Abstract
The replication initiator protein (gene II protein (gpII] of bacteriophage f1 is a multifunctional protein that plays central roles in initiation and termination of phage DNA replication. It introduces a nick at a specific site on the (+)-strand of supercoiled replicative form DNA. The 3'-hydroxyl end of the nick serves as the primer for (+)-strand rolling-circle replication. Upon completion of a round of synthesis, gpII cleaves and circulaizes the displaced single strand. When Mn2+ is included in the buffer instead of Mg2+, gpII cleaves both strands. In this paper, we investigate the mechanism of the Mn2+-dependent double-strand cleavage activity of gpII. This reaction, unlike nicking in the presence of Mg2+, does not require superhelicity. The reaction proceeds in two kinetic steps: first nicking of the (+)-strand, and then cleavage of the (-)-strand. The nucleotide sequence requirement for nicking is reduced compared to that in the presence of Mg2+. The product of the double-strand cleavage has an unusual structure. The left end is a telomere-like hairpin since the (+)- and (-)-strands are joined, as demonstrated by base sequencing. The right end has a onebase 3'-overhang. This reaction probably reflects the cleavage-joining activity of gpII in the termination event.
Highlights
The replication initiator protein because gpII requires asupercoiled substrate for)of bacteriophage f l is a multifunctional protein nicking (Meyer and Geider, 1979b). gpII functions a t a that plays central roles in initiation and termination step beyond nicking: DNA molecules that have been nicked of phage DNA replication
The 3”hydroxyl end of the nick serves as gpII functions in the termination reaction bycleaving and the primer for (+)-strandrolling-circlereplication
The reaction proceeds in two kineticsteps: first nickcircularizing the displaced single strand (Harth et al, 1981). gpII has site-specific topoisomerase activity since, in the presence of M e, the RFI DNAis converted by gpII i n vitro to a mixture of RFII and RFIV (Meyer and Geider,1979b)
Summary
I Replicolion enhancer that extended to the siteof cleavage by gene I1 protein was isolated on a formamide (70%)-polyacrylamide (8%) gel and purified by elec-. The core origin sequence and thereplication enhancer sequence are indicated
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.