| Summary: | 碩士 === 長庚大學 === 基礎醫學研究所 === 94 === Replication of DNA is fundamental to all actively growing cells. In Escherichia coli, DNA replication is initiated at specific site on the genomic DNA, termed oriC, and then proceeds bidirectionally to the terminus. The question of whether discontinuous DNA synthesis operates in only the lagging strand or in both strands at a replication fork remains unresolved. Data from most in vivo studies are clearly in favor of a discontinuous DNA replication model, and the DnaG primase binding sites are known to be abundant on both strands. For discontinuous synthesis to occur in the leading-strand, one would predict that RNA primers are synthesized in the leading-strand, and therefore, there should be a primosome complex for the leading-strand. We hypothesize that inactivation of genes that function only in the leading-strand primosome shall convert the discontinuous synthesis to continuous mode. In this work, we examined several candidate genes ( priA, B, C and rep ) for their possible role in the leading strand DNA synthesis. The size distribution of nascent DNA synthesized from pulse-labeling with 3H-thimidine was analyzed in wild-type, priA, B, C and rep cells. The majority of short pulse-labeled (10 second) DNAs from the wild-type cells are smaller than 5kb, which is consistent with the previous findings by Okazaki et al. A greater percentage of short pulse-labeled DNAs from the priA2,and priA300, but not from priB302, priC303 or rep, mutants was partitioned into high Mr. (eg, greater than 5kb) forms. To minimize the technical problems of analyzing the small amount of DNA synthesized during the very short pulse-labeling, the effects of these mutations on the nascent DNAs synthesized in a lig-7 or polA4113 strain were examined. Introduction of priC303 into a lig-7 or polA4113 strain did not appear to alter the size distribution of nascent DNA synthesized in these mutants, i.e., the bulk of nascent DNA was all Okazaki fragments. However, the nascent DNA synthesized in the priA lig7, priA2 polA4113, or rep polA4113 strains appeared to contain a greater fraction of DNA in high Mr. (eg. greater than 5 kb). Our results indicate that the priA and possibly rep genes may play an important role in the apparent discontinuous DNA replication in E. coli.
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