Publications by authors named "Julie K Horton"

52 Publications

Lysines in the lyase active site of DNA polymerase β destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition.

J Biol Chem 2020 08 9;295(34):12181-12187. Epub 2020 Jul 9.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina, USA. Electronic address:

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.
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http://dx.doi.org/10.1074/jbc.RA120.013547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443498PMC
August 2020

Requirements for PARP-1 covalent crosslinking to DNA (PARP-1 DPC).

DNA Repair (Amst) 2020 06 28;90:102850. Epub 2020 Apr 28.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, North Carolina, 27709, USA. Electronic address:

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http://dx.doi.org/10.1016/j.dnarep.2020.102850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299745PMC
June 2020

Mitochondrial dysfunction and DNA damage accompany enhanced levels of formaldehyde in cultured primary human fibroblasts.

Sci Rep 2020 03 27;10(1):5575. Epub 2020 Mar 27.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 27709, USA.

Formaldehyde (FA) is a simple biological aldehyde that is produced inside cells by several processes such as demethylation of DNA and proteins, amino acid metabolism, lipid peroxidation and one carbon metabolism (1-C). Although accumulation of excess FA in cells is known to be cytotoxic, it is unknown if an increase in FA level might be associated with mitochondrial dysfunction. We choose to use primary human fibroblasts cells in culture (foreskin, FSK) as a physiological model to gain insight into whether an increase in the level of FA might affect cellular physiology, especially with regard to the mitochondrial compartment. FSK cells were exposed to increasing concentrations of FA, and different cellular parameters were studied. Elevation in intracellular FA level was achieved and was found to be cytotoxic by virtue of both apoptosis and necrosis and was accompanied by both G2/M arrest and reduction in the time spent in S phase. A gene expression assessment by microarray analysis revealed FA affected FSK cells by altering expression of many genes including genes involved in mitochondrial function and electron transport. We were surprised to observe increased DNA double-strand breaks (DSBs) in mitochondria after exposure to FA, as revealed by accumulation of γH2A.X and 53BP1 at mitochondrial DNA foci. This was associated with mitochondrial structural rearrangements, loss of mitochondrial membrane potential and activation of mitophagy. Collectively, these results indicate that an increase in the cellular level of FA can trigger mitochondrial DNA double-strand breaks and dysfunction.
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http://dx.doi.org/10.1038/s41598-020-61477-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101401PMC
March 2020

Requirements for PARP-1 covalent crosslinking to DNA (PARP-1 DPC).

DNA Repair (Amst) 2020 05 17;89:102824. Epub 2020 Feb 17.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA. Electronic address:

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http://dx.doi.org/10.1016/j.dnarep.2020.102824DOI Listing
May 2020

Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase β and a DNA Methyltransferase.

Cells 2020 01 16;9(1). Epub 2020 Jan 16.

Biochemistry Ph.D. Program, Florida International University, Miami, FL 33199, USA.

DNA damage and base excision repair (BER) are actively involved in the modulation of DNA methylation and demethylation. However, the underlying molecular mechanisms remain unclear. In this study, we seek to understand the mechanisms by exploring the effects of oxidative DNA damage on the DNA methylation pattern of the tumor suppressor breast cancer 1 (BRCA1) gene in the human embryonic kidney (HEK) HEK293H cells. We found that oxidative DNA damage simultaneously induced DNA demethylation and generation of new methylation sites at the CpGs located at the promoter and transcribed regions of the gene ranging from -189 to +27 in human cells. We demonstrated that DNA damage-induced demethylation was mediated by nucleotide misincorporation by DNA polymerase β (pol β). Surprisingly, we found that the generation of new DNA methylation sites was mediated by coordination between pol β and the DNA methyltransferase, DNA methyltransferase 3b (DNMT3b), through the interaction between the two enzymes in the promoter and encoding regions of the BRCA1 gene. Our study provides the first evidence that oxidative DNA damage can cause dynamic changes in DNA methylation in the BRCA1 gene through the crosstalk between BER and DNA methylation.
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http://dx.doi.org/10.3390/cells9010225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016758PMC
January 2020

Shining light on the response to repair intermediates in DNA of living cells.

