Identification of genes Involved in Escherichia coli DNA replication

Abstract

Chromosomal DNA replication is a key step not only for genetic inheritance but also for the regulation of cell proliferation. Intensive biochemical studies revealed the role of initiator proteins in DNA replication. But, the molecular mechanisms underlying the regulation of initiation process is relatively unexplored. For identifying the genes involved in that regulation, DDRT-PCR was adapted. It was designed to detect the alternation of transcript during cell cycle, but prevailing method was not efficient for prokaryotic RNA. So the first aim of my study is to develop DDRT-PCR for sensitive analysis. This purpose was accomplished by modulating the polyadenylation step; In that step, trans-acting DNA primer restricted the poly(a) tail-length and recruit the poly(A) polymerase to unreacted RNA substrate in first cycle. Next, an effort to find a noble DNA binding protein was suggested as being important to gain a better insight in this regulation process. So, novel candidates were implied to have DNA binding ability. The second aim of my study is characterization of putative DNA binding protein. In this study, the B1625 protein, one of the candidates, was purified in the Thioredoxin(Trx)fusion form and its oriC specific binding properties were confirmed.