The processes by which minus-one frameshifts arise during replication of double-stranded DNA by a human replication apparatus were examined. Using M13mp2 DNA containing the simian virus 40 (SV40) origin of replication and a plus-one frameshift mutation in the lacZ alpha reporter gene, we performed replication reactions using a HeLa cell extract and the SV40 large T antigen. Frameshifts that restore the reading frame to give a blue-plaque phenotype include the loss of one of five consecutive A.T base pairs or any one of 36 non-reiterated base pairs. Although both types of deletions were generated at rates substantially above the background mutant frequency of unreplicated DNA, the rate was highest at the A.T run, suggesting the involvement of a misaligned replication intermediate at this homopolymeric sequence. The error rate for both types of deletions increased as the concentration of dNTPs was increased. A small increase in error rate at the run of A.T base pairs was also observed when a dNMP was added to the replication reaction. These results are consistent with the correction of frameshift intermediates during replication by exonucleolytic proofreading. To examine frameshift error rates on the leading and lagging strands, we compared reversion frequencies for two vectors containing the origin of replication close to, but on opposite sides of, the mutational target. To generate strand-specific errors, nucleotide substrate imbalances were used in replication reactions with these vectors. The results suggest that there is less than a 2-fold difference in the fidelity of leading- and lagging-strand synthesis for deletions at the run of A.T base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)