DNA Repair (Amst) 2020 01 12;85:102749. Epub 2019 Nov 12.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Fluorescently-tagged repair proteins have been widely used to probe recruitment to micro-irradiation-induced nuclear DNA damage in living cells. Here, we quantify APE1 dynamics after micro-irradiation. Markers of DNA damage are characterized and UV-A laser micro-irradiation energy conditions are selected for formation of oxidatively-induced DNA base damage and single strand breaks, but without detectable double strand breaks. Increased energy of laser micro-irradiation, compared with that used previously in our work, enables study of APE1 dynamics at the lesion site. APE1 shows rapid transient kinetics, with recruitment half-time of less than 1 s and dissociation half-time of less than 15 s. In cells co-transfected with APE1 and PARP1, the recruitment half-time of PARP1 was slower than that of APE1, indicating APE1 is a rapid responder to the damage site. While recruitment of APE1 is unchanged in the presence of co-transfected PARP1, APE1 dissociation is 3-fold slower, revealing PARP1 involvement in APE1 dynamics. Further, we find that APE1 dissociation kinetics are strongly modified in the absence of DNA polymerase β (pol β). After unchanged recruitment to the damage site, dissociation of APE1 became undetectable. This indicates a necessary role for pol β in APE1 release after its recruitment to the damage site. These observations represent an advance in our understanding of in vivo dynamics of base excision repair factors APE1, PARP1 and pol β.
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http://dx.doi.org/10.1016/j.dnarep.2019.102749DOI Listing
January 2020

Histone H3 Lysine 56 Acetylation Enhances AP Endonuclease 1-Mediated Repair of AP Sites in Nucleosome Core Particles.

Biochemistry 2019 09 16;58(35):3646-3655. Epub 2019 Aug 16.

Genome Integrity and Structural Biology Laboratory , National Institute of Environmental Health Sciences , Research Triangle Park , North Carolina 27709 , United States.

Deciphering factors modulating DNA repair in chromatin is of great interest because nucleosomal positioning influences mutation rates. H3K56 acetylation (Ac) is implicated in chromatin landscape regulation, impacting genomic stability, yet the effect of H3K56Ac on DNA base excision repair (BER) remains unclear. We determined whether H3K56Ac plays a role in regulating AP site incision by AP endonuclease 1 (APE1), an early step in BER. Our studies of acetylated, well-positioned nucleosome core particles (H3K56Ac-601-NCPs) demonstrate APE1 strand incision is enhanced compared with that of unacetylated WT-601-NCPs. The high-mobility group box 1 protein enhances APE1 activity in WT-601-NCPs, but this effect is not observed in H3K56Ac-601-NCPs. Therefore, our results suggest APE1 activity on NCPs can be modulated by H3K56Ac.
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http://dx.doi.org/10.1021/acs.biochem.9b00433DOI Listing
September 2019

Eukaryotic Base Excision Repair: New Approaches Shine Light on Mechanism.

Annu Rev Biochem 2019 06;88:137-162

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Science, National Institutes of Health, Research Triangle Park, North Carolina 27709-2233, USA; email:

Genomic DNA is susceptible to endogenous and environmental stresses that modify DNA structure and its coding potential. Correspondingly, cells have evolved intricate DNA repair systems to deter changes to their genetic material. Base excision DNA repair involves a number of enzymes and protein cofactors that hasten repair of damaged DNA bases. Recent advances have identified macromolecular complexes that assemble at the DNA lesion and mediate repair. The repair of base lesions generally requires five enzymatic activities: glycosylase, endonuclease, lyase, polymerase, and ligase. The protein cofactors and mechanisms for coordinating the sequential enzymatic steps of repair are being revealed through a range of experimental approaches. We discuss the enzymes and protein cofactors involved in eukaryotic base excision repair, emphasizing the challenge of integrating findings from multiple methodologies. The results provide an opportunity to assimilate biochemical findings with cell-based assays to uncover new insights into this deceptively complex repair pathway.
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http://dx.doi.org/10.1146/annurev-biochem-013118-111315DOI Listing
June 2019

Repair pathway for PARP-1 DNA-protein crosslinks.

