Single-stranded DNA binding protein Gp5 of Bacillus subtilis phage Φ29 is required for viral DNA replication in growth-temperature dependent fashion.

In the absence of viral single-stranded DNA binding protein gp5, Bacillus subtilis phage φ29 failed to grow and to replicate its genome at 45 °C, while it grew and replicated normally at 30 °C and 42 °C. This indicates that gp5 is dispensable for φ29 DNA replication at 42 °C and lower temperatures.

Functional linkages between replication proteins of genes 1, 3 and 5 of Bacillus subtilis phage φ29.

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 temperature-sensitive 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 single-stranded 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.

A variant protein from phi29 replication gene, gene 1, did not form homo-polymer due to a single amino acid substitution near the carboxyl terminus.

For study of the self-association of the product of psi29 gene 1, one variant which has a substitution at the 71(st) amino acid was used. By glycerol gradient sedimentation, the product of wild-type gene 1 existed both as large aggregate and oligomer, whereas the variant was detected as a single peak of monomer size. According to experiments using His-tagged proteins and Ni-NTA magnetic beads, the variant made only a little self-associated complex. From these results, a site essential for self-association was suggested to exist close to the carboxyl terminus of the product of psi29 gene 1.

Effects of single amino acid substitutions at the predicted coiled-coil or hydrophobic region on the self-assembly of phi29 replication protein, gp1.

Gp1, the product of one of the essential genes of phi29 replication, is an RNA binding protein and self-associates to form large complexes. Furthermore, gp1 suppresses the synthesis of phi29 DNA polymerase and primer protein in the post-transcriptional process. In this report, we have employed seven variants with single amino acid substitutions to analyze the self-assembly of gp1. Using chemical cross-linking and sedimentation assays, amino acid substitutions within the predicted coiled-coil or hydrophobic region were shown to strongly affect the formation of large complexes, suggesting that these two regions were required for the self-assembly of gp1. The self-association of gp1 was suggested to be necessary for the efficient binding to RNA and the translational repression.

Isolation of a series of single missense mutants of a dna gene of phage psi29, gene 1, utilizing their inhibitory effect on E. coli growth.

Gene 1 product (gp1) of Bacillus subtilis phage psi29 is known to promote DNA replication of the phage. Although its role in the DNA replication is not clear, gp1 is reported to exhibit multiple characteristics, including RNA binding, cell membrane localization, and self-association. To investigate these characteristics, we undertook the isolation of a series of missense mutants of gene 1 bearing substitutions at various regions. During cloning of gene 1, we found that its expression severely inhibited the growth of its host Escherichia coli cells. In this study, we utilized this growth-inhibition phenomenon to screen a random library muta-genized by error-prone PCR, expecting that mutants which could not inhibit cell growth would be affected in the authentic functions of gp1. Using this approach, we obtained 31 different mutants bearing single amino acid substitutions at 26 positions along the entire length of gp1. As a preliminary analysis of these mutants, we compared the deduced amino acid sequences of gp1s from psi29 and its related phages PZA, B103 and M2. Alignment of these sequences revealed three conserved regions, i.e. a hydrophobic region near the carboxyl terminus (assumed to be involved in the membrane localization and self-association of gp1), coiled-coil motif (essential for self-association), and a region of unknown function near the amino terminus. Interestingly, many of the substitutions in the isolated mutants occurred at strongly conserved residues in these regions and affected characteristic features of the regions (e.g. hydrophobicity of the hydrophobic region). These substitutions are expected to affect authentic functions of gp1, and the mutants will be useful for studies of the structure and functions of gp1.