| Bacteriophage T7 is one of several double-stranded DNA phages that have been found to inactivate the host RecBCD enzyme during infection. In this work, T7 gene 5.9 has been identified as a gene whose product inhibits all known activities of RecBCD enzyme in vitro and at least the nuclease activities of the enzyme in vivo. Gene 5.9 was identified by genetic rescue of a phage T4 gene 2 amber mutant that cannot grow on a host with an active RecBCD. Gene 5.9 was cloned in a T7 expression vector, and gene 5.9 protein was expressed and purified. The inhibitory activity of gene 5.9 protein was verified by in vitro tests: it inhibits the double-stranded and single-stranded DNA exonuclease, single-stranded DNA endonuclease, helicase, and DNA-dependent ATPase activities of RecBCD enzyme. Gene 5.9 protein was found to inhibit RecBCD enzyme by binding to the holoenzyme, but it does not bind to isolated subunits of RecBCD. The binding is stoichiometric and reversible. The gene 5.9 protein is not inactivated by boiling. A high ;Gene 5.9 mutants were constructed and studied. Gene 5.9 was found not to be essential to T7 infection. However, the gene 5.9 coding sequence appears to be translationally coupled to the gene 6 coding sequence, and reduction of gene 5.9 protein can have effects by reducing the amount of gene 6 protein. It is not clear what role the gene 5.9 protein plays in T7 infection.;T7 gene 5.5 was also studied in this work. Gene 5.5 was found to be the gene responsible for the rbl (repressed by lambda) phenomenon rather than a potential gene 20 or gene 5.7, as previously thought. A single base pair change in gene 5.5, which changes the 30th codon from leucine to proline, prevents the mutant phage from growing on lambda lysogens. Other results suggest that gene 5.5 may have some role in DNA metabolism, but what this role is or how the rbl mutation prevents growth on lambda lysogens remains uncertain. |