DNA Repair (Amst) 2019 01 12;73:71-77. Epub 2018 Nov 12.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, NC, 27709, USA. Electronic address:

Poly(ADP-ribose) polymerase-1 (PARP-1) is a regulatory enzyme involved in many different processes of DNA and RNA metabolism, including DNA repair. Previously, PARP-1 was found capable of forming a covalent DNA-protein crosslink (DPC) at the apurinic/apyrimidinic (AP) site in double-stranded DNA. The C1´ atom of the AP site participates in Schiff base formation with a lysine side chain in PARP-1, and a covalent bond is formed upon reduction of the Schiff base. The PARP-1 DPC is formed in vivo where DPC formation correlates with AP site induction by a monofunctional alkylating agent. Here, we examined repair of PARP-1 DPCs in mouse fibroblasts and found that a proteasome inhibitor, MG-132, reduces repair resulting in accumulation of PARP-1 DPCs and increased alkylating agent cytotoxicity. Using a model DNA substrate mimicking the PARP-1 DPC after proteasomal degradation, we found that repair is completed by a sub-pathway of base excision repair (BER). Tyrosyl-DNA phosphodiesterase 1 was proficient in removing the ring-open AP site sugar at the phosphodiester linkage, leaving an intermediate for processing by other BER enzymes. The results reveal proteasomal degradation of the PARP-1 DPC is active in mouse fibroblasts and that a model repair intermediate is processed by the BER machinery.
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http://dx.doi.org/10.1016/j.dnarep.2018.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312470PMC
January 2019

XRCC1 phosphorylation affects aprataxin recruitment and DNA deadenylation activity.

DNA Repair (Amst) 2018 04 15;64:26-33. Epub 2018 Feb 15.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Aprataxin (APTX) is a DNA-adenylate hydrolase that removes 5'-AMP blocking groups from abortive ligation repair intermediates. XRCC1, a multi-domain protein without catalytic activity, interacts with a number of known repair proteins including APTX, modulating and coordinating the various steps of DNA repair. CK2-phosphorylation of XRCC1 is thought to be crucial for its interaction with the FHA domain of APTX. In light of conflicting reports, the importance of XRCC1 phosphorylation and APTX function is not clear. In this study, a phosphorylation mutant of XRCC1 designed to eliminate APTX binding was stably expressed in Xrcc1 cells. Analysis of APTX-GFP accumulation at micro-irradiation damage confirmed that phosphorylated XRCC1 is required for APTX recruitment. APTX-mediated DNA deadenylation activity (i.e., 5'-AMP removal) was measured in extracts of cells expressing wild-type XRCC1 or the XRCC1 phosphorylation mutant, and compared with activity in APTX-deficient and APTX-complemented human cells. APTX activity was lower in extracts from Xrcc1 and XRCC1 phosphorylation mutant cells compared to the robust activity in extract from wild-type XRCC1 expressing cells. Taken together, results verify that interaction with phosphorylated XRCC1 is a requirement for significant APTX recruitment to cellular DNA damage and enzymatic activity in cell extracts.
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http://dx.doi.org/10.1016/j.dnarep.2018.02.004DOI Listing
April 2018

DNA polymerase β: A missing link of the base excision repair machinery in mammalian mitochondria.

DNA Repair (Amst) 2017 12 28;60:77-88. Epub 2017 Oct 28.

Genome Integrity and Structural Biology Laboratory, National Institutes of Health, NIEHS, 111 T.W. Alexander Drive, P.O. Box 12233, Research Triangle Park, NC 27709, USA. Electronic address:

Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) β. Pol β was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol β. Mitochondria from pol β-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol β-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol β and thus pol β plays a role in preserving mitochondrial genome stability.
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http://dx.doi.org/10.1016/j.dnarep.2017.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919216PMC
December 2017

XRCC1-mediated repair of strand breaks independent of PNKP binding.

DNA Repair (Amst) 2017 12 19;60:52-63. Epub 2017 Oct 19.

