Recombination-dependent DNA replication in bacteriophage T4: An evolutionary study.
AuthorMcCreary, Ronald Patrick.
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PublisherThe University of Arizona.
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AbstractGenetic recombination is an ubiquitious process in living organisms and is one of the most elaborate activities that DNA undergoes. As such, the recombination process potentially interacts with all of the major metabolic events involving DNA. This study examined one of these interactions, specifically that occuring between recombination and DNA replication in phage T4. This interaction was investigated from both the mechanisitic and evolutionary viewpoints. In phage and bacteria, the central reactions of homologous recombination are those of homologous pairing and strand insertion. These reactions are generally catalyzed by a single enzyme. The E. coli recA gene product serves as the paradigm for this class of enzyme. Four cloned wild type "recA" genes from the bacterial species E. coli, A. caviae, B. pertussis, and the uvsX gene from phage T4, were used to test for complementation of both E. coli recA⁻ and phage T4 uvsX⁻ mutants. The B. pertussis recA⁺ gene was able to complement an E. coli recA⁻ mutant with respect to the repair of UV induced DNA damage and general recombination. In a phage Mode 2 DNA synthesis assay, all three bacterial recA genes complemented a uvsX⁻ mutant to some extent, but the B. pertussis RecA protein allowed "runaway" DNA synthesis to occur. In contrast, none of the three bacterial genes were able to increase DNA synthesis of a uvsX⁺, uvsY⁻ phage T4. In a one step growth experiment, only the B. pertussis RecA protein complemented a phage uvsX⁻ mutant. However, none of the three bacterial recA genes complemented the same phage mutant in a UV survival assay. These results imply that normal wild type Mode 2 phage DNA synthesis is an example of a specific interaction between the DNA replication and homologous recombination "machines" of phage T4. Multiplicity reactivation (MR) is a type of recombinational repair. MR experiments were performed using a gene 42(ts) mutant phage to study the effect of this member of the phage's DNA replication machinery on recombinational DNA repair. The data show that MR is dependent on the gene 42 gene product suggesting that the phage DNA replication and recombination metabolic pathways are coupled through a protein-protein interaction involving gpuvsX and gp42. A hypothesis concerning the molecular coevolution of DNA replication and recombination pathways is developed. This hypothesis is used to explain the significantly different ways in which each of the enzymatically equivalent "recA" proteins substitutes for the E. coli RecA protein and gpuvsX.