Publications by authors named "Samuel H Wilson"

245 Publications

Genomic and evolutionary classification of lung cancer in never smokers.

Nat Genet 2021 Sep 6;53(9):1348-1359. Epub 2021 Sep 6.

Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA.

Lung cancer in never smokers (LCINS) is a common cause of cancer mortality but its genomic landscape is poorly characterized. Here high-coverage whole-genome sequencing of 232 LCINS showed 3 subtypes defined by copy number aberrations. The dominant subtype (piano), which is rare in lung cancer in smokers, features somatic UBA1 mutations, germline AR variants and stem cell-like properties, including low mutational burden, high intratumor heterogeneity, long telomeres, frequent KRAS mutations and slow growth, as suggested by the occurrence of cancer drivers' progenitor cells many years before tumor diagnosis. The other subtypes are characterized by specific amplifications and EGFR mutations (mezzo-forte) and whole-genome doubling (forte). No strong tobacco smoking signatures were detected, even in cases with exposure to secondhand tobacco smoke. Genes within the receptor tyrosine kinase-Ras pathway had distinct impacts on survival; five genomic alterations independently doubled mortality. These findings create avenues for personalized treatment in LCINS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-021-00920-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432745PMC
September 2021

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair.

Nat Commun 2021 08 20;12(1):5055. Epub 2021 Aug 20.

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

Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-24486-xDOI Listing
August 2021

Perspectives on formaldehyde dysregulation: Mitochondrial DNA damage and repair in mammalian cells.

DNA Repair (Amst) 2021 Sep 11;105:103134. Epub 2021 May 11.

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

Maintaining genome stability involves coordination between different subcellular compartments providing cells with DNA repair systems that safeguard against environmental and endogenous stresses. Organisms produce the chemically reactive molecule formaldehyde as a component of one-carbon metabolism, and cells maintain systems to regulate endogenous levels of formaldehyde under physiological conditions, preventing genotoxicity, among other adverse effects. Dysregulation of formaldehyde is associated with several diseases, including cancer and neurodegenerative disorders. In the present review, we discuss the complex topic of endogenous formaldehyde metabolism and summarize advances in research on fo dysregulation, along with future research perspectives.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2021.103134DOI Listing
September 2021

Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide.

Nat Commun 2021 04 6;12(1):2059. Epub 2021 Apr 6.

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

Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (A). Rotation of A into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-21354-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024293PMC
April 2021

Using Human Primary Foreskin Fibroblasts to Study Cellular Damage and Mitochondrial Dysfunction.

Curr Protoc Toxicol 2020 12;86(1):e99

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

Several cell lines of different origin are routinely used in research and drug development as important models to study human health and disease. Studying cells in culture represents an easy and convenient tool to approach complex biological questions, but the disadvantage is that they may not necessarily reflect what is effectively occurring in vivo. Human primary cells can help address this limitation, as they are isolated directly from human biological samples and can preserve the morphological and functional features of their tissue of origin. In addition, these can offer more relevant data and better solutions to investigators because they are not genetically manipulated. Human foreskin tissue discarded after surgery, for instance, represents a precious source for isolating such cells, including human foreskin fibroblasts (FSK), which are used in several areas of research and medicine. The overall health of cells is determined by the mitochondria. Alterations of cellular metabolism and cell death pathways depend, in part, on the number, size, distribution, and structure of mitochondria, and these can change under different cellular and pathological conditions. This highlights the need to develop accurate approaches to study mitochondria and evaluate their function. Here, we describe three easy, step-by-step protocols to study cellular viability and mitochondrial functionality in FSK. We describe how to use circumcision tissue obtained from the clinic to isolate FSK cells by mechanical and enzymatic disaggregation, how to use a cationic dye, crystal violet, which is retained by proliferating cells, to determine cell viability, and how to prepare samples to assess the metabolic status of cells by evaluating different mitochondrial parameters with transmission electron microscopy. We have successfully used the approaches outlined here to recapitulate physiological conditions in these cells in order to study the effects of increased intracellular levels of formaldehyde. © 2020 U.S. Government. Basic Protocol 1: Isolation and maintenance of human primary foreskin fibroblasts (FSK) Basic Protocol 2: Determination of cell viability by crystal violet staining Basic Protocol 3: Transmission electron microscopy to study cellular damage and mitochondrial dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cptx.99DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757388PMC
December 2020

RNA abasic sites in yeast and human cells.

Proc Natl Acad Sci U S A 2020 08 11;117(34):20689-20695. Epub 2020 Aug 11.

