XRCC1 protects against the lethality of induced oxidative DNA damage in nondividing neural cells

XRCC1 is a critical scaffold protein that orchestrates efficient single-strand break repair (SSBR). Recent data has found an association of XRCC1 with proteins causally linked to human spinocerebellar ataxias—aprataxin and tyrosyl-DNA phosphodiesterase 1—implicating SSBR in protection against neuronal cell loss and neurodegenerative disease. We demonstrate herein that shRNA lentiviral-mediated XRCC1 knockdown in human SH-SY5Y neuroblastoma cells results in a largely selective increase in sensitivity of the nondividing (i.e. terminally differentiated) cell population to the redox-cycling agents, menadione and paraquat; this reduced survival was accompanied by an accumulation of DNA strand breaks. Using hypoxanthine–xanthine oxidase as the oxidizing method, XRCC1 deficiency affected both dividing and nondividing SH-SY5Y cells, with a greater effect on survival seen in the former case, suggesting that the spectrum of oxidative DNA damage created dictates the specific contribution of XRCC1 to cellular resistance. Primary XRCC1 heterozygous mouse cerebellar granule cells exhibit increased strand break accumulation and reduced survival due to increased apoptosis following menadione treatment. Moreover, knockdown of XRCC1 in primary human fetal brain neurons leads to enhanced sensitivity to menadione, as indicated by increased levels of DNA strand breaks relative to control cells. The cumulative results implicate XRCC1, and more broadly SSBR, in the protection of nondividing neuronal cells from the genotoxic consequences of oxidative stress

Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R mutation abolished the interaction with POLβ, but did not disrupt the interactions with PARP-1, LIG3α and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLβ interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S) and two frequent (R194W and R399Q) amino acid population variants had little or no effect on XRCC1 protein stability or the interactions with POLβ, PARP-1, LIG3α, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLβ, PARP-1, LIG3α and PCNA, but did reduce DNA-binding ability. When expressed in HeLa cells, the XRCC1 variants—excluding E98K, which was largely nucleolar, and C389Y, which exhibited reduced expression—exhibited normal nuclear distribution. Most of the protein variants, including the V86R POLβ-interaction mutant, displayed normal relocalization kinetics to/from sites of laser-induced DNA damage: except for E98K and C389Y, and the polymorphic variant R280H, which exhibited a slightly shorter retention time at DNA breaks

Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1

APE1 is the major nuclease for excising abasic (AP) sites and particular 3′-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC1280), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals

Location specific recognition of the recombination hot-spot "Chi" by the E. coli RecBCD enzyme

RecBCD is an ATP-dependent helicase and exonuclease, which generates 3'-single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. Critical to this function is its encounter with an regulatory DNA octamer called the Chi sequence, 5'-GCTGGTGG-3', which is known to stimulate recombination by the RecBCD pathway. RecBCD undergoes an unexplained modification following Chi encounter that results in attenuation of its 3'-5'exonuclease activity. There are several aspects of the Chi-RecBCD interactions that are still not understood, and most studies addressing these issues have done so by employing Chi- bearing duplexes in various biochemical assays for RecBCD functions. The recognition of Chi on exclusively single-stranded substrates has never been tested before, even though Chi is known to be recognized as the single stranded sequence 5' GCTGGTGG 3'. This study tests the effect of ssDNA oligonucleotides, having a Chi sequence (Chi+) or a single base mutant of the Chi sequence (Chio), on the enzymatic activities of RecBCD. The results obtained show that Chi is specifically recognized by RecBCD even when part of a single-stranded substrate. However its location within the single strand is important; specific recognition and distinction from the mutant sequence occurs only when Chi is in the middle of the construct, flanked by DNA at either end. The activities affected included the helicase, and consequently the exonuclease and Chi-recognition activity itself. The effects observed were also found to be dependent on the length of the oligonucleotides. The longer oligonucleotides affect the enzymatic function of RecBCD better than the shorter ones. Preincubation of DNA substrate with RecBCD abolishes the inhibitory effect of the oligonucleotides on RecBCD function however, preincubation of the oligonucleotides themselves with RecBCD does not enhance inhibition by them. This study also presents some preliminary results from photocrosslinking of oligonucleotides to RecBCD, which suggest that RecC may play an important role in binding and recognizing Chi. The results lead to the proposition of a model that explains the location-specific (flank-dependent) recognition of the Chi sequence by RecBCD and discusses the possibility of the existence of a specific site on RecBCD for Chi

