Modified SV40 for analysis of mismatch repair in simian and human cells

We have developed a way of introducing specific mispairs into the genome of simian virus 40 and of determining the fate of the mispaired bases in simian and human cells. Mispairs are introduced into viral DNA within the intron of the gene coding for the large T antigen. Each DNA molecule harbors a single mispair in a defined orientation. Transfection of mismatch-containing SV40 DNA into host cells yields plaques, each corresponding to a productive infection initiated by a single viral DNA molecule. Isolation of DNA derived from individual plaques and determination of the DNA sequence at the site of the mispair reveals whether correction occurred and what the repair products are. Here we describe repair patterns for G/T and A/C mispairs in CV-1 African green monkey kidney cells, and for G/T mispairs in human fibroblasts derived from 3 normal individuals, 1 patient with xeroderma pigmentosum (complementation group A), and 3 patients with Bloom's syndrome. G/T mispairs, which arise in resting DNA through the deamination of 5-methylcytosine (mC) to form thymine, are corrected in all cases with extremely high efficiency and nearly always in favor of guanine. In contrast, A/C mispairs are corrected randomly and relatively inefficiently in simian cells.