Gene 1 product (gp1) of Bacillus subtilis phage ϕ29 has been shown to be involved in viral DNA replication in vivo, but the essential role is still unknown. As part of an ongoing effort to understand the role of gp1 in viral DNA replication, we investigated genetic interaction between gene 1 and other viral genes. Because ϕ29 mutants which do not produce functional gp1 show temperaturesensitive growth, we isolated temperature-resistant phages from the ϕ29 gene 1 mutants, and eventually, obtained nine extragenic suppressors. These suppressor mutations were located in two essential genes for ϕ29 DNA replication in vivo: gene 3 encoding terminal/primer protein (gp3) or gene 5 encoding viral singlestranded DNA binding protein (gp5). Most of these mutations resulted in single amino acid substitutions in the products. By trans-complementation assay, we confirmed that the absence of gp1 at non-permissive temperature can be compensated by the suppressors which have the single amino acid substitution in either gp5 or gp3. These results indicate that gp1 has functional relationship to gp5 and gp3. From the positions of amino acid substitutions in gp3, we propose its new regulatory subdomain at which other molecules including gp1 would interact with and regulate functions of gp3.
EndoSceI is a eucaryotic site-specific endoDNase of 120 kDa that causes double-stranded scission at welldefined sites, but is distinguishable from procaryotic restriction endonucleases by its mode of sequence recognition and lack of related specific DNA modification. In purified preparations of endoSce1, only two polypeptide species of 75 kDa (75-kDa peptide) and 50 kDa (50-kDa peptide) are detected in apparently equal amounts. We prepared mouse monoclonal IgGs that bound specifically to the 75-kDa peptide (but not the 50-kDa peptide) without inhibiting the endoSceI activity. Immunoprecipitation experiments with these IgGs revealed that the 75-kDa peptide and the 50-kDa peptide are physically associated with each other and with the endonucleolytic activity. Full endoSceI activity was recovered by mixing the purified 75-kDa peptide and the partially purified 50-kDa peptide, each of which exhibited.little or no endonuclease activity alone. These observations indicate that endoSceI consists of two non-identical subunits of 75 kDa and 50 kDa, and that both subunits are required for full enzyme activity.Extensive genetic studies on the homologous recombination during meiosis in fungi and other organisms have suggested that meiotic recombination, including both gene conversion and crossing-over, is initiated by the cleavage of DNA strands at specific sites (see [l -31 for reviews). Prompted by this suggestion, we found and purified endoSceI from Saccharomyces cerevisiae, the first example of a eucaryotic site-specific endonuclease [4 -61. Like bacterial type I1 and type I11 restriction endonucleases, endoSce1 causes double-stranded scission at well-defined sites [5]. However, endoSce1 has unique characteristics that distinguish it from restriction endonucleases [5, 71: (a) it is active on endogenous DNA [6]; (b) it differs from restriction endonucleases in its mode of recognition of the cleavage sites. No common sequence consisting of five base pairs or more is found around the cleavage sites for endoSceI on the substrate DNA, but, instead, there is a very asymmetric consensus sequence consisting of 26 base pairs [7]. Therefore, the mode of recognition of cleavage sites by endoSceI resembles that of target sequences by regulatory proteins, such as RNA polymerases and protein factors for transcriptional regulation. Recently three new site-specific eucaryotic endonucleases were found in S. cerevisiae : HO-endonuclease (originally named YZendonuclease; [S, 9]), endoSceII [8, 91 and an endonuclease (rl-endonuclease) encoded by the intron of mitochondria1 21s rRNA (rl intron) [lo]. The HO-endonuclease and rlendonuclease have been shown to be involved in the initiation of gene conversion, a type of recombination; the former is involved in mating-type switching in homothallic yeasts and the latter in the transposition of rl intron into the w -site. A comparison of the structural features around the cleavage sites of endoSce1, endoSceII and HO-endonuclease suggests Correspondence to T. Shibata, Laboratory of Microbiology, Ri...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.