Interstrand cross-links induce DNA synthesis in damaged and undamaged plasmids in mammalian cell extracts

Mammalian cell extracts have been shown to carry out damage-specific DNA re pair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-li nk induces DNA synthesis in both the damaged plasmid and a second homologou s unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psor alen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with p revious evidence indicating a recombinational mode of repair for interstran d cross-links. DNA synthesis is compromised in extracts from mutants (defic ient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross- linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G ) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the compleme ntation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extrac ts is restored by addition of purified ERCC1-XPF heterodimer. This system p rovides a biochemical assay for investigating mechanisms of interstrand cro ss-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.