Genomic Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Repair of DNA-protein crosslinks and oxidatively damaged DNA base lesions generates intermediates with nicks or gaps with abnormal and blocked 3'-phosphate and 5'-OH ends that prevent the activity of DNA polymerases and ligases. End cleaning in mammalian cells by Tdp1 and PNKP produces the conventional 3'-OH and 5'-phosphate DNA ends suitable for completion of repair. This repair function of PNKP is facilitated by its binding to the scaffold protein XRCC1, and phosphorylation of XRCC1 by CK2 at several consensus sites enables PNKP binding and recruitment to DNA damage. To evaluate this documented repair process, a phosphorylation mutant of XRCC1, designed to eliminate PNKP binding, was stably expressed in Xrcc1 mouse fibroblast cells. Analysis of PNKP-GFP accumulation at micro-irradiation induced damage confirmed that the XRCC1 phosphorylation mutant failed to support efficient PNKP recruitment, whereas there was rapid recruitment in cells expressing wild-type XRCC1. Recruitment of additional fluorescently-tagged repair factors PARP-1-YFP, GFF-XRCC1, PNKP-GFP and Tdp1-GFP to micro-irradiation induced damage was assessed in wild-type XRCC1-expressing cells. PARP-1-YFP recruitment was best fit to two exponentials, whereas kinetics for the other proteins were fit to a single exponential. The similar half-times of recruitment suggest that XRCC1 may be recruited with other proteins possibly as a pre-formed complex. Xrcc1 cells are hypersensitive to the DNA-protein cross-link inducing agent camptothecin (CPT) and the DNA oxidative agent HO due in part to compromised PNKP-mediated repair. However, cells expressing the PNKP interaction mutant of XRCC1 demonstrated marked reversal of CPT hypersensitivity. This reversal represents XRCC1-dependent repair in the absence of the phosphorylation-dependent PNKP recruitment and suggests either an XRCC1-independent mechanism of PNKP recruitment or a functional back-up pathway for cleaning of blocked DNA ends.
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http://dx.doi.org/10.1016/j.dnarep.2017.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696015PMC
December 2017

Role of the oxidized form of XRCC1 in protection against extreme oxidative stress.

Free Radic Biol Med 2017 06 4;107:292-300. Epub 2017 Feb 4.

Genomic Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

The multi-domain protein XRCC1 is without catalytic activity, but can interact with a number of known repair proteins. The interaction between the N-terminal domain (NTD) of XRCC1 and DNA polymerase β (pol β) is critical for recruitment of pol β to sites of DNA damage and repair. Crystallographic and NMR approaches have identified oxidized and reduced forms of the XRCC1 NTD, and the corresponding forms of XRCC1 have been identified in cultured mouse fibroblast cells. Both forms of NTD interact with pol β, but the interaction is much stronger with the oxidized form. The potential for formation of the C12-C20 oxidized conformation can be removed by alanine substitution at C12 (C12A) leading to stabilized reduced XRCC1 with a lower pol β binding affinity. Here, we compare cells expressing C12A XRCC1 (XRE8) with those expressing wild-type XRCC1 (XC5). Reduced C12A XRCC1 is detected at sites of micro-irradiation DNA damage, but provides slower recruitment of pol β. Expression of reduced XRCC1 does not affect sensitivity to MMS or HO In contrast, further oxidative stress imposed by glutathione depletion results in increased sensitization of reduced XRCC1-expressing cells to HO compared with wild-type XRCC1-expressing cells. There is no indication of enhanced HO-generated free radicals or DNA strand breaks in XRE8 cells. However, elevated cellular PAR is found following HO exposure, suggesting BER deficiency of HO-induced damage in the C12A expressing cells.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457714PMC
June 2017

Oxidized nucleotide insertion by pol β confounds ligation during base excision repair.

Nat Commun 2017 01 9;8:14045. Epub 2017 Jan 9.

Genome Integrity and Structural Biology Laboratory, National Institutes of Health, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.

Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase β (pol β) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol β insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol β expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides.
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http://dx.doi.org/10.1038/ncomms14045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228075PMC
January 2017

DNA polymerase β contains a functional nuclear localization signal at its N-terminus.

Nucleic Acids Res 2017 02;45(4):1958-1970

National Institute of Environmental Health Sciences, Genome Integrity and Structural Biology Laboratory, National Institutes of Health, Research Triangle Park, NC 27709, USA.

