Genes that Control DNA Repair and Genetic Recombination in Escherichia coli

Cytotoxicity of free Cu(II) ions and its complexes of EDTA, NTA or IDA in Escherichia coli (AB 4401) wild type cells were assessed by cell inactivation assay. In order to understand the toxic effects of these additives, membrane status by AFM vis-à-vis K⁺ ion efflux were followed in the absence and in the presence of Cu(II) ions or its complexes. This was coupled with the determinations of cellular copper concentrations by atomic absorption spectrometry. The observed results show that free copper ions are more cytotoxic and cause considerable plasma membrane damage compared to that of Cu–EDTA, Cu–NTA and Cu–IDA. Determination of cellular copper reveals that Cu²⁺ and Cu–NTA are able to enter inside the cells while Cu–EDTA and Cu–IDA fail to do that. This may be attributed to the electron affinity of free Cu²⁺ ions and Cu–NTA, which help in binding with histidine present in copper transport proteins. In addition to cytotoxicity, genotoxicity of free copper and its complexes were also assessed on E. coli isogenic DNA repair proficient and repair-deficient strains. In contrast to free Cu²⁺ ions, Cu–NTA does not cause any significant cytotoxicity but render greater genotoxicity.

DMC1 is a new meiosis-specific yeast gene. Dmc1 protein is structurally similar to bacterial RecA proteins. dmc1 mutants are defective in reciprocal recombination, accumulate double-strand break (DSB) recombination intermediates, fail to form normal synaptonemal complex (SC), and arrest late in meiotic prophase. dmc1 phenotypes are consistent with a functional relationship between Dmc1 and RecA, and thus eukaryotic and prokaryotic mechanisms for homology recognition and strand exchange may be related. dmc1 phenotypes provide further evidence that recombination and SC formation are interrelated processes and are consistent with a requirement for DNA-DNA interactions during SC formation. dmc1 mutations confer prophase arrest. Additional evidence suggests that arrest occurs at a meiosis-specific cell cycle “checkpoint” in response to a primary defect in prophase chromosome metabolism. DMC1 is homologous to yeast's RAD51 gene, supporting the view that mitotic DSB repair has been recruited for use in meiotic chromosome metabolism.

In synchronous cultures of S. cerevisiae undergoing meiosis, an early event in the meiotic recombination pathway, site-specific double strand breaks (DSBs), occurs early in prophase, in some instances well before tripartite synaptonemal complex (SC) begins to form. This observation, together with previous results, supports the view that events involving DSBs are required for SC formation. We discuss the possibility that the mitotic pathway for recombinational repair of DSBs served as the primordial mechanism for connecting homologous chromosomes during the evolution of meiosis. DSBs disappear during the period when tripartite SC structure is forming and elongating (zygotene); presumably, they are converted to another type of recombination intermediate. Neither DSBs nor mature recombinant molecules are present when SCs are full length (pachytene). Mature reciprocally recombinant molecules arise at the end of or just after pachytene. We suggest that the SC might coordinate recombinant maturation with other events of meiosis.

Phage G4 origin of complementary DNA strand synthesis (ori_c) consists of three stable stem-loop structures (I, II, and III). Mutant ori_c sequences with alterations in the structure of stem-loop II, stem-loop III, and the stem-loop II–III spacer region have been constructed and cloned into the filamentous phage vectors to assay their functional activity. Changes in the lowermost GC base pair in the stem of stem-loop III, in the 9-bp spacer region between the stems of stem-loops II and III, and in the loop of stem-loop II, impair or abolish in vivo ori_c function. The results suggest that recognition sequences for dnaG primase must be present in the loop of stem-loop II, and in the spacer region between the stems of stem-loops II and III.

Using the plaque assay system for screening the single-strand (ss) initiation determinant (ssi) sequences, we have found that 119-bp region in pACYC184, a derivative of the plasmid P15A of Escherichia coli, can direct such ss DNA initiation. This region is located downstream from the P15A origin of replication and conserves consensus sequences of the ssi signals found in the other plasmids. Signals for ss DNA initiation are defined as nucleotide sequences present on ss DNA templates and required for priming DNA synthesis. The direction of chain elongation in DNA synthesis is opposite to that of the leading strand. In this region, we found a potential stem-and-loop structure. The 119-bp DNA segment of plasmid pACYC184 cloned in f1R199 filamentous phage could direct rifampicin-resistant conversion of the ss DNA to the double-stranded replicative form.

The efficiency of Weigle reactivation of ultraviolet light-irradiated single and double-stranded φX174 DNA by wild-type and excision repair-defective E. coli hosts was determined. After limited exposure to ultraviolet light, the efficiency of Weigle reactivation by an ultraviolet light-irradiated wild-type host was greater for double-stranded φX174 DNA than for its single-stranded counterpart. However, the efficiency of inducible until it became constant at a value 1.5 times less than that for single-stranded form of φX174 DNA. The efficiency of Weigle reactivation of the single-stranded DNA molecule by the same host, however, was independent of the dose to the DNA, as were the efficiencies of reactivation for both forms of φX174 DNA by ultraviolet light-irradiated excision repair-deficient hosts. In excision repair-defective hosts the efficiency of Weigle reactivation of double-stranded φX174 DNA was also 1.5 time less than that for the single-stranded molecule.

These results suggest that the Weigle reactivation of double-stranded φX174 DNA is mediated in part by an excision repair process, and that this component of Weigle reactivation eventually can be saturated by ultraviolet light-induced DNA damage leaving other repair processes, such as trans-damage synthesis, responsible for the remaining inducible reactivation.