Alvin J. Clark

Genetic analysis of the recF pathway to genetic recombination in Escherichia coli k12: Isolation and characterization of mutants☆

A recombination proficient strain ofEscherichia coli which is recB− recC− sbcB− has been subjected to mutagenesis by nitrosoguanidine. Among the recombination deficient mutants isolated one was sbcB+, three were recA∼ and 11 were mutants in at least four newrec genes: recF, recJ, recK and recL. recF143 and recL152 are cotransducible with ilv but they lie on opposite sides of the ilv operons as determined by F

Homologous Genetic Recombination: The Pieces Begin to Fall into Place

studies. recF, recL and recK are not involved in the RecBC pathway of recombination since a recB+ recC+ sbcB− strain carrying a mutation in one of these genes is recombination proficient. Hence the hypothesis that a RecF pathway of recombination can operate as a partially independent substitute for the RecBC pathway of recombination is supported. recF− recB+ and recF+ recB− single mutants are sensitive to u.v. irradiation while the recF−recB− double mutant is more sensitive than either single mutant. The sensitivity of the recB− recC− sbcB− recF− strain approaches the sensitivity of a recA− single mutant. This is interpreted to mean that there are partially independent RecF and RecBC pathways for the repair of u.v. damage. recJ and mutations were not mapped precisely; hence the mutant properties they confer can not be stated conclusively.

Overexpression of archaeal proteins in Escherichia coli

One of the authors (AJC) acknowledges with gratitude the important role Fernando Bastarrachea played in the authors discovery that E. coli could carry out homologous genetic recombination by multiple pathways. This in turn led to the discovery of several genes, including recF, recO, and recR, whose role in recombination would not otherwise have been detected. Subsequent genetic and biochemical studies have led to a general formulation in which there are multiple nucleolytic ways to achieve a presynaptic intermediate bound to RecA protein. Postsynaptic events in the general formulation occur by means of multiple branch migration enzymes to form Holliday DNA structures and a specific nuclease to cleave them. The general formulation is built on synapsis catalyzed by RecA protein. A second RecA-independent synapsis catalyzed by RecT (and RecE?) protein is now under study and a third type independent of both RecA and RecT has apparently been discovered. How these will affect the general formulation remains to be seen. Some proteins, most prominently RecF, RecO, and RecR, have no role in the general formulation. The hypothesis is presented that these proteins act as a switch between replication and recombination by helping to convert replication to recombination intermediates. Universality of the general formulation is supported by the widespread occurrence of recA, recB, recC, and recD genes among bacteria. Recent discovery of recA-like genes in several eukaryotes further supports its universality. We have contributed additional support by sequencing a recA-like gene from an archaeal species, thus making it plausible that the mechanism of synapsis worked out for E. coli RecA protein will hold for all three organismal domains. The boundaries of the puzzle of homologous genetic recombination therefore seem complete and the pieces to the complex picture they encompass are falling into place.

Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichia coli K12.

Six archaeal proteins containing a high number of Escherichia coli rare codons in their genes were not well expressed in E. coli. These genes showed a five to twenty-fold increase in production when expressed in the presence of a plasmid harboring and expressing the argU and ileX genes encoding rare tRNAs (tRNA arg(de)AGA/AGG and tRNA ile(de)AUA.

Evolutionary Comparisons of RecA-Like Proteins Across All Major Kingdoms of Living Organisms

A given dose of ultraviolet irradiation produces as many pyrimidine-dimer-containing photoproducts in the DNA of the Escherichia coli Rec − strain JC1569 as it does in the related Rec + strain JC1557. Both strains excise dimer-containing photoproducts from their DNA to the same extent. In comparison with JC1557, approximately 30 times as much DNA of the Rec − strain is degraded per unit dose of ultraviolet light. Perhaps as a result of this excess ultraviolet-induced degradation, irradiated Rec − cells do not incorporate appreciable amounts of exogenous thymidine or thymine into DNA and have few survivors following exposure to an ultraviolet dose which almost all Rec + cells survive. A numerical argument is offered to establish the ability of Rec − cells successfully to replace excised pyrimidine-dimer-containing regions of the DNA with normal nucleotide residues. No effect of the rec − mutation in JC1569 was found on the level of activity of the following enzymes: endonuclease I, exonuclease I, exonuclease III and DNA phosphatase, and DNA polymerase and exonuclease II.

