Continuation is planned of a long-term study of plasmid molecular biology, focusing on the model rolling circle plasmid, pTl8l. Studies are proposed of the mechanisms of initiation and termination of leading strand replication, with special attention to the possible involvement of a type C cruciform structure containing the specific nick site. A comprehensive mutational analysis of the plasmid-coded initiator (Rep) protein is proposed, with the aim of isolating and localizing mutations affecting each of the protein's nine known functions. Attention will be devoted to the effects of local and distal secondary structures and other special sequence elements on the function of the pTl8l leading strand origin, with special reference to cis-inhibitory sequences. We have predicted and verified the replication-specific inactivation of the Rep protein and have demonstrated that the inactive form of the protein is modified. The nature of the modification and the mechanism of its formation will be determined.

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National Institute of General Medical Sciences (NIGMS)
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New York University
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Jin, R; Novick, R P (2001) Role of the double-strand origin cruciform in pT181 replication. Plasmid 46:95-105
Jin, R; Fernandez-Beros, M E; Novick, R P (1997) Why is the initiation nick site of an AT-rich rolling circle plasmid at the tip of a GC-rich cruciform? EMBO J 16:4456-66
Jin, R; Zhou, X; Novick, R P (1996) The inactive pT181 initiator heterodimer, RepC/C, binds but fails to induce melting of the plasmid replication origin. J Biol Chem 271:31086-91
Rasooly, A; Projan, S J; Novick, R P (1994) Plasmids of the pT181 family show replication-specific initiator protein modification. J Bacteriol 176:2450-3
Rasooly, A; Wang, P Z; Novick, R P (1994) Replication-specific conversion of the Staphylococcus aureus pT181 initiator protein from an active homodimer to an inactive heterodimer. EMBO J 13:5245-51
Wang, P Z; Projan, S J; Henriquez, V et al. (1993) Origin recognition specificity in pT181 plasmids is determined by a functionally asymmetric palindromic DNA element. EMBO J 12:45-52
Bargonetti, J; Wang, P Z; Novick, R P (1993) Measurement of gene expression by translational coupling: effect of copy mutations on pT181 initiator synthesis. EMBO J 12:3659-67
Rasooly, A; Novick, R P (1993) Replication-specific inactivation of the pT181 plasmid initiator protein. Science 262:1048-50
Projan, S J; Novick, R P (1992) cis-inhibitory elements in the pT181 replication system. Plasmid 27:81-92
Wang, P Z; Projan, S J; Henriquez, V et al. (1992) Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element. J Mol Biol 223:145-58

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