Studies on the regulation of DNA replication of Rtsl and P

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A.The joint research with Dr.S.Austin groupThe Rtsl RepA protein, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rtsl RepA with the initiator protein of phage P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA (RepAXn), and the N-terminal from P1 RepA and the C-terminal from Rts1 RepA (RepALXn). We already reported the RepAXn functions in J.Bacteriol. 177 : 4028-4035, 1995, suggesting that the aminoacid residues 113 to 129 was important for ori binding in vitro, and the residues 177 to 206 region was required for ori activation in vivo as well as the ori binding domain.Four RepALXn proteins were constructed ; RepALX12 (P1 RepA 256 aa/Rts1 RepA 31 aa), RepALX13 (205/82), RepALX15 (144/143), and RepALX17 (112/175) RepALX15 and RepALX17 activated Rts1 ori efficiently. This evidenced our previous finding that the 177 to 206 aa region is essential for Rts1 ori activation. The P1 ori was activated by RepALX12 and RepALX13, which suggests a quite similar domain structure of P1 RepA to rts1 RepA.B.The joint research with Dr.A.Kaji groupWe determined the locus related to the inhibition of the host cell growth on the Rts1 genome. The locus, named hig, consists of higA and higB genes, and their functions were analyzed. The HigB product (92 aa) displayd a killer activity to the host cells and the HigA protein (104 aa) suppressed the HigB function. The results will be appeared in Biochem. Biophys.Res.Commun. (in press).