Eukaryotic DNA polymerase alpha pauses at some sites on the natural DNA template of M13mp2. Terminal misincorporations of dA or dG, in place of dT, by DNA polymerase alpha have been reported to be within one of the pause sites, pause site II (positions 6269 and 6270 (Fry, M., and Loeb, L.A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 763-767)). The DNA products arrested within pause site II (position 6270) were separated, annealed with synthetic templates, and further elongated by DNA polymerase alpha. It was confirmed that a considerable amount of terminal misincorporation of dG in place of dT occurred at this position. When M13mp2 DNA was coated with various amounts of replication protein-A (RP-A), however, DNA polymerase alpha was able to overcome the pause site II, whereas pause bands at other sites barely decreased. In contrast, Escherichia coli single-stranded DNA-binding protein did not specifically abolish the arrested band at pause site II, though it generally suppressed the reaction. Since RP-A hardly increased the elongation frequency from the primer carrying a 3'-mismatched terminal deoxynucleotide, the reduction of arrested products by RP-A may be attributed to the change in the incorporation mode from noncomplementary to complementary deoxynucleotides within pause site II and may not be due to the reinitiation from the mismatched 3'-terminal deoxynucleotide. To confirm this, we amplified the reaction products at pause site III by means of a polymerase chain reaction method and showed that the complementary strand to pause site II, which was elongated in the presence of RP-A, did not carry any detectable misinsertion. Therefore, the errorprone step of the DNA synthesis catalyzed by DNA polymerase alpha may be readily avoided by RP-A.