Each day, approximately 20,000 oxidative lesions form in the DNA of every nucleated human cell. The base excision repair (BER) enzymes that repair these lesions must function in a chromatin milieu. We have determined that the DNA glycosylase hNTH1, apurinic endonuclease (APE), and DNA polymerase ␤ (Pol ␤), which catalyze the first three steps in BER, are able to process their substrates in both 601-and 5S ribosomal DNA (rDNA)-based nucleosomes. hNTH1 formed a discrete ternary complex that was displaced by the addition of APE, suggesting an orderly handoff of substrates from one enzyme to the next. In contrast, DNA ligase III␣-XRCC1, which completes BER, was appreciably active only at concentrations that led to nucleosome disruption. Ligase III␣-XRCC1 was also able to bind and disrupt nucleosomes containing a single base gap and, because of this property, enhanced both its own activity and that of Pol ␤ on nucleosome substrates. Collectively, these findings provide insights into ratelimiting steps that govern BER in chromatin and reveal a unique role for ligase III␣-XRCC1 in enhancing the efficiency of the final two steps in the BER of lesions in nucleosomes.Reactive oxygen species (ROS), generated as by-products of normal aerobic cellular metabolism or from exposure to exogenous agents, such as gamma irradiation, generate approximately 20,000 DNA damage events per day in each nucleated human cell. The DNA lesions produced include numerous oxidative base damages, apurinic/apyrimidinic (AP) sites, and single-strand DNA breaks (6). Base excision repair (BER) enzymes recognize and replace oxidized bases with the corresponding undamaged bases. In its simplest ("short-patch") form, BER entails four enzymatic steps (1,10,21,23,51,53) (Fig. 1A), beginning with the recognition and excision of a damaged base by either a mono-or bifunctional DNA glycosylase. Bifunctional glycosylases first cleave the glycosidic bond between the damaged base and the deoxyribose and then cleave the phosphodiester bond 3Ј of the resulting AP site. AP endonuclease (APE) removes a residual moiety to generate a single nucleotide gap, with a 3Ј-OH group that can be filled by DNA polymerase ␤ (Pol ␤). Finally, DNA ligase III-␣ (LigIII␣), in association with XRCC1, catalyzes the formation of a phosphodiester bond between the 3Ј-OH of the newly added nucleotide and the adjacent downstream 5Ј-phosphate.The nucleosomes that package most of the nuclear DNA in eukaryotes provide only minimal protection from ROS (14, 31); a small degree of protection from hydroxyl radicals is evident in DNA segments where the minor groove faces into the histone octamer (20), and histones themselves may act as a sink for ROS, thereby reducing the frequency of free-radicalinflicted DNA damage (28). Clearly, however, nucleosomal DNA is vulnerable to oxidative damage that must be made available to BER enzymes. Chromatin remodeling agents and histone chaperones facilitate most processes involving chromatin, and the other DNA repair pathways-nucleotide excision repair, mismatc...

Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase III␣/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase III␣ and the association between MRN and DNA ligase III␣/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase III␣/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.The repair of DNA double-strand breaks (DSBs) 3 is of the utmost importance for cell viability and genomic stability. Notably, defects in DSB repair result in genomic rearrangements that promote cancer formation and progression (1-3). Although there are multiple DSB repair pathways, these pathways can be divided into two groups depending on whether the repair reaction is dependent or not upon substantial DNA sequence homology between the recombining molecules (4, 5). The key step of the major homology-dependent pathway is the invasion of a single strand into a homologous duplex. In contrast, the key step of nonhomologous end joining (NHEJ) involves the end-to-end alignment of broken DNA molecules (4, 5).Unlike the major homology-dependent pathway, repair of DSBs by the major or classic NHEJ (C-NHEJ) pathway is errorprone, frequently resulting in the loss or addition of a few nucleotides at the break site (4, 5). Despite the mutagenic consequences of NHEJ, this is a major DSB repair pathway in mammalian cells. In addition, there is emerging evidence for alternative versions of NHEJ (alt-NHEJs) that are more errorprone than C-NHEJ. Although alt-NHEJ is more evident in cells that are deficient in C-NHEJ (6, 7), it is detectable in wild-type cells (8). Repair of DSBs by alt-NHEJ is characterized by the presence of short tracts of sequence homology (microhomologies) at the repair site, large deletions, and chromosome translocations (6 -10). Furthermore, alt-NHEJ appears to be responsible for chromosomal translocations found in cancer cells (11)(12)(13)(14)(15) and to make a much greater contribution to DSB repair in cancer cells compared with normal cells (10).A combination of biochemical and molecular genetic studies by many laboratories has identified the key components of C-NHEJ (4, 5). In contrast, the factors involved in and the mechanisms of alt-NHEJ are not well defined. Poly(ADP-ribose) polymerase-1 (PARP-1), the initiating protein for DNA single-strand break (SSB) repair, also binds to DSBs (16) and has been implicated in DNA PK-independent NHEJ (17, 18). The h...

Background: XRCC1 interacts with multiple DNA repair proteins. Results: Identification of mutant versions of XRCC1 that are defective in binding with a different single partner. Conclusion:Interaction between XRCC1 and polynucleotide kinase 3Ј-phosphatase is critical for the retention of XRCC1 at DNA damage sites and DNA damage repair. Significance: Insights into function of one of three DNA end processing factors that bind to the same region of XRCC1.

The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIIIβ and the hLigIIIα/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/ XRCC4 molecules.

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