DNA damage-dependent inactivation of complementary strand synthesis in Xenopus laevis egg or HeLa cell lysates

Genotoxic lesions frequently arrest DNA synthesis and, as a consequence, result in the accumulation of incompletely replicated chromosomal segments containing long single-stranded regions of parental DNA. Here, we exploited complementary strand synthesis in Xenopus laevis egg or HeLa cell lysates to test how the eukaryotic replication machinery responds to such damaged single-stranded intermediates. Although both cell lysates promoted efficient conversion of M13 or phi X174 single-stranded templates to double-stranded products, their replication activity was inhibited by DNA damage arising from ultraviolet (UV) radiation or exposure to the alkylating agent N-methyl-N-nitrosourea (MNU). When M13 single-stranded DNA containing UV-or MNU-induced lesions was coincubated with single-stranded substrates containing no lesions, we observed suppression of DNA synthesis on both damaged and undamaged templates. In contrast, complementary strand synthesis remained unaffected in coincubation reactions containing damaged DNA in the double-stranded form. Effective inhibition of complementary strand synthesis on undamaged templates required the presence of at least stoichiometric amounts of UV-or MNU-treated single-stranded DNA, indicating that a DNA polymerase or accessory protein is excluded from DNA synthesis by immobilization at or near the lesion sites. In support of this competitive mode of inhibition, we found that inactivation of DNA synthesis by coincubation with damaged single-stranded DNA was relieved by the addition of an exogenous DNA polymerase that catalyzes processive strand elongation. In summary, this study reveals sequestration of critical components of the eukaryotic replication machinery in a DNA damage-dependent and single-strand-specific manner, thereby providing a potential mechanism to sense arrested replication intermediates during an early recognition step of S phase checkpoint responses.