Genetic evidence for physical interactions between enzymes of nucleotide synthesis and proteins involved in DNA replication in bacteriophage T4.

We have found that mutations in phage T4 genes 41 (five of five) and 61 (three of three) cause resistance to the folate analogue pyrimethamine that inhibits T4 dihydrofolate (FH2) reductase. These genes code for subunits of a T4 primase and are part of a putative T4 replication complex. In contrast to many previously isolated folate analogue-resistant (Far) T4 mutants, these T4 primase mutants do not overproduce FH2 reductase nor do they alter its primary structure. A new mutant with a single lesion in gene 41 was isolated which proved resistant to the folate analogue at 30 degrees and was lethal at 42 degrees. This mutant induced normal levels of FH2 reductase (encoded by the frd gene) and appeared to have normal expression of other T4 genes at 30 degrees. Like other mutations in gene 41, tsP129 reduced phage-induced DNA synthesis to about 15% that of wild-type T4 as measured by thymidine incorporation under restrictive conditions. Double mutants carrying mutations in genes 41 and 61, 41 and frd or 61 and frd showed allele-specific suppression suggesting that the products of these genes interact. We suggest that abnormal interactions between components of the replication complex and a DNA precursor synthesizing complex cause folate analog resistance by allosterically altering the T4 FH2 reductase.