Functional linkages between replication proteins of genes 1, 3 and 5 of <i>Bacillus subtilis</i> phage <b>φ</b>29

Abstract

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.

Isolation of suppressors of the temperature-sensitive growth caused by a nonsense mutation in gene 1 of <i>Bacillus subtilis</i> phage ø29 using hydroxylamine

“…Gp5 has also a helix-destabilizing activity and greatly stimulates dNTP incorporation in vitro ø29 DNA replication (Gutiérrez et al, 1991;Martín et al, 1989;Soengas et al, 1995). The suppressor analyses suggest functional relationships between gp1, gp3, and gp5 and further investigations of these suppressors would be helpful to understand the role of gp1 in viral DNA replication (Tone et al, 2012a).…”

“…By mapping the suppressor mutations in gene 3 on the structure of gp3-DNA polymerase, we proposed the existence of a regulatory subdomain in the intermediate domain of gp3 (Tone et al, 2012a); in this model, gp1 regulates gp3's interactions by acting as a bridging molecule or as a competitive inhibitor, or by inducing a conformational change of gp3. On the other hand, the structure of gp5 is not yet determined.…”

“…As a parental strain, a ø29 gene 1 mutant ns1, which has an ochre codon at position 6 in gene 1 and does not produce gp1 in infected cells (Tone et al, 2012a), was used (Viral and bacterial strains used in this study are listed in Table 1). To avoid accumulation of numerous mutations Bacillus subtilis phage ø29 has a linear double-stranded DNA genome with a terminal protein covalently linked to each 5′ end (Hirokawa, 1972;Ortín et al, 1971;Yanofsky et al, 1976;, and has been studied extensively as a model system of the protein-priming DNA replication in which a viral-encoded protein provides the hydroxyl group needed by viral DNA polymerase to initiate the DNA synthesis (Matsumoto et al, 1983(Matsumoto et al, , 1984Peñalva and Salas, 1982;Watabe et al, 1982Watabe et al, , 1984.…”

“…The detailed mechanism and proteins involved in the reaction has been clarified in vitro (Blanco et al, 1986(Blanco et al, , 1992(Blanco et al, , 1994Gutiérrez et al, 1991). ø29 gene 1 mutants which do not produce functional gene 1 product (gp1) can replicate their genome at 30°C but not at 42°C, indicating that gp1 is required for ø29 DNA replication in a growth-temperature dependent fashion (Bravo and Salas, 1998;Carrascosa et al, 1976;Tone et al, 2012a). Although several characteristics of gp1 have been reported Salas, 1997, 1998;Bravo et al, 2000;Takeuchi et al, 1998), its role in viral DNA replication is still unclear.…”

“…Although several characteristics of gp1 have been reported Salas, 1997, 1998;Bravo et al, 2000;Takeuchi et al, 1998), its role in viral DNA replication is still unclear. By isolating temperature-resistant revertants with spontaneous mutations from ø29 gene 1 mutants, we have identified several second-site mutations that suppressed the defect of the gene 1 mutations at 42°C (Tone et al, 2012a); these suppressor mutations were located in gene 3 and gene 5. ø29 gene 3 product (gp3) is a key protein for ø29 DNA replication: it acts as a protein primer for the initiation of DNA replication and is also the component of the replication origin as a terminal protein (Matsumoto et al, 1983(Matsumoto et al, , 1984Peñalva and Salas, 1982;Watabe et al, 1982Watabe et al, , 1984. On the other hand, ø29 gene 5 product (gp5) is a single-stranded DNA binding (SSB) protein required for viral DNA replication in a growth-temperature dependent fashion (Martín et al, 1989; in the ø29 genomes, ø29 ns1 was incubated with 0.2 M hydroxylamine at 37°C for 20 hours to give a survival of 10 -2 ; on average, one mutation per genome is introduced in this condition (Matsumoto et al, 1986).…”