HHMI, University of Michigan, Ann Arbor, MI 48109;

RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2011511117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456069PMC
August 2020

Telomeres come to life thanks to an exciting review article in this issue.

Authors:
Samuel H Wilson

DNA Repair (Amst) 2020 10 29;94:102904. Epub 2020 Jun 29.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2020.102904DOI Listing
October 2020

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.013547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443498PMC
August 2020

Preferential DNA Polymerase β Reverse Reaction with Imidodiphosphate.

ACS Omega 2020 Jun 19;5(25):15317-15324. Epub 2020 Jun 19.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, North Carolina 27709-2233, United States.

DNA replication and repair reactions involve the addition of a deoxynucleoside monophosphate onto a growing DNA strand with the loss of pyrophosphate. This chemical reaction is also reversible; the addition of pyrophosphate generates a deoxynucleoside triphosphate, thereby shortening the DNA by one nucleotide. The forward DNA synthesis and reverse pyrophosphorolysis reactions strictly require the presence of divalent metals, usually magnesium, at the reactive center as cofactors. The overall equilibrium enzymatic reaction strongly favors DNA synthesis over pyrophosphorolysis with natural substrates. The DNA polymerase β chemical reaction has been structurally and kinetically characterized, employing natural and chemically modified substrates. Substituting an imido-moiety (NH) for the bridging oxygen between Pβ and Pγ of dGTP dramatically decreased the overall enzymatic activity and resulted in a chemical equilibrium that strongly favors the reverse reaction (i.e., ≪ 1). Using QM/MM calculations in conjunction with the utilization of parameters such as quantum mechanically derived atomic charges, we have examined the chemical foundation for the altered equilibrium with this central biological reaction. The calculations indicate that the rapid reverse reaction is likely due, in part, to the increased nucleophilicity of the reactive oxygen on the tautomeric form of imidodiphosphate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsomega.0c01345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331038PMC
June 2020

Structure of a DNA polymerase abortive complex with the 8OG:dA base pair at the primer terminus.

Commun Biol 2020 07 3;3(1):348. Epub 2020 Jul 3.

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

Adenine frequently pairs with the Hoogsteen edge of an oxidized guanine base (8OG) causing G to T transversions. The (syn)8OG:dA base pair is indistinguishable from the cognant base pair and can be extended by DNA polymerases with reduced efficiency. To examine the structural basis of this reduced efficiency, we sought to obtain the structure of the "product" complex of DNA polymerase (pol) β with the (syn)8OG:dA base pair at the primer terminus by soaking the binary complex crystals with a hydrolysable dCTP analogue complementary to the template base G. Crystallographic refinement of the structure revealed that the adenine of the (syn)8OG:dA base pair had been expelled from the primer terminus and a dCMP was inserted opposite 8OG in a reverse orientation; another uninserted molecule of the analogue was bound to the templating base G. This leads to an abortive complex that could form the basis of oxidatively-induced pol β stalling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-020-1080-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334213PMC
July 2020

Topoisomerase I-driven repair of UV-induced damage in NER-deficient cells.

Proc Natl Acad Sci U S A 2020 06 8;117(25):14412-14420. Epub 2020 Jun 8.

Department of Radiation Genetics, Kyoto University, Graduate School of Medicine, 606-8501 Kyoto, Japan;

Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1920165117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321995PMC
June 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:

View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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:

View Article and Find Full Text PDF

Download full-text PDF

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

Revealing an Internal Stabilization Deficiency in the DNA Polymerase β K289M Cancer Variant through the Combined Use of Chemical Biology and X-ray Crystallography.

Biochemistry 2020 03 12;59(8):955-963. Epub 2020 Feb 12.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Drive, Research Triangle Park, North Carolina 27709, United States.

The human DNA polymerase (pol) β cancer variant K289M has altered polymerase activity , and the structure of wild-type pol β reveals that the K289 side chain contributes to a network of stabilizing interactions in a C-terminal region of the enzyme distal to the active site. Here, we probed the capacity of the K289M variant to tolerate strain introduced within the C-terminal region and active site. Strain was imposed by making use of a dGTP analogue containing a CF group substitution for the β-γ bridging oxygen atom. The ternary complex structure of the K289M variant displays an alteration in the C-terminal region, whereas the structure of wild-type pol β is not altered in the presence of the dGTP CF analogue. The alteration in the K289M variant impacts the active site, because the enzyme in the ternary complex fails to adopt the normal open to closed conformational change and assembly of the catalytically competent active site. These results reveal the importance of the K289-mediated stabilizing network in the C-terminal region of pol β and suggest an explanation for why the K289M cancer variant is deficient in polymerase activity even though the position 289 side chain is distal to the active site.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.9b01072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263314PMC
March 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9010225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016758PMC
January 2020

Oxidative DNA-protein crosslinks formed in mammalian cells by abasic site lyases involved in DNA repair.