DNA polymerase β (pol β) requires nuclear localization to fulfil its DNA repair function. Although its small size has been interpreted to imply the absence of a need for active nuclear import, sequence and structural analysis suggests that a monopartite nuclear localization signal (NLS) may reside in the N-terminal lyase domain. Binding of this domain to Importin α1 (Impα1) was confirmed by gel filtration and NMR studies. Affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide corresponding to pol β residues 2-13. These studies indicate high affinity binding, characterized by a low micromolar Kd, that is selective for the murine Importin α1 (mImpα1) minor site, with the Kd strengthening to ∼140 nM for the full lyase domain (residues 2-87). A further reduction in Kd obtains in binding studies with human Importin α5 (hImpα5), which in some cases has been demonstrated to bind small domains connected to the NLS. The role of this NLS was confirmed by fluorescent imaging of wild-type and NLS-mutated pol β(R4S,K5S) in mouse embryonic fibroblasts lacking endogenous pol β. Together these data demonstrate that pol β contains a specific NLS sequence in the N-terminal lyase domain that promotes transport of the protein independent of its interaction partners. Active nuclear uptake allows development of a nuclear/cytosolic concentration gradient against a background of passive diffusion.
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http://dx.doi.org/10.1093/nar/gkw1257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389473PMC
February 2017

Bisphenol a promotes cell survival following oxidative DNA damage in mouse fibroblasts.

PLoS One 2015 18;10(2):e0118819. Epub 2015 Feb 18.

Genomic Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, United States of America.

Bisphenol A (BPA) is a biologically active industrial chemical used in production of consumer products. BPA has become a target of intense public scrutiny following concerns about its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer. Recent studies link BPA with the generation of reactive oxygen species, and base excision repair (BER) is responsible for removing oxidatively induced DNA lesions. Yet, the relationship between BPA and BER has yet to be examined. Further, the ubiquitous nature of BPA allows continuous exposure of the human genome concurrent with the normal endogenous and exogenous insults to the genome, and this co-exposure may impact the DNA damage response and repair. To determine the effect of BPA exposure on base excision repair of oxidatively induced DNA damage, cells compromised in double-strand break repair were treated with BPA alone or co-exposed with either potassium bromate (KBrO3) or laser irradiation as oxidative damaging agents. In experiments with KBrO3, co-treatment with BPA partially reversed the KBrO3-induced cytotoxicity observed in these cells, and this was coincident with an increase in guanine base lesions in genomic DNA. The improvement in cell survival and the increase in oxidatively induced DNA base lesions were reminiscent of previous results with alkyl adenine DNA glycosylase-deficient cells, suggesting that BPA may prevent initiation of repair of oxidized base lesions. With laser irradiation-induced DNA damage, treatment with BPA suppressed DNA repair as revealed by several indicators. These results are consistent with the hypothesis that BPA can induce a suppression of oxidized base lesion DNA repair by the base excision repair pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0118819PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334494PMC
December 2015

Complementation of aprataxin deficiency by base excision repair enzymes.

Nucleic Acids Res 2015 Feb 6;43(4):2271-81. Epub 2015 Feb 6.

Laboratory of Structural Biology, NIEHS, National Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA

Abortive ligation during base excision repair (BER) leads to blocked repair intermediates containing a 5'-adenylated-deoxyribose phosphate (5'-AMP-dRP) group. Aprataxin (APTX) is able to remove the AMP group allowing repair to proceed. Earlier results had indicated that purified DNA polymerase β (pol β) removes the entire 5'-AMP-dRP group through its lyase activity and flap endonuclease 1 (FEN1) excises the 5'-AMP-dRP group along with one or two nucleotides. Here, using cell extracts from APTX-deficient cell lines, human Ataxia with Oculomotor Apraxia Type 1 (AOA1) and DT40 chicken B cell, we found that pol β and FEN1 enzymatic activities were prominent and strong enough to complement APTX deficiency. In addition, pol β, APTX and FEN1 coordinate with each other in processing of the 5'-adenylated dRP-containing BER intermediate. Finally, other DNA polymerases and a repair factor with dRP lyase activity (pol λ, pol ι, pol θ and Ku70) were found to remove the 5'-adenylated-dRP group from the BER intermediate. However, the activities of these enzymes were weak compared with those of pol β and FEN1.
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http://dx.doi.org/10.1093/nar/gkv079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344515PMC
February 2015

Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells.

