We report here the first genome-wide functional genomic screen for longevity genes. We systematically surveyed Caenorhabditis elegans genes using large-scale RNA interference (RNAi), and found that RNAi inactivation of 89 genes extend C. elegans lifespan. Components of the daf-2/insulin-like signaling pathway are recovered, as well as genes that regulate metabolism, signal transduction, protein turnover, and gene expression. Many of these candidate longevity genes are conserved across animal phylogeny. Genetic interaction analyses with the new longevity genes indicate that some act upstream of the daf-16/FOXO transcription factor or the sir2.1 protein deacetylase, and others function independently of daf-16/FOXO and sir2.1, and might define new pathways to regulate lifespan.[Keywords: Longevity; aging; C. elegans; insulin signaling; RNAi screen; genomic] Supplemental material is available at http://www.genesdev.org.

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...

Most of the DNA in eukaryotes is packaged in tandemly arrayed nucleosomes that, together with numerous DNA- and nucleosome-associated enzymes and regulatory factors, make up chromatin. Chromatin modifying and remodeling agents help regulate access to selected DNA segments in chromatin, thereby facilitating transcription and DNA replication and repair. Studies of nucleotide excision repair (NER), single strand break repair (SSBR), and the homology-directed (HDR) and non-homologous end-joining (NHEJ) double strand break repair pathways have led to an ‘access-repair-restore’ paradigm, in which chromatin in the vicinity of damaged DNA is disrupted, thereby enabling efficient repair and the subsequent repackaging of DNA into nucleosomes. When damage is extensive, these repair processes are accompanied by cell cycle checkpoint activation, which provides cells with sufficient time to either complete the repair or initiate apoptosis. It is not clear, however, if base excision repair (BER) of the ~20,000 or more oxidative DNA damages that occur daily in each nucleated human cell can be viewed through this same lens. Until recently, we did not know if BER requires or is accompanied by nucleosome disruption, and it is not yet clear that anything short of overwhelming oxidative damage (resulting in the shunting of DNA substrates into other repair pathways) results in checkpoint activation. This review highlights studies of how oxidatively damaged DNA in nucleosomes is discovered and repaired, and offers a working model of events associated with BER in chromatin that we hope will have heuristic value.

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Although DNA in eukaryotes is packaged in nucleosomes, it remains vulnerable to oxidative damage that can result from normal cellular metabolism, ionizing radiation, and various chemical agents. Oxidatively damaged DNA is repaired in a stepwise fashion via the base excision repair (BER) pathway, which begins with the excision of damaged bases by DNA glycosylases. We reported recently that the human DNA glycosylase hNTH1 (human Endonuclease III), a member of the HhH GpG superfamily of glycosylases, can excise thymine glycol lesions from nucleosomes without requiring or inducing nucleosome disruption; optimally oriented lesions are excised with an efficiency approaching that seen for naked DNA [1]. To determine if this property is shared by human DNA glycoylases in the Fpg/Nei family, we investigated the activity of NEIL1 on defined nucleosome substrates. We report here that the cellular concentrations and apparent kcat/KM ratios for hNTH1 and NEIL1 are similar. Additionally, after adjustment for non-specific DNA binding, hNTH1 and NEIL1 proved to have similar intrinsic activities towards nucleosome substrates. However, NEIL1 and hNTH1 differ in that NEIL1 binds undamaged DNA far more avidly than hNTH1. As a result, hNTH1 is able to excise both accessible and sterically occluded lesions from nucleosomes at physiological concentrations, while the high non-specific DNA affinity of NEIL1 would likely hinder its ability to process sterically occluded lesions in cells. These results suggest that, in vivo, NEIL1 functions either at nucleosome-free regions (such as those near replication forks) or with cofactors that limit its non-specific binding to DNA.