Propanodeoxyguanosine (PdG) is a model for several unstable exocyclic adducts formed by reaction of DNA with bifunctional carbonyl compounds generated by lipid peroxidation. The effect of PdG on DNA synthesis by human DNA polymerase beta was evaluated using template-primers containing PdG at defined sites. DNA synthesis was conducted in vitro and the products were analyzed by polyacrylamide gel electrophoresis and DNA sequencing. The extent of PdG bypass was low and the products comprised a mixture of base pair substitutions and deletions. Sequence analysis of all of the products indicated that the deoxynucleoside monophosphate incorporated "opposite" PdG was complementary to the base 5' to PdG in the template strand. These findings are very similar to recent results of Efrati et al. (Efrati, E., Tocco, G., Eritja, R., Wilson, S. H., and Goodman, M. F. (1997) J. Biol. Chem. 272, 2559-2569) obtained in DNA replication of template-primers containing abasic sites and suggest that PdG is a non-informational lesion when acted upon by polymerase (pol) beta. In addition to base pair substitutions and one- or two-base deletions, a four-base deletion was observed and the mechanism of its formation was probed by site-specific mutagenesis. The results indicated that this deletion occurred by one-base insertion followed by slippage to form a four-base loop followed by extension. All of the observations on pol beta replication of PdG-containing template-primers are consistent with a mechanism of lesion bypass that involves template slippage and dNTP stabilization followed by deoxynucleoside monophosphate incorporation and extension. This mechanism of PdG bypass is completely different than that previously determined for the Klenow fragment of DNA polymerase I and is consistent with recent structural models for DNA synthesis by pol beta.