Repair of large insertion/deletion heterologies in human nuclear extracts is directed by a 5 ' single-strand break and is independent of the mismatchrepair system

The repair of 12-, 27-, 62-, and 216-nucleotide unpaired insertion/deletion heterologies has been demonstrated in nuclear extracts of human cells. Whe n present in covalently closed circular heteroduplexes or heteroduplexes co ntaining a single-strand break 3' to the heterology, such structures are su bject to a low level repair reaction that occurs with little strand bias. H owever, the presence of a single-strand break 5' to the insertion/deletion heterology greatly increases the efficiency of rectification and directs re pair to the incised DNA strand. Because nick direction of repair is indepen dent of the strand in which a particular heterology is placed, the observed strand bias is not due to asymmetry imposed on the heteroduplex by the ext rahelical DNA segment. Strand-specific repair by this system requires ATP a nd the four dNTPs and is inhibited by aphidicolin, Repair is independent of the mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 and occurs by a me chanism that is distinct from that of the conventional mismatch repair syst em. Large heterology repair in nuclear extracts of human cells is also inde pendent of the XPF gene product, and extracts of Chinese hamster ovary cell s deficient in the ERCC1 and ERCC4 gene products also support the reaction.