Characterization of REP-mutants and their interaction with P2 phage

Abstract. Protein sequences with similarities to Escherichia coli RecA were compared across the major kingdoms of eubacteria, archaebacteria, and eukaryotes. The archaeal sequences branch monophyletically and are most closely related to the eukaryotic paralogous Rad51 and Dmc1 groups. A multiple alignment of the sequences suggests a modular structure of RecA-like proteins consisting of distinct segments, some of which are conserved only within subgroups of sequences. The eukaryotic and archaeal sequences share an N-terminal domain which may play a role in interactions with other factors and nucleic acids. Several positions in the alignment blocks are highly conserved within the eubacteria as one group and within the eukaryotes and archaebacteria as a second group, but compared between the groups these positions display nonconservative amino acid substitutions. Conservation within the RecA-like core domain identifies possible key residues involved in ATP-induced conformational changes. We propose that RecA-like proteins derive evolutionarily from an assortment of independent domains and that the functional homologs of RecA in noneubacteria comprise an array of RecA-like proteins acting in series or cooperatively.

dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12

Abstract Three rep 5 mutations in Escherichia coli have been mapped between metE and ilv. rep is recessive. Using REP− strains derived by transduction, we have obtained no evidence that three rep mutations confer recombination deficiency, although they do do appear to confer a slight increase in UV sensitivity. A number of temperate coliphages cannot use REP− mutants as a host in lytic multiplication. The interaction of REP− mutants and the non-inducible temperate phage P2 is examined in detail, with the following findings: (1) P2 can inject its DNA into REP− hosts, and the phage DNA can circularize, but replication of the DNA is blocked. (2) P2 can lysogenize REP− mutants at approximately normal frequency. (3) REP− mutants are resistant to killing by P2.

Interaction of the recombination pathways of bacteriophage λ and its host Escherichia coli K12: Effects on exonuclease V activity☆

PriA, PriB and PriC were originally discovered as proteins essential for the ΦX174 in vitro DNA replication system. Recent studies have shown that PriA mutants are poorly viable, have high basal levels of SOS expression (SOSH), are recombination deficient (Rec−), sensitive to UV irradiation (UVS) and sensitive to rich media. These data suggest that priAs role may be more complex than previously thought and may involve both DNA replication and homologous recombination. Based on the ΦX174 system, mutations in priB and priC should cause phenotypes like those seen in priA2::kan mutants. To test this, mutations in priB and priC were constructed. We found that, contrary to the ΦX174 model, del(priB)302 and priC303::kan mutants have almost wild‐type phenotypes. Most unexpectedly, we then found that the priBC double mutant had very poor viability and/or a slow growth rate (even less than a priA2::kan mutant). This suggests that priB and priC have a redundant and important role in Escherichia coli. The priA2::kan suppressor, dnaC809, partially suppressed the poor viability/slow growth phenotype of the priBC double mutant. The resulting triple mutant (priBC dnaC809 ) had small colony size, recombination deficiency and levels of SOS expression similar to a priA2::kan mutant. The priBC dnaC809 mutant, however, was moderately UVR and had good viability, unlike a priA2::kan mutant. Additional mutations in the triple mutant were selected to suppress the slow growth phenotype. One suppressor restored all phenotypes tested to nearly wild‐type levels. This mutation was identified as dnaC820 (K178N) [mapping just downstream of dnaC809 (E176G)]. Experiments suggest that dnaC820 makes dnaC809 suppression of priA and or priBC mutants priB and or priC independent. A model is proposed for the roles of these proteins in terms of restarting collapsed replication forks from recombinational intermediates.

The dependence of postreplication repair on uvrB in a recF mutant of Escherichia coli K-12

Infection of recB+ host cells of Escherichia coli with lambda bacteriophage converts the cells to RecB− phenocopies. Two of the four activities of exonuclease V, the product of the recB and recC genes, have been tested and both disappear soon after infection by lambda or induction of a lambda lysogen. A functional phage gam+ gene is required to produce this effect. Exonuclease I activity is not reduced following lambda infection, indicating that the effect of the gam+ product may be specific to exonuclease V.

recO and recR mutations delay induction of the SOS response in Escherichia coli

SummaryMutants carrying recF143 or recF144 show wild type levels of host cell reactivation of UV-irradiated λvir and wild type rates of excision gap closure in repairing UV damage to their own DNA. The same mutants showed reduced rates of postreplication repair strand joining. When uvrA- recF- or uvrB- recF- strains are tested, postreplication repair strand joining is incomplete or does not occur at fluences above 1 J/m2. We suggest that there may be a UvrAB and a RecF pathway of postreplication repair or that the repair functions controlled or determined by uvrA uvrB and by recF may be similar. An intermediate in postreplication repair may accumulate in the uvr- recF- strain.