Deoxyuridylate-hydroxymethylase of bacteriophage SPO1.

Phage SPO1 of Bacillus subtilis carries hydroxymethyl-deoxyuridylate in place of thymidylate in its DNA. The enzyme, responsible for the conversion of dUMP to HmdUMP, is a dUMP hydroxymethylase, encoded by the SPO1 gene 29. Here we describe the cloning and sequencing of the gene and the overexpression of the gene product. DNA hybridization using the DNA of bacteriophage T4 dCMP-hydroxymethylase gene as a probe, allowed us to identify and map g29 on a 3.9-kb restriction fragment, EcoRI*11. We determined the nucleotide sequence. One of the open reading frames detected, coding for a putative 44.6-kDa protein, showed significant amino acid homologies with all known thymidylate synthases. Gp29 was overexpressed in the pT7 system. Extracts prepared from induced cells show hydroxymethylase activity in a tritium release assay.