Tokio Kogoma

Recombination by Replication

The evidence summarized above strongly suggests that E. coli cells deal with exposed double-stranded ends by a process that requires both homologous recombination and DNA replication functions. This process could lead to recombinant formation. Importantly, inhibition of recombination-dependent replication in priA null mutants does not completely block homologous recombination, indicating the presence of other modes of homologous recombination that do not require extensive DNA replication. These nonreplicative modes of recombination could account for the multiple crosses seen within a given stretch of the chromosome. Nevertheless, a majority of recombinants appear to be formed by the replicative recombination mode in E. coli. Strong interdependence has also been documented for bacteriophage T4 DNA replication and homologous recombination processes. In T4-infected cells, DNA replication can be initiated from a recombination intermediate and such replication results in generation of recombinant molecules (reviewed byKreuzer and Morrical 1994xSee all ReferencesKreuzer and Morrical 1994). Under certain circumstances, plasmid replication can occur in a recombination-dependent manner in bacteria (Viret et al. 1991xViret, J.-F, Bravo, A, and Alonzo, J.C. Microbiol. Rev. 1991; 55: 675–683PubMedSee all ReferencesViret et al. 1991).In yeast and mammalian cells, there is evidence that ends generated by double-stranded breaks can initiate or stimulate homologous recombination (Thaler and Stahl 1988xThaler, D.S and Stahl, F.W. Annu. Rev. Genet. 1988; 22: 169–197Crossref | PubMedSee all ReferencesThaler and Stahl 1988). Transient meiosis-specific double-stranded breaks are known to occur at many locations in the yeast genome, which may initiate meiotic homologous recombination (Schwacha and Kleckner, 1995xSchwacha, A and Kleckner, N. Cell. 1995; 83: 783–791Abstract | Full Text PDF | PubMed | Scopus (330)See all ReferencesSchwacha and Kleckner, 1995 and references therein). Furthermore, double-stranded break repair is implicated in immunoglobulin gene recombination in mice and humans (Jeggo et al. 1995xJeggo, P.A, Taccioli, G.E, and Jackson, S.P. BioEssay. 1995; 17: 949–957Crossref | PubMed | Scopus (236)See all ReferencesJeggo et al. 1995). Thus, homologous recombination triggered by double-stranded breaks appears to be a widespread strategy for the generation of genetic diversity and repair of damaged DNA. Evidence suggests that DNA synthesis might be involved in immuloglobulin class switch recombination (e.g.,Dunnick et al. 1993xDunnick, W, Hertz, G.Z, Scappino, L, and Gritzmacher, C. Nucleic Acids Res. 1993; 3: 365–372Crossref | Scopus (204)See all ReferencesDunnick et al. 1993). Whether homologous recombination and double-stranded break repair in eukaryotic cells generally involve extensive DNA replication remains to be seen.

DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

Homologous recombination-dependent DNA replication (RDR) of a lambda cos site-carrying plasmid is demonstrated in E. coli cells when the cells express lambda terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot chi. Chi is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the theta mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (oriMs) on the chromosome.

The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12.

The sdrA224 mutants of Escherichia coli K‐12, capable of continued DNA replication in the absence of protein synthesis (stable DNA replication), tolerate inactivation of the dnaA gene by insertion of transposon Tn10. Furthermore, oriC, the origin of E. coli chromosome replication, can be deleted from the chromosome of sdrA mutants without loss of viability. The results suggest the presence of a second, normally repressed, initiation system for chromosome replication alternative to the ‘normal’ dnaA+ oriC+‐dependent initiation mechanism.

Multiple origin usage for DNA replication in sdrA(rnh) mutants of Escherichia coli K-12. Initiation in the absence of oriC.

In stable DNA replication (sdrA/rnh) mutants of Escherichia coli, initiation of rounds of DNA replication occurs in the absence of the normal origin of replication, oriC. To determine whether or not the initiation occurs at a fixed site(s) on the chromosome in sdrA mutants, the DNA from exponentially growing sdrA mutant cells with or without the oriC site (delta oriC) was analyzed for the relative copy numbers of various genes along the chromosome. The results suggest that there are at least four fixed sites or regions of the sdrA delta oriC chromosome from which DNA replication can be initiated in the absence of the oriC sequence.

Induction of UV-resistant DNA replication in Escherichia coli: Induced stable DNA replication as an SOS function

SummaryThe striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA+lexA+-dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication.The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with nalidixic acid was severely suppressed in tif-1 lexA mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-51 mutation, an intragenic suppressor of lexA3.Induced stable DNA replication was found to be considerably more resistant to UV irradiation than nromal replication both in a uvrA6 strain and a uvr+ strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed.

A novel Escherichia coli mutant capable of DNA replication in the absence of protein synthesis.

