Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5' incision by ERCC1-XPF precedes the 3' incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.

two pathways are completely distinct and their major and Tomas Lindahl 1,5 forms in mammalian cells share no enzymes or other 1 Imperial Cancer Research Fund protein factors (Wood, 1996;). De-Clare Hall Laboratories fects in key activities of the BER process, such as AP South Mimms, Hertfordshire EN6 3LD endonuclease, DNA polymerase ␤, or the XRCC1-DNA United Kingdom ligase III heterodimer, lead to embryonic lethal pheno-2 Department of Genetics and Microbiology types in the mouse, indicating that repair of endogenous Centre Me ´dical Universitaire (CMU) DNA lesions is essential during development (Wilson 1211 Geneva 4 and Thompson, 1997). In contrast, NER defects gener-Switzerland ally are nonlethal, and mutations in any of the 7 key 3 Department of Biochemistry genes XPA to XPG can be the cause of the inherited and Division of Cancer Biology cancer-prone disease xeroderma pigmentosum in man. Department of Radiation Oncology Deamination of cytosine to uracil in DNA is counter-Emory University School of Medicine acted by BER; the repair process involves DNA polymer-Atlanta, Georgia 30322 ase ␤-catalyzed substitution of a single dCMP residue 4 Department of Chemistry in DNA to replace the excised uracil and deoxyribose Wesleyan University phosphate moieties and has been reconstituted with Middletown, Connecticut 06459 purified human factors (Kubota et al., 1996; Nicholl et al., 1997; Srivastava et al., 1998). Oxidized DNA bases such as thymine glycol (Tg) and 8-oxoguanine are be-Summary lieved to be repaired in a similar way, although the initial step is carried out by bifunctional enzymes, which can Oxidized pyrimidines in DNA are removed by a distinct both release a damaged base by DNA glycosylase activbase excision repair pathway initiated by the DNA glyity and cleave the DNA chain at the abasic site by AP cosylase-AP lyase hNth1 in human cells. We have lyase activity. Excision of various ring-saturated and reconstituted this single-residue replacement pathring-fragmented oxidized derivatives of thymine and cytosine is due to a Tg-DNA glycosylase-AP lyase activity, way with recombinant proteins, including the AP endothe human counterpart of E. coli endonuclease III or nuclease HAP1/APE, DNA polymerase ␤, and DNA li-Nth. The three-dimensional structure of the bacterial gase III-XRCC1 heterodimer. With these proteins, the enzyme has been established (Kuo et al., 1992); the nucleotide excision repair enzyme XPG serves as a homologous human enzyme hNth1 retains relevant key cofactor for the efficient function of hNth1. XPG profeatures and has been expressed in active form from a tein promotes binding of hNth1 to damaged DNA. The cloned cDNA (Aspinwall et al., 1997; Hilbert et al., 1997). stimulation of hNth1 activity is retained in XPG cata-Characteristic structural properties include a conserved lytic site mutants inactive in nucleotide excision repair. helix-hairpin-helix region that accounts for binding of The data support the model that development of Cockthe damaged pyrimidine and also contains an active ayn...

In normal human cells, damage due to ultraviolet light is preferentially removed from active genes by nucleotide excision repair (NER) in a transcription-coupled repair (TCR) process that requires the gene products defective in Cockayne syndrome (CS). Oxidative damage, including thymine glycols, is shown to be removed by TCR in cells from normal individuals and from xeroderma pigmentosum (XP)-A, XP-F, and XP-G patients who have NER defects but not from XP-G patients who have severe CS. Thus, TCR of oxidative damage requires an XPG function distinct from its NER endonuclease activity. These results raise the possibility that defective TCR of oxidative damage contributes to the developmental defects associated with CS.

The human XPG endonuclease cuts on the 3 side of a DNA lesion during nucleotide excision repair. Mutations in XPG can lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of structure-specific nucleases and exonucleases. To begin defining its catalytic mechanism, we changed highly conserved residues and determined the effects on the endonuclease activity of isolated XPG, its function in open complex formation and dual incision reconstituted with purified proteins, and its ability to restore cellular resistance to UV light. The substitution A792V present in two XP complementation group G (XP-G) individuals reduced but did not abolish endonuclease activity, explaining their mild clinical phenotype. Isolated XPG proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In the reconstituted repair system, alanine substitutions at these positions permitted open complex formation but were inactive for 3 cleavage, whereas D77E and E791D proteins retained considerable activity. The function of each mutant protein in the reconstituted system was mirrored by its ability to restore UV resistance to XP-G cell lines. Hydrodynamic measurements indicated that XPG exists as a monomer in high salt conditions, but immunoprecipitation of intact and truncated XPG proteins showed that XPG polypeptides can interact with each other, suggesting dimerization as an element of XPG function. The mutation results define critical residues in the catalytic center of XPG and strongly suggest that key features of the strand cleavage mechanism and active site structure are shared by members of the nuclease family.The XPG protein is a DNA endonuclease with remarkable structure-specific properties, cleaving near the junctions between duplex and single-stranded DNA with a defined polarity.In its N-terminal region (N region) 1 and internal region (I region), XPG shares similarity in sequence with a family of other nucleases. These include the bacteriophage T4 RNase H and T5 D15 proteins (1), as well as the 5Ј to 3Ј exonuclease domains of eubacterial DNA polymerases (2-4). Eukaryotic family members include a family of small replication and repair nucleases (mammalian FEN-1/DNase IV, Saccharomyces cerevisiae Rad27, and Schizosaccharomyces pombe rad2) and larger proteins (vertebrate XPG, S. cerevisiae Rad2, and S. pombe rad13) involved in nucleotide excision repair (NER). The FEN-1 group is active on flap structures (5), whereas the NER enzymes cleave bubbles, splayed arms, and flaps (5-7).In human cells, XPG functions to cleave on the 3Ј side of a damaged site in DNA during NER, the process that removes injuries induced by UV light and many chemical agents. Individuals with mutations in XPG are almost equally divided between those with the inherited syndrome xeroderma pigmentosum (XP) and those with a combination of both XP and Cockayne syndrome (CS). The XP phenotype includes acute sun sensitivity with a high incidence of cancers and, in some individuals, progres...

Xeroderma pigmentosum (XP) patients have defects in nucleotide excision repair (NER), the versatile repair pathway that removes UV-induced damage and other bulky DNA adducts. Patients with Cockayne syndrome (CS), another rare sun-sensitive disorder, are specifically defective in the preferential removal of damage from the transcribed strand of active genes, a process known as transcription-coupled repair. These two disorders are usually clinically and genetically distinct, but complementation analyses have assigned a few CS patients to the rare XP groups B, D, or G. The XPG gene encodes a structure-specific endonuclease that nicks damaged DNA 3′ to the lesion during NER. Here we show that three XPG/CS patients had mutations that would produce severely truncated XPG proteins. In contrast, two sibling XPG patients without CS are able to make full-length XPG, but with a missense mutation that inactivates its function in NER. These results suggest that XPG/CS mutations abolish interactions required for a second important XPG function and that it is the loss of this second function that leads to the CS clinical phenotype.