Mutagenesis of bacteriophage T7 in vitro by incorporation of O6-methylguanine during DNA synthesis.

Abstract

An in vitro system in which bacteriophage T7 DNA is replicated and efficiently packaged into procapsids to form viable phage has been used to examine mutagenesis. The fidelity of replication was assayed both by measuring-reversion of an amber mutation in an essential gene and by generation of temperature-sensitive mutants among the phage produced in vitro. Under standard reaction conditions, the fidelity of DNA replication is about equal to that normally found in vivo. However, when O6_ methyldeoxyguanosine triphosphate is included in the reaction, 06-methylguanine is incorporated into newly synthesized DNA and the mutation frequencies increase 10-to 70-fold over the control. These experiments demonstrate in vitr mutagenesis with the T7 DNA replication-packaging system and provide more direct evidence for the premutagenic role of 06-methylguanine.Studies on the fidelity ofin vitro DNA synthesis have frequently been carried out with synthetic DNA templates and purified DNA polymerases (1). These measurements provide useful information but represent a significant departure from the in vivo situation. Experiments using natural DNA templates and welldefined enzyme systems have allowed valuable extensions of these studies (1). Even closer adherence to the in vivo situation is now possible by. using an in vitro system that replicates intact chromosomes of duplex T7 DNA with efficiency sufficient to replicate 2-5 times as much product DNA as the amount of exogenous template provided (2, 3). Also, biological activity of T7 DNA can be effectively monitored with an in vitro system able to encapsulate T7 DNA into proheads with good efficiency (4, 5). Thus, the ability to synthesize T7 DNA in vitro and to package that DNA into phage particles means that the entire process ofT7 self-replication can be carried out in vitro (6). In this paper, we report'mutagenesis with this in vitro system in which bacteriophage T7 DNA is replicated and then packaged into capsids to form viable phage. Using this system, we have examined the consequences of incorporating _6-methylguanine (m6Gua) into T7 DNA.Only indirect evidence supports the original proposal (7) that m6Gua is mutagenic because it may mispair with thymine and thus cause C -+ T transitions. For example, in vivo experiments with Escherichia coli show that a majority of the mutations induced in a particular gene by methylnitronitrosognanidine are of the G-C -) A-T type (8), and in vitro measurements suggest that, during DNA synthesis (9) or transcription (10), m6Gua can base pair with thymine (or uridine). Furthermore, studies ofin vitro DNA synthesis with prokaryotic DNA polymerases indicate that the presence of m6Gua in the template significantly favors incorporation of thymine rather than cytosine in the product (unpublished observation). Also, the mutagenicity of alkylating agents appears to correlate with their tendency to react at the oxygen sites in DNA, including the O6 of guanine (11). Collectively, these observations argue that direct alkylation ofDNA can prod...

Visualization of Repair of Double-Strand Breaks in the Bacteriophage T7 Genome without Normal DNA Replication

“…The lower yield of phage found by using intact DNA and extracts from T7 3 Ϫ 5 Ϫ 6 Ϫ rather than from T7 3 Ϫ was not unexpected. Previous studies have shown that the absence of gene 6 reduces the ability of the packaging system to produce viable phage (4). This may be due to a role for the gene 6 product in maturation of the T7 genomes prior to packaging (28).…”

“…Our laboratory has been examining the repair of double-strand breaks by using an in vitro system based on extracts made from Escherichia coli infected with bacteriophage T7 (13,21,25,55). In this system, DNA replication closely mimics the in vivo replication of T7 DNA (4,28). Moreover, the in vitro system is able to carry out at least some steps of homologous recombination (22,23,27,38,39).…”

T7 Single Strand DNA Binding Protein but Not T7 Helicase Is Required for DNA Double Strand Break Repair

“…Table 1 shows similar recovery of intact genomes whether uninfected extracts or no extract was used. The T7-infected extract gave a significantly higher yield of phage from the intact genomes, because of replication of the genomes during the reactions and because incubation in the T7-infected extracts improves the ability of the phage genomes to be packaged (6). A double strand break reduced yield of viable phage by 2 to 3 orders of magnitude if there was no extract in the reaction or uninfected E. coli was the source of extract.…”

Extrachromosomal Probes for Mutagenesis by Carcinogens: Studies on the Mutagenic Activity of O 6 -Methylguanine Built into a Unique Site in a Viral Genome

“…Second, the reaction of alkylating agents with the 06-atom of guanine fixes the enol tautomer of this base, in which guanine has been predicted to base-pair with thymine as well as cytosine (14). Much data from in vitro systems support this prediction (15)(16)(17)(18) (Fig. 1) (19).…”