The Involvement of DNA-Damage and -Repair Defects in Neurological Dysfunction

A genetic link between defects in DNA repair and neurological abnormalities has been well established through studies of inherited disorders such as ataxia telangiectasia and xeroderma pigmentosum. In this review, we present a comprehensive summary of the major types of DNA damage, the molecular pathways that function in their repair, and the connection between defective DNA-repair responses and specific neurological disease. Particular attention is given to describing the nature of the repair defect and its relationship to the manifestation of the associated neurological dysfunction. Finally, the review touches upon the role of oxidative stress, a leading precursor to DNA damage, in the development of certain neurodegenerative pathologies, such as Alzheimer's and Parkinson's

Specific inhibition of the E.coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxyribonucleotides

RecBCD is an ATP-dependent helicase and exonuclease which generates 3′ single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. The exonuclease activity is altered when RecBCD encounters a Chi sequence (5′-GCTGGTGG-3′) in double-stranded DNA (ds DNA), an event critical to the generation of the 3′-ssDNA. This study tests the effect of ssDNA oligonucleotides having a Chi sequence (Chi(+)) or a single base change that abolishes the Chi sequence (Chi(o)), on the enzymatic activities of RecBCD. Our results show that a 14 and a 20mer with Chi(+) in the center of the molecule inhibit the exonuclease and helicase activities of RecBCD to a greater extent than the corresponding Chi(o) oligonucleotides. Oligonucleotides with the Chi sequence at one end, or the Chi sequence alone in an 8mer, failed to show Chi-specific inhibition of RecBCD. Thus, Chi recognition requires that Chi be flanked by DNA at either end. Further experiments indicated that the oligonucleotides inhibit RecBCD from binding to its dsDNA substrate. These results suggest that a specific site for Chi recognition exists on RecBCD, which binds Chi with greater affinity than a non-Chi sequence and is probably adjacent to non-specific DNA binding sites

Effects of temperature and equivalence ratio on mass balance and energy analysis in loblolly pine oxygen gasification

The purpose of this study was to evaluate the effects of temperature and equivalence ratios (ERs) on the distribution of products (primary gases carbon monoxide [CO], H2, CH4, CO2), gas phase contaminants (tar, NH3, HCN, H2S, HCl), char, carbon, and inorganics), and energy flows on an oxygen-blown bubbling fluidized bed gasifier system using loblolly pine. The goal and value of this study was to provide quantitative and qualitative performance analysis and data for process engineering and optimization of these fledgling biomass conversion systems. As temperature and ER increased, mass balance closures also increased from 94.73% to 96.72% for temperature and 89.82–96.93% for ER. In addition, the carbon closures ranged from 80.77% to 92.29% and from 79.09% to 87.13% as temperature and ER increased, respectively. Carbon conversion efficiency to gas product ranged from 72.26% to 84.32% as temperature increased and from 72.26% to 84.66% as ER increased. Carbon flow analysis showed that the char product streams retained 10.26–6.94% and 8.82–2.13% of the carbon fed to the gasifier as temperature and ER increased, respectively. The carbon content in the liquid condensate was minimal compared to the carbon in other product streams and accounted for less than 0.1% of the carbon input to the gasifier at all conditions. The cold and hot gas efficiencies increased from 56.12% to 67.45% and from 67.51% to 83.83% as temperature increased due to higher production of CO and hydrogen (H2). In contrast, cold and hot gas efficiencies decreased from 63.85% to 52.84% and from 78.06% to 73.00% as ER increased, respectively, due to enhanced oxidation of gas products resulting in a net decrease in heating value

Effect of Telomere Proximity on Telomere Position Effect, Chromosome Healing, and Sensitivity to DNA Double-Strand Breaks in a Human Tumor Cell Line ▿

The ends of chromosomes, called telomeres, are composed of a DNA repeat sequence and associated proteins, which prevent DNA degradation and chromosome fusion. We have previously used plasmid sequences integrated adjacent to a telomere to demonstrate that mammalian telomeres suppress gene expression, called telomere position effect (TPE). We have also shown that subtelomeric regions are highly sensitive to double-strand breaks, leading to chromosome instability, and that this instability can be prevented by the addition of a new telomere to the break, a process called chromosome healing. We have now targeted the same plasmid sequences to a site 100 kb from a telomere in a human carcinoma cell line to address the effect of telomere proximity on telomere position effect, chromosome healing, and sensitivity to double-strand breaks. The results demonstrate a substantial decrease in TPE 100 kb from the telomere, demonstrating that TPE is very limited in range. Chromosome healing was also diminished 100 kb from the telomere, consistent with our model that chromosome healing serves as a repair process for restoring lost telomeres. Conversely, the region 100 kb from the telomere was highly sensitive to double-strand breaks, demonstrating that the sensitive region is a relatively large target for ionizing radiation-induced chromosome instability