Endogenously formed reactive oxygen species continuously damage cellular constituents including DNA. These challenges, coupled with exogenous exposure to agents that generate reactive oxygen species, are both associated with normal aging processes and linked to cardiovascular disease, cancer, cataract formation, and fatty liver disease. Although not all of these diseases have been definitively shown to originate from mutations in nuclear DNA or mitochondrial DNA, repair of oxidized, saturated, and ring-fragmented bases via the base excision repair pathway is known to be critical for maintaining genomic stability. One enzyme that initiates base excision repair of ring-fragmented purines and some saturated pyrimidines is NEIL1, a mammalian homolog to Escherichia coli endonuclease VIII. To investigate the organismal consequences of a deficiency in NEIL1, a knockout mouse model was created. In the absence of exogenous oxidative stress, neil1 knockout (neil1 ؊/؊ ) and heterozygotic (neil1 ؉/؊ ) mice develop severe obesity, dyslipidemia, and fatty liver disease and also have a tendency to develop hyperinsulinemia. In humans, this combination of clinical manifestations, including hypertension, is known as the metabolic syndrome and is estimated to affect >40 million people in the United States. Additionally, mitochondrial DNA from neil1 ؊/؊ mice show increased levels of steady-state DNA damage and deletions relative to wild-type controls. These data suggest an important role for NEIL1 in the prevention of the diseases associated with the metabolic syndrome.DNA repair ͉ fatty liver disease ͉ mitochondria ͉ obesity ͉ oxidative stress A fter exposure to reactive oxygen species (ROS), the major purine lesions are 8-oxoguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine (1-5). To reverse the potentially deleterious effects of oxidative DNA base lesions, cells primarily use the base excision repair (BER) pathway to restore the DNA to its undamaged state (6). The BER pathway is initiated by lesion-specific DNA glycosylases that catalyze bond scission and release the damaged base from the deoxyribose sugar.Unlike nucleotide excision repair, which functions only on nuclear DNA, the BER pathway is operative on both nuclear DNA and mitochondrial DNA (mtDNA). Although BER in the mitochondria repairs normal endogenous oxidant-induced DNA damage, expression of mitochondrially targeted DNA glycosylases that are specific for the repair of oxidatively induced DNA lesions leads to enhanced repair and increased survival after ROS challenge (7-10). These data emphasize that maintenance of the mitochondrial genome is a delicate balance of mtDNA copy number and BER capacity.To initiate repair of oxidative lesions, mammalian cells primarily use the products of the ogg1, nth1, neil1, and neil2 genes (11). These corresponding proteins have somewhat overlapping substrate specificities that may explain, at least partially, the absence of obvious phenotypes in ogg1-and nth1-null mice. NEIL1 has a strong sub...

Alpha-synuclein is a presynaptic protein that forms abnormal cytoplasmic aggregates in Lewy body disorders. Although nuclear alpha-synuclein localization has been described, its function in the nucleus is not well understood. We demonstrate that alpha-synuclein modulates DNA repair. First, alpha-synuclein colocalizes with DNA damage response components within discrete foci in human cells and mouse brain. Removal of alpha-synuclein in human cells leads to increased DNA double-strand break (DSB) levels after bleomycin treatment and a reduced ability to repair these DSBs. Similarly, alpha-synuclein knock-out mice show increased neuronal DSBs that can be rescued by transgenic reintroduction of human alpha-synuclein. Alpha-synuclein binds double-stranded DNA and helps to facilitate the non-homologous end-joining reaction. Using a new, in vivo imaging approach that we developed, we find that serine-129-phosphorylated alpha-synuclein is rapidly recruited to DNA damage sites in living mouse cortex. We find that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-synuclein reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss-of-function. This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms.

Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB 1 ) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB 1 -induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB 1 -deoxyguanosine adduct (AFB 1 -Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB 1 show significant increases in the levels of AFB 1 -Fapy-dG in Neil1 −/− vs. wild-type liver DNA. Further, Neil1 −/− mice are highly susceptible to AFB 1 -induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1 −/− . The magnitude of this effect in Neil1 −/− mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB 1 -associated HCCs.aflatoxin | base excision repair | ring-fragmented purines | liver cancer | environmental toxicant L iver cancers pose an international public health concern as the second leading cause of cancer-related deaths worldwide, with >700,000 estimated deaths per year (1-3). This mortality approaches its annual incidence throughout the world, highlighting the need for development of effective treatments and early diagnostic tools. HCCs represent the major histological subtype among liver cancers. The global distribution of HCCs is dominated by its incidence in developing countries, especially in eastern Asia and Africa, where two major chronic etiological factors drive this disease: (i) routine dietary exposures to grains and nuts that are contaminated with molds, Aspergillus flavus and Aspergillus parasiticus, which produce aflatoxins, and (ii) extremely high rates of hepatitis B (HBV) and C viral infections. In geographical regions of China where aflatoxin contamination of human food products is highest, there is a large shift in not only the age of onset of HCCs, but also in the incidence rate. Within Qidong, a significant number of HCCs occur in males beginning in their early 20s, with the frequency of HCCs peaking between the ages of 40 and 50 (4). These data are in contrast to HCC frequencies in portions of China, such as Beijing, where aflatoxin exposures are minimal. The kinetics of HCC formation in aflatoxin-affected areas are similar to that observed in early onset breast and ovarian cancers in women who are carriers (heterozygotic) for inactivating mutations in BRCA1 or 2.Although there are several different aflatoxin structures, AFB 1 has been demonstrated to be the most potent hepatoc...

The human MutY homolog, hMYH, is an adenine-specific DNA glycosylase that removes adenines or 2-hydroxyadenines mispaired with guanines or 8-oxoguanines. In order to prevent mutations, this activity must be directed to the newly synthesized strand and not the template strand during DNA synthesis. The subcellular localization and expression of hMYH has been studied in serum-stimulated, proliferating MRC5 cells. Using specific antibodies, we demonstrate that endogenous hMYH protein localized both to nuclei and mitochondria. hMYH in the nuclei is distinctly distributed and co-localized with BrdU at replication foci and with proliferating cell nuclear antigen (PCNA). The levels of hMYH in the nucleus increased 3- to 4-fold during progression of the cell cycle and reached maximum levels in S phase compared to early G(1). Similar results were obtained for PCNA, while there were no notable changes in expression of 8-oxoguanine glycosylase or the human MutT homolog, MTH1, throughout the cell cycle. The cell cycle-dependent expression and localization of hMYH at sites of DNA replication suggest a role for this glycosylase in immediate post-replication DNA base excision repair.

Although it is well established that DNA-protein crosslinks are formed as a consequence of cellular exposure to agents such as formaldehyde, transplatin, ionizing and ultraviolet radiation, the biochemical pathways that promote cellular survival via repair or tolerance of these lesions are poorly understood. To investigate the mechanisms that function to limit DNA-protein crosslink-induced cytotoxicity, the Saccharomyces cerevisiae non-essential gene deletion library was screened for increased sensitivity to formaldehyde exposure. Following low-dose, chronic exposure, strains containing deletions in genes mediating homologous recombination showed the greatest sensitivity, while under the same exposure conditions, deletions in genes associated with nucleotide excision repair conferred only low to moderate sensitivities. However, when the exposure regime was changed to a high-dose acute (short-term) formaldehyde treatment, the genes that conferred maximal survival switched to the nucleotide excision repair pathway, with little contribution of the homologous recombination genes. Data are presented which suggest that following acute formaldehyde exposure, repair and/or tolerance of DNA-protein crosslinks proceeds via formation of nucleotide excision repair-dependent single-strand break intermediates and without a detectable accumulation of doublestrand breaks. These data clearly demonstrate a differential pathway response to chronic versus acute formaldehyde exposures and may have significance and implications for risk extrapolation in human exposure studies.