An Escherichia coli mutant capable of continued DNA synthesis in the presence of chloramphenicol has been isolated by an autoradiographic technique. The DNA synthesis represents semiconservative replication of E. coli DNA. It can occur in the presence of chloramphenicol or in the absence of essential amino acids, but not in the presence of an RNA synthesis inhibitor, rifampin. The mutant, termed constitutive stable DNA replication (Sdrc) mutant, appears to grow normally at 37 °C with a slightly slower growth rate than that of the parental strain. DNA replication in the mutant occurs at a reduced rate after 60 minutes in the absence of protein synthesis and continues linearly for several hours thereafter. This distinct slowdown in the DNA replication rate is due to a reduced rate of DNA synthesis in all the cells in the population. Constitutive stable DNA replication appears to require the dnaA and dnaC gene products. The sdrc mutation has been mapped near the pro-lac region of the E. coli chromosome. The mutation is recessive. Autoradiographic experiments have ruled out the possibility of multiple initiations during a cell cycle. The implication of the above findings is discussed in terms of the regulation of chromosome replication in E. coli.

Requirement of homologous recombination functions for viability of the Escherichia coli cell that lacks RNase HI and exonuclease V activities

rnhA224 and rnhA339::cat mutants which lack RNase HI activity were found to constitutively express the sfiA::lacZ operon fusion in a recA+ lexA(+)-dependent manner. The sfiA::lacZ expression (indicating SOS induction) in rnhA mutants was increased to higher levels by the introduction of the recD1903 or recB21 mutation. The SOS induction in these cells was further enhanced by nutritional shift up from casamino acid medium to Luria broth. Although the extent by which the recD and recB mutations increased the sfiA expression in rnhA mutants was similar, the rnhA224 recB21 double mutant had plating efficiencies that were 25-fold lower on casamino acid plates and 5 x 10(5)-fold lower on Luria broth plates than the respective plating efficiencies of either rnhA224 recD or rnhA::cat recD double mutants. Whereas the recD mutation inactivates the exonuclease activity of the RecBCD (Exo V) enzyme without reducing the recombination proficiency of the mutant, the recB21 mutation abolishes both the exonuclease activity and recombination capability. Therefore, in the absence of both RNase HI and Exo V activities, homologous recombination functions become crucial for viability, particularly in Luria broth. Introduction of mutations in recA, recJ and recN exacerbated the phenotypes. It is proposed that R-loops which persist due to the lack of RNase HI activity can be removed by two alternative routes of DNA repair: one involving Exo V, Exo I and DNA polymerase I, and the other involving both the RecBCD and RecF pathways of homologous recombination activities. The isolation of RNA polymerase mutants that constitutively express the SOS response at high levels and exhibit remarkable broth-sensitivity lend strong support to the contention that increased amounts of the persisting R-loop in rnhA mutants growing in Luria broth give rise to a stronger SOS response.

DNA damage-inducible origins of DNA replication in Escherichia coli

Upon induction of the SOS response in Escherichia coli, the mode of initiation of DNA replication is altered such that it can occur in the absence of normally required protein synthesis. This type of DNA replication has been termed induced stable DNA replication (iSDR). We examined the origin usage during iSDR and found that the initiation of iSDR occurs primarily in the oriC and terC regions of the chromosome in a manner completely independent of transcription, translation and DnaA protein. Minichromosomes (oriC plasmids) pOC23 and pOC81 were induced to replicate in the absence of DnaA protein and transcription after SOS induction. The results localized one of the iSDR origin activities in a 596 bp region which includes the minimal oriC.

Function of ribonuclease H in initiation of DNA replication in Escherichia coli K-12

SummaryEscherichia coli rnh mutants lacking ribonuclease H (RNase H) activity can tolerate deletion of the origin of DNA Replication (ΔoriC) and transposon-insertional inactivation of an initiator gene (dnaA:Tn10). Introduction of the recA200 allele encoding a thermolabile RecA protein intornh− dnaA: Tn10 and rnh−ΔoriC mutants strains rendered DNA synthesis and colony formation of these mutants temperature sensitive. The temperature sensitivity and the broth sensitivity (Srm−) of the rnh− dnaA: Tn10 recA200 strain was suppressed by the presenceof plasmids (pBR322 derivatives) carrying dnaA+only when the intact oriC site was present on the chromosome. Lack of RNase H activity neither promoted replication of minichromosomes (pOC24 and pλasn20) in the absence of required DnaA+ protein nor inhibited dnaA+−dependent minichromosome replication. These results led to the conclusion that RNase H is not directly involved in the events leading to initiation of DNA replication at oriC. Rather, it functions as a specificity factor by eliminating certain forms of RNA-DNA hybrids which could otherwise be used to prime DNA replication at sites other than oriC.

dnaA suppressor (dasF) mutants of Escherichia coli are stable DNA replication (sdrA/rnh) mutants

SummaryThe possible allelic relationship between dasF (dnaA suppressor) and sdrA/rnh (stable DNA replication/RNase H) mutations was examined. dasF mutations could not only suppress various dnaA(ts) mutations, but also the insertional inactivation of the dnaA gene or deletion of the oriC sequence, as could sdrA mutations. dasF mutants were found to exhibit the stable DNA replication phenotype, and the sensitivity to rich media, of sdrA mutants. The dasF and sdrA mutations were mapped very closely between metD and proA on the E. coli genetic map. The mutations were recessive to the wild-type allele for all the above phenotypes. It was concluded that dasF is allelic to sdrA/mh.