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The mammalian abasic endonuclease, APE1, has two distinct roles in the repair of oxidative DNA damage and in gene regulation. Here we show that both functions are essential for cell survival. Deletion of the APE1 gene causes embryonic lethality in mice, and no nullizygous embryo fibroblasts have been isolated. We have now established nullizygous embryo fibroblast lines from APE1 ؊/؊ mouse embryos that are transgenic with the ''floxed'' human APE1 (hAPE1) gene. Removal of hAPE1 by Cre expression through nuclear microinjection elicited apoptosis in these cells within 24 h, which was blocked by coinjection of the wild-type hAPE1 gene. In contrast, mutant hAPE1 alleles, lacking either the DNA repair or acetylation-mediated gene regulatory function, could not prevent apoptosis, although the combination of these two mutants complemented APE deficiency induced by Cre. These results indicate that distinct and separable functions of APE1 are both essential for mammalian cells even in vitro and provide the evidence that mammalian cells, unlike yeast or Escherichia coli, absolutely require APE for survival, presumably to protect against spontaneous oxidative DNA damage.conditional gene inactivation ͉ DNA repair ͉ endogenous DNA damage ͉ base excision repair A basic endonuclease (APE), a ubiquitous enzyme, plays a central role in repairing toxic and mutagenic abasic (AP) sites generated in genomes during the repair of oxidation and alkylation damage through the base excision repair (BER) pathway (1). Oxidative DNA lesions, including AP sites, are also spontaneously generated at an estimated rate of 1.5 ϫ 10 5 residues⅐cell Ϫ1 ⅐day Ϫ1 (2). Unlike two distinct APEs present in Escherichia coli and Saccharomyces cerevisiae, only one active APE, APE1, an ortholog of E. coli xth and yeast APN2, has been identified in mammalian cells (3). Based on sequence homology, a second APE-like gene, APE2, was cloned from mammalian cells. However, we could not detect APE activity in the recombinant human APE2 (4), and hAPE2, unlike hAPE1, could not complement yeast APE mutants (5). Although APE-negative bacteria and yeast are viable, very early death (3.5-7.5 days after fertilization) was observed in APE1 nullizygous mouse embryos (6-8). Unlike other BER proteins, e.g., DNA polymerase ␤ and X-ray cross complementation group 1, which are essential for embryonic survival but not for mouse embryonic fibroblasts (MEFs) cultured in vitro (9, 10), APE1-null MEF mutant lines have not been established. The mammalian APE1, independently identified as redox-enhancing factor 1 (Ref1), has a distinct regulatory function in reductively activating C-Jun, p53, and other transcription factors (3, 11) for which Cys-65 (Cys-64 in mouse APE1) was identified as the active site (12). The N-terminal region of the 36-kDa polypeptide, including Cys-65, is not conserved in the E. coli homolog exonuclease III. An additional regulatory function of APE1͞Ref1 was identified in Ca 2ϩ -dependent down-regulation of the parathyroid hormone and renin genes containing negativ...

Proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor that is required for DNA replication during S phase of the cell cycle and for resynthesis during nucleotide excision repair of damaged DNA. PCNA binds to flap endonuclease 1 (FEN-1), a structure-specific endonuclease involved in DNA replication. Here we report the direct physical interaction of PCNA with xeroderma pigmentosum (XP) G, a structurespecific repair endonuclease that is homologous to FEN-1. We have identified a 28-amino acid region of human FEN-1 (residues 328 -355) and a 29-amino acid region of human XPG (residues 981-1009) that contains the PCNA binding activity. These regions share key hydrophobic residues with the PCNA-binding domain of the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 , and all three competed with one another for binding to PCNA. A conserved arginine in FEN-1 (Arg 339 ) and XPG (Arg 992 ) was found to be crucial for PCNA binding activity. R992A and R992E mutant forms of XPG failed to fully reconstitute nucleotide excision repair in an in vivo complementation assay. These results raise the possibility of a mechanistic linkage between excision and repair synthesis that is mediated by PCNA.Exposure to UV light causes damage to DNA primarily in the form of cyclobutane pyrimidine dimers and (6-4) photoproducts. These types of DNA lesions, as well as bulky adducts produced by some chemical mutagens, are processed by nucleotide excision repair (NER). 1 The human genetic disorder xeroderma pigmentosum (XP) is the result of defects in this DNA damage repair pathway. Symptoms of XP include extreme sensitivity to sunlight exposure and a greatly elevated risk of skin cancer. In the past few years, much progress has been made in understanding the molecular events associated with NER (1). The DNA-binding protein XPA is involved in damage recognition. In concert with replication protein A, which binds singlestranded DNA, and helicases XPB and XPD, a ϳ27-29-base oligonucleotide segment containing the lesion is excised as the result of dual incision by structure-specific endonucleases XPF-ERCC1 and XPG. The XPF-ERCC1 complex cleaves the damaged strand at a 5Ј site about 23 nucleotides from the lesion, whereas XPG cleaves the strand approximately 5 nucleotides to the 3Ј side of the damage. The resultant gap is filled in by the action of DNA polymerase ␦ or ⑀, and then DNA ligase seals the nick to complete repair. The resynthesis step requires proliferating cell nuclear antigen (PCNA; Refs. 2 and 3), a ring-shaped homotrimeric protein that encircles DNA and acts as a "sliding clamp" that links the polymerase to the DNA template (4). PCNA performs the same essential function in replicative DNA synthesis during S phase of the cell cycle. PCNA requires replication factor C, a primer recognition protein that loads the PCNA trimer onto DNA in an ATP-dependent manner (5-7).XPG is homologous to another structure-specific endonuclease, FEN-1. FEN-1 is involved in Okazaki fragment processing during DNA replication (8), and it i...

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