Bio Protoc 2015;5(11). Epub 2015 Jun 5.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.

We previously reported enzymatic activity assays for the base excision repair (BER) enzymes DNA polymerase β (pol β), aprataxin (APTX), and flap endonuclease 1 (FEN1) in cell extracts from (Çağlayan and Wilson, 2014). Here, we describe a method to prepare cell extracts from vertebrate cells to investigate these enzymatic activities for the processing of the 5'-adenylated-sugar phosphate-containing BER intermediate. This new protocol complements our previous publication. The cell lines used are wild-type and APTX-deficient human lymphoblast cells from an Ataxia with Oculomotor Apraxia Type 1 (AOA1) disease patient, wild-type and APTX-null DT40 chicken B cells, and mouse embryonic fibroblast (MEF) cells. This protocol is a quick and efficient way to make vertebrate cell extracts without using commercial kits.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933510PMC
http://dx.doi.org/10.21769/bioprotoc.1493DOI Listing
June 2015

DNA polymerase β-dependent cell survival independent of XRCC1 expression.

DNA Repair (Amst) 2015 Feb 3;26:23-9. Epub 2014 Dec 3.

Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1(+/+) and Xrcc1(-/-) mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants.
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http://dx.doi.org/10.1016/j.dnarep.2014.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4308486PMC
February 2015

DNA polymerase β-dependent cell survival independent of XRCC1 expression.

DNA Repair (Amst) 2015 Feb 3;26:23-9. Epub 2014 Dec 3.

Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1(+/+) and Xrcc1(-/-) mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants.
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http://dx.doi.org/10.1016/j.dnarep.2014.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4308486PMC
February 2015

Base excision repair defects invoke hypersensitivity to PARP inhibition.

Mol Cancer Res 2014 Aug 25;12(8):1128-39. Epub 2014 Apr 25.

Laboratory of Structural Biology, NIEHS, NIH, Research Triangle Park, North Carolina

Unlabelled: PARP-1 is important for the recognition of both endogenous and exogenous DNA damage, and binds to DNA strand breaks including intermediates of base excision repair (BER). Once DNA-bound, PARP-1 becomes catalytically activated synthesizing PAR polymers onto itself and other repair factors (PARylation). As a result, BER repair proteins such as XRCC1 and DNA polymerase β (pol β) are more efficiently and rapidly recruited to sites of DNA damage. In the presence of an inhibitor of PARP activity (PARPi), PARP-1 binds to sites of DNA damage, but PARylation is prevented. BER enzyme recruitment is hindered, but binding of PARP-1 to DNA is stabilized, impeding DNA repair and leading to double-strand DNA breaks (DSB). Deficiencies in pol β(-/-) and Xrcc1(-/-) cells resulted in hypersensitivity to the PARP inhibitor 4-AN and reexpression of pol β or XRCC1, in these contexts, reversed the 4-AN hypersensitivity phenotype. BER deficiencies also showed evidence of replication defects that lead to DSB-induced apoptosis upon PARPi treatment. Finally, the clinically relevant PARP inhibitors olaparib and veliparib also exhibited hypersensitivity in both pol β(-/-) and Xrcc1(-/-) BER-deficient cells. These results reveal heightened sensitivity to PARPi as a function of BER deficiency.

Implications: BER deficiency represents a new therapeutic opportunity to enhance PARPi efficacy.
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http://dx.doi.org/10.1158/1541-7786.MCR-13-0502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135006PMC
August 2014

Strategic Combination of DNA-Damaging Agent and PARP Inhibitor Results in Enhanced Cytotoxicity.

Front Oncol 2013 Sep 30;3:257. Epub 2013 Sep 30.

Laboratory of Structural Biology, NIEHS, National Institutes of Health , Research Triangle Park, NC , USA.

