The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.

Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of the tyrosyl-3 0 phosphate linkage found in topoisomerase I-DNA covalent complexes. The inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1), is caused by a H493R mutation in Tdp1. Contrary to earlier proposals that this disease results from a loss-of-function mutation, we show here that this mutation reduces enzyme activity B25-fold and importantly causes the accumulation of the Tdp1-DNA covalent reaction intermediate. Thus, the attempted repair of topoisomerase I-DNA complexes by Tdp1 unexpectedly generates a new protein-DNA complex with an apparent half-life of B13 min that, in addition to the unrepaired topoisomerase I-DNA complex, may interfere with transcription and replication in human cells and contribute to the SCAN1 phenotype. The analysis of Tdp1 mutant cell lines derived from SCAN1 patients reveals that they are hypersensitive to the topoisomerase I-specific anticancer drug camptothecin (CPT), implicating Tdp1 in the repair of CPT-induced topoisomerase I damage in human cells. This finding suggests that inhibitors of Tdp1 could act synergistically with CPT in anticancer therapy.

Human DNA topoisomerase I is an essential enzyme involved in resolving the torsional stress associated with DNA replication, transcription, and chromatin condensation. The catalytic cycle of the enzyme consists of DNA cleavage to form a covalent enzyme-DNA intermediate, DNA relaxation, and finally, re-ligation of the phosphate backbone to restore the continuity of the DNA. Structure/function studies have elucidated a flexible enzyme that relaxes DNA through coordinated, controlled movements of distinct enzyme domains. The cellular roles of topoisomerase I are apparent throughout the nucleus, but the concentration of processes acting on ribosomal DNA results in topoisomerase I accumulation in the nucleolus. Although the activity of topoisomerase I is required in these processes, the enzyme can also have a deleterious effect on cells. In the event that the final re-ligation step of the reaction cycle is prevented, the covalent topoisomerase I-DNA intermediate becomes a toxic DNA lesion that must be repaired. The complexities of the relaxation reaction, the cellular roles, and the pathways that must exist to repair topoisomerase I-mediated DNA damage highlight the importance of continued study of this essential enzyme.

The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase III␣, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase III␣ but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase III␣-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADPribose) polymer.Three human genes, LIG1, LIG3, and LIG4, that encode DNA ligases have been identified (30). Unlike the LIG1 and LIG4 genes, which appear to be conserved among all eukaryotes, the LIG3 gene has been found only in the genomes of mammals and of the amphibian Xenopus laevis (6,22,32). Intriguingly, the LIG3 gene is more closely related to poxvirus DNA ligase genes than to those for the other eukaryotic DNA ligases (6, 10). Furthermore, the LIG3 gene is more complex than the other LIG genes in that it encodes multiple products that appear to have distinct biological functions.Alternative splicing of the LIG3 gene transcript generates two species of mRNA, designated ␣ and ␤, that encode polypeptides with different C termini (17,22). DNA ligase III␣ mRNA is ubiquitously expressed, whereas DNA ligase III␤ mRNA has been detected only in germ cells (17,22). The unique C terminus of DNA ligase III␣, which exhibits homology with the BRCT motif initially identified in the product of the breast cancer susceptibility gene BRCA1 (5, 11), mediates formation of a stable complex with the DNA repair protein XRCC1 (3,4,17,21,29). In contrast, no protein partner or biochemical activity has been ascribed to the unique C terminus of DNA ligase III␤. Further heterogeneity of products from the LIG3 gene is generated by translation initiation at different ATG codons within DNA ligase III mRNA, generating mitochondrial and nuclear forms of DNA ligase III (14,15,22).A unique feature of the DNA ligases encoded by the LIG3 gene is the zinc finger motif situated at the N termini of these polypeptides (32). Interestingly, this motif is closely related to the two tandem-arrayed zinc fingers that constitute the DNA binding domain of poly(ADP-ribose) polymerase 1 (PARP-1), a nuclea...