Deoxyribonucleotide Pool Asymmetry and Genome Variation

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Analysis of homologous gene sequences among closely related mammalian species reveals that different genes within a genome evolve at different rates. The rate of gene variation is related to the nucleotide composition of the gene, specifically, to the guanine+cytosine content. In mammalian cells dGTP is almost invariably the least abundant deoxyribonucleoside triphosphate (dNTP) in the proximal DNA precursor pool. If dGTP levels are subsaturating for replicative DNA polymerases, replication fidelity can be affected, both by competition with more abundant nucleotides for insertion opposite template cytosines, and by increased proofreading upstream from dGMP in newly synthesized DNA. We will analyze replication fidelity in vitro in a forward mutation assay system developed by Roberts and Kunkel (Proc. Natl. Acad. Sci. USA 85, 7064 (1988). This system uses a modified M13 phage template that contains an SV40 viral replication origin and a portion of the E. coli lacZ gene and control region. Mutations in the lac sequence target, arising during replication by a HeLa cell extract, are scored by a plaque assay in the presence of a chromogenic substrate. First we will ask whether this system, when provided with dNTP mixtures that approximate the intranuclear environment, yields mutation frequencies significantly different from those seen with an equimolar dNTP mixture. The critical test, however, will be to systematically vary the GC content of the target DNA sequence without changing its coding properties, and to ask whether mutation frequencies vary as a function of GC content when replicated in the presence of asymmetric dNTP mixtures. The GC content variation will involve substitution of chemically synthesized oligonucleotides for portions of the natural lacZ sequence. %%% It is important to know the parameters which are associated with mutations in mammalian cells in order to better control them. The information from this study will advance our ability to do so.