PARP inhibitors (PARPi) are under clinical trial for combination cancer chemotherapy. In the presence of a PARPi, PARP-1 binds DNA strand breaks but cannot produce poly(ADP-ribose) polymers or undergo auto-poly(ADP-ribosyl)ation. DNA binding is persistent, hindering DNA repair. Methylated bases formed as a result of cellular exposure to DNA-methylating agents are repaired by DNA polymerase β (pol β)-dependent base excision repair (BER) producing a 5'-deoxyribose phosphate (5'-dRP) repair intermediate. PARP-1 binds and is activated by the 5'-dRP, and PARPi-mediated sensitization to methylating agents is considerable, especially in pol β-deficient cells. Cells deficient in the BER factor XRCC1 are less sensitized by PARPi than are wild-type cells. PARPi sensitization is reduced in cells expressing forms of XRCC1 deficient in interaction with either pol β or PARP-1. In contrast, agents producing oxidative DNA damage and 3'- rather than 5'-repair intermediates are modestly PARPi sensitized. We summarize PARPi experiments in mouse fibroblasts and confirm the importance of the 5'-dRP repair intermediate and functional pol β and XRCC1 proteins. Understanding the chemistry of repair is key to enhancing the clinical success of PARPi.
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http://dx.doi.org/10.3389/fonc.2013.00257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3786324PMC
September 2013

Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses.

DNA Repair (Amst) 2013 Sep 18;12(9):774-85. Epub 2013 Jul 18.

Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 29909-2233, USA.

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12-C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1(-/-) mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.
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http://dx.doi.org/10.1016/j.dnarep.2013.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924596PMC
September 2013

Interaction between DNA Polymerase β and BRCA1.

PLoS One 2013 27;8(6):e66801. Epub 2013 Jun 27.

Laboratory of Structural Biology, NIEHS, National Institutes of Health, North Carolina, United States of America.

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066801PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694962PMC
October 2017

Predicting enhanced cell killing through PARP inhibition.

Mol Cancer Res 2013 Jan 27;11(1):13-8. Epub 2012 Nov 27.

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, 111 T.W. Alexander Dr., MD F1-12, Research Triangle Park, NC 27709, USA.

PARP inhibitors show promise as combination and single agents in cancer chemotherapy. Here, we evaluate results obtained with mouse fibroblasts and the common laboratory PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) and analyze the potential for enhanced cytotoxicity following the combination of a DNA-damaging agent and a PARP inhibitor. Methylated DNA bases are repaired by the monofunctional glycosylase-initiated single-nucleotide base excision repair (BER) pathway. An intermediate of this process has a single-nucleotide gap in double-stranded DNA containing the 5'-deoxyribose phosphate (dRP) group at one margin. This 5'-dRP group is removed by the lyase activity of pol β prior to gap filling; then completion of repair is by DNA ligation. PARP-1 binds to and is activated by the 5'-dRP group-containing intermediate, and poly(ADP-ribos)ylation is important for efficient repair. 4-AN-mediated sensitization to the methylating chemotherapeutic agent temozolomide is extreme, producing a level of cytotoxicity not seen with either agent alone. In contrast, with agents producing oxidative DNA damage repaired by bifunctional glycosylase-initiated BER, there is only weak sensitization by cotreatment with PARP inhibitor. Other clinically used DNA-damaging agents repaired by different DNA repair pathways also reveal minimal 4-AN-mediated sensitization. This information has potentially important implications for strategic use of PARP inhibitors in chemotherapy.
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http://dx.doi.org/10.1158/1541-7786.MCR-12-0512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552016PMC
January 2013

Hyperactivation of PARP triggers nonhomologous end-joining in repair-deficient mouse fibroblasts.

PLoS One 2012 7;7(11):e49301. Epub 2012 Nov 7.

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America.

Regulation of poly(ADP-ribose) (PAR) synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose) polymerase-1 (PARP-1) occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase β (pol β). The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS), or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol β nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol β null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ) protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049301PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492265PMC
June 2013

HMGN1 protein regulates poly(ADP-ribose) polymerase-1 (PARP-1) self-PARylation in mouse fibroblasts.

J Biol Chem 2012 Aug 26;287(33):27648-58. Epub 2012 Jun 26.

Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709-2233, USA.

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.
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http://dx.doi.org/10.1074/jbc.M112.370759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431713PMC
August 2012

Increased PARP-1 association with DNA in alkylation damaged, PARP-inhibited mouse fibroblasts.

Mol Cancer Res 2012 Mar 13;10(3):360-8. Epub 2012 Jan 13.

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.

