Tadayoshi Bessho

Reconstitution of human excision nuclease with recombinant XPF-ERCC1 complex

The human XPF-ERCC1 protein complex is one of several factors known to be required for general nucleotide excision repair. Genetic data indicate that both proteins of this complex are necessary for the repair of interstrand cross-links, perhaps via recombination. To determine whether XPF-ERCC1 completes a set of six proteins that are sufficient to carry out excision repair, the human XPF and ERCC1 cDNAs were coexpressed in Sf21 insect cells from a baculovirus vector. The purified complex contained the anticipated 5′ junction-specific endonuclease activity that is stimulated through a direct interaction between XPF and replication protein A (RPA). The recombinant complex also complemented extracts of XP-F cells and Chinese hamster ovary mutants assigned to complementation groups 1, 4, and 11. Furthermore, reconstitution of the human excision nuclease was observed with a mixture of five repair factors (XPA, XPC, XPG, TFIIH, and RPA) and the recombinant XPF-ERCC1, thus verifying that no additional protein factors are needed for the specific dual incisions characteristic of human excision repair.

DNA Interstrand Cross-Links Induce Futile Repair Synthesis in Mammalian Cell Extracts

ABSTRACT DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5′ to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3′-to-5′ exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.

Nucleotide Excision Repair in Man

Nucleotide excision repair (excision repair) is a general repair system for damaged (modified) bases. It is considered the repair system for bulky adducts, but it is also capable of eliminating all base lesions, big or small, from DNA. Damage removal is accomplished by dual incisions bracketing the lesion by a multisubunit enzyme system referred to as excision nuclease or excinuclease. The prokaryotic excinuclease incises the 8th phosphodiester bond 5′ and the 4th or 5th phosphodiester bond 3′ to the lesion and thus excises the damaged nucleotide(s) in 12- to 13-nucleotide-long oligomers (Sancar and Rupp 1983). The human excinuclease incises the 22nd phosphodiester bond 5′ and the 4th to 6th phosphodiester bond 3′ to the damaged nucleotide(s) and releases the damage in 27- to 29-nucleotide-long oligomers (Huang et al. 1992). Following excision the resulting gap is filled and ligated by DNA polymerases and ligases. In contrast to the excision step the gap filling step, which is referred to as repair synthesis, is carried out by proteins whose primary function is replication. Hence, defect in the repair synthesis step is usually a side effect of a more general problem in cellular physiology.