| Summary: | 碩士 === 長庚大學 === 基礎醫學研究所 === 93 === The question of whether discontinuous DNA synthesis operates in only one strand or in both strands of DNA at a replication fork in Escherichia coli remains unresolved. Although in vitro data suggest a semi-discontinuous DNA replication model, most in vivo studies support a discontinuous DNA replication model. In this work, we reexamine the validity of discontinuous DNA replication in E. coli. First, by analyzing the size distribution of nascent DNA synthesized by pulse-labeling with 3H-thimidine, we confirm that wild-type E. coli cells replicate DNA discontinuously. Similarly, we observed that discontinuous DNA replication also operates in E. coli strains deficient in genes thought to be involved in replication fork reactivation (e.g. recF, priA and rep). Therefore, all of E. coli strains examined so far appear to employ discontinuous DNA synthesis to replicate their DNA.
To gain further insights about the nature of discontinuous DNA synthesis at a replication fork, we isolated 0.5~2 kb Okazaki DNA fragments and found these ssDNAs could self-reanneal to form duplex DNAs with lengths ranging from 0.5 to 2.6 kb. Based on this result, we suggest that the synthesis of Okazaki DNA fragments in leading and lagging strand are coupled at a given replication fork. Finally, we investigated the effects of UV irradiation on the DNA synthesis kinetics in excision-defective uvrB cells of E. coli. After low-doses of UV-irradiation in uvrB cells, the DNA synthesis was not affected at all. However, in some cells that had a defective repair of daughter strand gap, e.g., recA and recF mutants, the ability of replicating UV-damaged DNA were severely inhibited. These results are consistent with the idea that E. coli cells replicate UV-damaged DNA discontinuously, leaving daughter-strand gaps on the replicated chromosome. Taken together, our results are in accordance with the discontinuous DNA replication model originally proposed by Okazaki et al.
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