Treatment of base excision repair-proficient mouse fibroblasts with the DNA alkylating agent methyl methanesulfonate (MMS) and a small molecule inhibitor of PARP-1 results in a striking cell killing phenotype, as previously reported. Earlier studies showed that the mechanism of cell death is apoptosis and requires DNA replication, expression of PARP-1, and an intact S-phase checkpoint cell signaling system. It is proposed that activity-inhibited PARP-1 becomes immobilized at DNA repair intermediates, and that this blocks DNA repair and interferes with DNA replication, eventually promoting an S-phase checkpoint and G(2)-M block. Here we report studies designed to evaluate the prediction that inhibited PARP-1 remains DNA associated in cells undergoing repair of alkylation-induced damage. Using chromatin immunoprecipitation with anti-PARP-1 antibody and qPCR for DNA quantification, a higher level of DNA was found associated with PARP-1 in cells treated with MMS plus PARP inhibitor than in cells without inhibitor treatment. These results have implications for explaining the extreme hypersensitivity phenotype after combination treatment with MMS and a PARP inhibitor.
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http://dx.doi.org/10.1158/1541-7786.MCR-11-0477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3307909PMC
March 2012

Requirement for NBS1 in the S phase checkpoint response to DNA methylation combined with PARP inhibition.

DNA Repair (Amst) 2011 Feb 3;10(2):225-34. Epub 2010 Dec 3.

Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA.

Treatment of PARP-1-expressing cells with the combination of a DNA methylating agent (MMS) and the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) leads to an ATR/Chk1-dependent S phase checkpoint and cell death by apoptosis. Activation of ATM/Chk2 is involved in sustaining the S phase checkpoint, and double strand break (DSB) accumulation was demonstrated. NBS1, part of the MRN complex that responds to DSBs, is known to modulate ATR- and ATM-dependent checkpoint responses to UV and IR, but a role in the response to PARP inhibition has not been addressed. Here we show that the S phase checkpoint observed 4-8h after MMS+4-AN treatment was absent in cells deficient in NBS1, but was present in NBS1-complemented (i.e., functionally wild-type) cells, indicating a critical role for NBS1 in this checkpoint response. NBS1 was phosphorylated in response to MMS+4-AN treatment, and this was partially ATR- and ATM-dependent, suggesting involvement of both upstream kinases. NBS1 expression had little effect on ATR-mediated phosphorylation of Chk1 and ATM-mediated phosphorylation of Chk2 in response to MMS+4-AN. Phosphorylation of SMC1 was also observed in response to MMS+4-AN treatment. In the absence of ATM and NBS1, phosphorylation of SMC1 was weak, especially at early times after MMS+4-AN treatment. In the absence of ATR activation, reduced SMC1 phosphorylation was seen over a 24h time course. These results suggested that both ATR and ATM phosphorylate SMC1 in response to MMS+4-AN and that this phosphorylation is enhanced by phospho-NBS1. The loss of the MMS+4-AN-induced S phase checkpoint in NBS1-deficient cells may be due to a reduced cellular level of the critical downstream effector, phospho-SMC1.
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http://dx.doi.org/10.1016/j.dnarep.2010.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3050562PMC
February 2011

Base excision repair and design of small molecule inhibitors of human DNA polymerase β.

Cell Mol Life Sci 2010 Nov 16;67(21):3633-47. Epub 2010 Sep 16.

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.

Base excision repair (BER) can protect a cell after endogenous or exogenous genotoxic stress, and a deficiency in BER can render a cell hypersensitive to stress-induced apoptotic and necrotic cell death, mutagenesis, and chromosomal rearrangements. However, understanding of the mammalian BER system is not yet complete as it is extraordinarily complex and has many back-up processes that complement a deficiency in any one step. Due of this lack of information, we are unable to make accurate predictions on therapeutic approaches targeting BER. A deeper understanding of BER will eventually allow us to conduct more meaningful clinical interventions. In this review, we will cover historical and recent information on mammalian BER and DNA polymerase β and discuss approaches toward development and use of small molecule inhibitors to manipulate BER. With apologies to others, we will emphasize results obtained in our laboratory and those of our collaborators.
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http://dx.doi.org/10.1007/s00018-010-0489-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324036PMC
November 2010
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