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 deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex 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 replication 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 protein-protein interactions in the complex by immobilizing individual enzymes and identifying radiolabeled T4 proteins that are retained by columns of these respective affinity ligands. Elsewhere we have described interactions involving three T4 enzymes found in the complex. In this paper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide reductase, and E. coli nucleoside diphosphokinase,. All eight proteins analyzed to date retain single-strand DNA-binding protein (gp32), the product of T4 gene 32. At least one T4 protein, thymidylate synthase, binds directly to gp32, as shown by affinity chromatographic analysis of the two purified proteins. Among its several roles, gp32 stabilizes single-strand template DNA ahead of a replicating DNA polymerase. Our data suggest a model in which dNTP synthetase complexes, probably more than one per growing DNA chain, are drawn to replication forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of growing DNA 3' termini.
Wheeler
L J
LJ
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-7305, USA.
Ray
N B
NB
Ungermann
C
C
Hendricks
S P
SP
Bernard
M A
MA
Hanson
E S
ES
Mathews
C K
CK
D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000378:metabolism*; D008961:Models, Structural; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000302:isolation & purification / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase; D014764:Viral Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D004800:Enzymes, Immobilized; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C063448:T4 deoxyribonucleoside 5'-triphosphate synthetase complex; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D012264:Ribonucleotide Reductases; D013762:Tetrahydrofolate Dehydrogenase; D017856:Phosphotransferases (Phosphate Group Acceptor); C020015:(deoxy)nucleoside-phosphate kinase; D009701:Nucleoside-Diphosphate Kinase; D011755:Pyrophosphatases; C020007:dCTP pyrophosphatase; C020020:dUTP pyrophosphatase