The aspect of DNA replication in phage T4 that is least understood is how a replicative fork is initiated at a natural origin. We had concluded in previous work that the products of genes 39, 52 and 60 interact to form a complex required for initiation, and that this complex could be partially substituted for by the host gyrase. Others showed that this complex is a type II topoisomerase. We now plan to see if the phage topoisomerase binds to one or more specific restriction fragments of phage DNA, presumably containing origins of replication. if topoisomerase protects portions of these fragments from nuclease action, we plan to determine the nucleotide sequence of such regions.
Our aim i s to identify features of the sequence that could provide clues to the functional characteristics of the origin. Work by us and others indicated that the host gyrA protein plays a role in phage T4 infections, and that it might interact with the phage topoisomerase. Therefore we plan to combine the gyrA protein with the phage topoisomerase, and look for an acquired ability by the topoisomerase to introduce negative superhelicity in DNA, and activity predicted but so far undetected. We recently found that an E. coli dnaG mutation reduces the rate of DNA synthesis by wild-type infecting phage T4, suggesting a role for dnaG in phage DNA replication. Using DNA figer autoradiography, we plan to determine if the dnaG product acts in initiation or elongation. Additional experiments are planned to determine which other host functions might be required for initiation of phage replication. Our other area of research deals with recombinational repair. We recently proposed, and evaluated the evidence for, the idea that the principal evolutionary advantage of germ line recombination, and hence of sexual reproduction, is repair of DNA damage. However, little is known at the molecular level about how damages are overcome by this process. Using psoralen lesions as a model, we plan to quantitate their removal by recombinational repair, to characterize some of the key steps in this repair, and to determine the gene products needed for these steps. We hope, on this basis, to formulate a pathway for recombinational repair at the molecular level. In general, knowledge of the mechanisms of DNA replication and repair are essential for understanding many biological problems, including such health problems as aging and carcinogenesis.
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