the phi X174 site, whereas three share extensive homology with the previously characterized n'-pas of ColE1, ssiA(ColE1). All the n'-pas have a potential to form stem and loop structures, although sequence homology between the two classes is absent. In addition to the phi X174 type priming, three ssi do not require either dnaB or dnaC function for replication, and use a G4 type priming, requiring only SSB and primase. The 5' ends of primer RNA synthesized by primase are localized within the vicinity of one of the three blocks of highly conserved nucleotide sequences. Deletions of parts of these conserved sequences result in loss of priming activity, suggesting that they are important for priming on the G4 type ssi, which are termed G site. The general significance of these two types of priming in initiation of lagging or leading strand synthesis as well as various modes of initiation at origins of replication are proposed.

Initiation of Escherichia coli minichromosome replication at oriC and at protein n' recognition sites. Two modes for initiating DNA synthesis in vitro.

The start sites for leading and lagging DNA strands were determined in vitro with minichromosomes as templates. Fragments from replication intermediates were analyzed by hybridization to single-stranded probes. Leading strand synthesis in the counterclockwise direction was found to originate in or close to (position 248 to -44) the minimal origin. Complementary lagging strand synthesis started several positions to the left outside of oriC. The results suggest in addition a concerted synthesis of leading and lagging strands following the dnaA directed assembly of initiation proteins at double-stranded oricC DNA (pre-replisome). In addition, DNA synthesis could initiate at protein n' recognition sequences located within and clockwise to the asnA gene. Initiation at n' sites was dependent on protein i activity, whereas leading and lagging strand initiation in the oriC region was not affected by protein i. Our results argue against an involvement of the phi X174-type primosome in the initiation of discontinuous DNA synthesis at oriC. An alternative function is suggested.