Abstract
Bacteriophage T7 has long been used as a model for studying DNA replication. One of the interesting features of this system is the ability of T7 DNA polymerase to efficiently incorporate dideoxynucleotides into DNA, resulting in chain termination. We show that dideoxythymidine (ddT) present in the media at levels not toxic to the host Escherichia coli results in inhibition of phage T7 growth. We have isolated 100 T7 phage that are resistant to ddT. Surprisingly, all contain a mutation in T7 gene 1.7, a nonessential gene of unknown function. When T7 gene 1.7 is expressed from a plasmid, suppressor T7 phage that are resistant to ddT still arise; analysis of 36 of these mutants reveals that all have mutations in gene 5, that encodes the T7 DNA polymerase. The strongest suppressors change tyrosine 526 of gene 5 to phenylalanine. This mutation is known to dramatically increase the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. These results suggest that in the presence of the T7 gene 1.7 protein, ddT is inhibitory to phage T7 because it acts as a chain terminator for DNA synthesis. In support of this, whereas DNA synthesis in E. coli infected with wild‐type T7 is inhibited by ddT, no inhibition is observed in the absence of gene 1.7. Overexpression of gene 1.7 from a plasmid renders E. coli cells sensitive to ddT in the absence of T7 phage, indicating that gene 1.7 protein does not require other T7 proteins to confer sensitivity to ddT. We propose that gene 1.7 protein plays a critical role in the conversion of ddT to ddTTP.
Highlights
IntroductionWhen gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase
DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein
It has been shown that relatively high concentrations of ddA inhibit DNA synthesis in E. coli and lead to cell death whereas ddC has no effect on cell growth [18]
Summary
When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. The host DNA is degraded to 5Ј-deoxynucleoside monophosphates (dNMPs) by the joint action of the gene 3 endonuclease [5] and gene 6 exonuclease [6] It is not known whether the phage encodes its own enzymes to convert dNMPs to the dNTP precursors for DNA synthesis. Neither E. coli nor phage T7 will readily use exogenous thymine for DNA synthesis; in contrast, phage T4 efficiently incorporates exogenous thymine into its DNA [13]
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