T4-PHAGE GENE-32 PROTEIN AS A CANDIDATE ORGANIZING FACTOR FOR THE DEOXYRIBONUCLEOSIDE TRIPHOSPHATE SYNTHETASE COMPLEX

After T4 bacteriophage infection of Escherichia coli, the enzymes of d eoxyribonucleoside triphosphate biosynthesis form a multienzyme comple x that we call T4 deoxyribonucleoside triphosphate (dNTP) synthetase, At least eight phage-coded enzymes and two enzymes of host origin are found in this 1.5-mDa complex. The complex may shuttle dNTPs to DNA re plication sites, because replication draws from small pools, which are probably highly localized. Several specific protein-protein contacts within the complex are described in this paper. We have studied protei n-protein interactions in the complex by immobilizing individual enzym es and identifying radiolabeled T4 proteins that are retained by colum ns of these respective affinity ligands. Elsewhere we have described i nteractions involving three T4 enzymes found in the complex. In this p aper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ri bonucleotide reductase, and E. coli nucleoside diphosphokinase,. All e ight proteins analyzed to date retain single-strand DNA-binding protei n (gp32), the product of T4 gene 32. At least one T4 protein, thymidyl ate synthase, binds directly to gp32, as shown by affinity chromatogra phic analysis of the two purified proteins. Among its several roles, g p32 stabilizes single-strand template DNA ahead of a replicating DNA p olymerase, Our data suggest a model in which dNTP synthetase complexes , probably more than one per growing DNA chain, are drawn to replicati on forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of grow ing DNA 3' termini.