Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates.

Abstract

For the past several years we have been studying the replication of phage T7 DNA as a model for elucidating the molecular mechanisms by which a linear duplex DNA molecule is replicated. From in vivo and in vitro studies it is clear that two proteins, the phage T7-induced DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) and the protein encoded by gene 4 of phage T7, play a major role in replication. Genetic (2-4) and biochemical (5, 6) analyses show these two proteins to be essential for T7 DNA replication. The ¶7-induced DNA polymerase (7, 8) is composed of Escherichia coli thioredoxin and the gene 5 protein of the phage (1, 9, 10). The gene 4 protein has been purified with the aid of a complementation assay (11-13) to near homogeneity JFig. 2).Whereas neither the T7 DNA polymerase nor the gene 4 protein alone can synthesize DNA on duplex templates, the two proteins together catalyze extensive DNA synthesis (12). The reaction is specific for T7 DNA polymerase; neither T4 DNA polymerase nor E. coli DNA polymerases I, II, or III are effective (12). The reaction is stimulated by, but is not dependent on, the presence of rNTPs (12), as is replication in the in vitro system (5, 6). Although earlier studies (12, 14) with gene 4 protein suggested that the stimulation of DNA synthesis on duplex DNA resulted from a role of the gene 4 protein in initiation of DNA strands, our recent studies do not support such an interpretation. Our results (unpublished data) show that, in reaction mixtures containing the two proteins, synthesis initiates at single-strand breaks in duplex molecules with elongation of the polynucleotide chain proceeding in a 5' -3' direction, displacing the parental DNA strands. The number of replicative forks approximates the number of polymerase molecules, with polymerization proceeding at rates up to 6000 nucleotides per min per fork. At later times in the reaction the polymerase is displaced from the template by branch migration (15, 16) allowing it, along with part of the newly synthesized strand, to jump to the displaced strand and copy it in a gene 4 independent reaction. In this way T7 DNA polymerase and gene 4 protein synthesize self-complementary product molecules up to 120,000 nucleotides in length.Because the replication of duplex DNA by T7 DNA polymerase and gene 4 protein requires only 10 gene 4 protein molecules per polymerase molecule, it seems plausible that the gene 4 protein acts catalytically in conjunction with the polymerase to effect unwinding of the duplex DNA template. Inasmuch as this is likely to be an energy-requiring reaction, we looked for the hydrolysis of nucleoside 5'-triphosphates during the course of synthesis on duplex DNA. In this communication we show that the gene 4 protein is not only a single-strand-DNA-dependent nucleoside triphosphatase (NTPase; unspecific diphosphate phosphohydrolase, EC 3.6.1.15) but that hydrolysis of NTPs by gene 4 protein is coupled to DNA synthesis on duplex DNA. MAT...

Deletion mutagenesis independent of recombination in bacteriophage T7

“…The gene 4 product is a primase and a helicase (22,43). Even at the permissive temperature at which these experiments were done, this critical protein may not function the same as a truly wild-type gene 4 product.…”

“…On the basis of what is known about DNA metabolism in T7, there is no reason to suspect that either of the two marker genes used to assay recombination would by themselves affect recombination between direct repeats. But, since the products of both gene 1.2 and gene 4 are involved in DNA synthesis (22,31,32,37,43), it was important to ascertain whether either of these mutations might cause significant effects on the frequency of deletion of either the longer or shorter inserts. Therefore, we measured the deletion frequency of the two newly constructed strains of each of the insertions.…”

“…The small size of the repeats distinguishes illegitimate recombination events (such as those associated with transposition, slip mispairing, and deletion) from homologous recombination, which is dependent upon extensive regions of homology (2,6,16). However, some studies show that deletions are RecA dependent (1, 3, 4), while others show no RecA dependence (6,7,11,14,15,33,53 (22,32,37,43). The only known host protein important to T7 DNA replication is thioredoxin, which associates with the phage gene 5 product and confers extensive processivity upon the DNA polymerase (37).…”

“…The T7 DNA replication system is simpler than those of most phage or bacterial systems. T7 DNA can be synthesized in vitro with only the products of T7 gene 2.5 (single-stranded-DNA-binding protein), gene 4 (a helicase/primase), and gene 5 (DNA polymerase) (22,32,37,43). The only known host protein important to T7 DNA replication is thioredoxin, which associates with the phage gene 5 product and confers extensive processivity upon the DNA polymerase (37).…”

It Seems Like Only Yesterday

“…David Hinkle then purified the gene 4 protein by using a complementation assay (95). Richard Kolodner showed that it allowed T7 DNA polymerase to copy duplex DNA, the first of the DNA replication helicases to be described (96)(97)(98). Later, Steve Matson studied the nucleotide and DNA specificity (99)(100)(101)(102).…”