DNA Repair (Amst) 2020 03 9;87:102773. Epub 2020 Jan 9.

Department of Pharmacological Sciences, Stony Brook University, Renaissance School of Medicine, Basic Science Tower 8-140, Stony Brook, New York, 11794, USA. Electronic address:

Free radical attack on C1' of deoxyribose forms the oxidized abasic (AP) site 2-deoxyribonolactone (dL). In vitro, dL traps the major base excision DNA repair enzyme DNA polymerase beta (Polβ) in covalent DNA-protein crosslinks (DPC) via the enzyme's N-terminal lyase activity acting on 5'-deoxyribose-5-phosphate residues. We previously demonstrated formation of Polβ-DPC in cells challenged with oxidants generating significant levels of dL. Proteasome inhibition under 1,10-copper-ortho-phenanthroline (CuOP) treatment significantly increased Polβ-DPC accumulation and trapped ubiquitin in the DPC, with Polβ accounting for 60-70 % of the total ubiquitin signal. However, the identity of the remaining oxidative ubiquityl-DPC remained unknown. In this report, we surveyed whether additional AP lyases are trapped in oxidative DPC in mammalian cells in culture. Poly(ADP-ribose) polymerase 1 (PARP1), Ku proteins, DNA polymerase λ (Polλ), and the bifunctional 8-oxoguanine DNA glycosylase 1 (OGG1), were all trapped in oxidative DPC in mammalian cells. We also observed significant trapping of Polλ, PARP1, and OGG1 in cells treated with the alkylating agent methylmethane sulfonate (MMS), in addition to dL-inducing agents. Ku proteins, in contrast, followed a pattern of trapping similar to that for Polβ: MMS failed to produce Ku-DPC, while treatment with CuOP or (less effectively) HO gave rise to significant Ku-DPC. Unexpectedly, NEIL1 and NEIL3 were trapped following HO treatment, but not detectably in cells exposed to CuOP. The half-life of all the AP lyase-DPC ranged from 15-60 min, consistent with their active repair. Accordingly, CuOP treatment under proteasome inhibition significantly increased the observed levels of DPC in cultured mammalian cells containing PARP1, Ku protein, Polλ, and OGG1 proteins. As seen for Polβ, blocking the proteasome led to the accumulation of DPC containing ubiquitin. Thus, the ubiquitin-dependent proteolytic mechanisms that control Polβ-DPC removal may also apply to a broad array of oxidative AP lyase-DPC, preventing their toxic accumulation in cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2019.102773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065521PMC
March 2020

DNA polymerase β nucleotide-stabilized template misalignment fidelity depends on local sequence context.

J Biol Chem 2020 01 4;295(2):529-538. Epub 2019 Dec 4.

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

DNA polymerase β has two DNA-binding domains that interact with the opposite sides of short DNA gaps. These domains contribute two activities that modify the 5' and 3' margins of gapped DNA during base excision repair. DNA gaps greater than 1 nucleotide (nt) pose an architectural and logistical problem for the two domains to interact with their respective DNA termini. Here, crystallographic and kinetic analyses of 2-nt gap-filling DNA synthesis revealed that the fidelity of DNA synthesis depends on local sequence context. This was due to template dynamics that altered which of the two template nucleotides in the gap served as the coding nucleotide. We observed that, when a purine nucleotide was in the first coding position, DNA synthesis fidelity was similar to that observed with a 1-nt gap. However, when the initial templating nucleotide was a pyrimidine, fidelity was decreased. If the first templating nucleotide was a cytidine, there was a significantly higher probability that the downstream template nucleotide coded for the incoming nucleotide. This dNTP-stabilized misalignment reduced base substitution and frameshift deletion fidelities. A crystal structure of a binary DNA product complex revealed that the cytidine in the first templating site was in an extrahelical position, permitting the downstream template nucleotide to occupy the coding position. These results indicate that DNA polymerase β can induce a strain in the DNA that modulates the position of the coding nucleotide and thereby impacts the identity of the incoming nucleotide. Our findings demonstrate that "correct" DNA synthesis can result in errors when template dynamics induce coding ambiguity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA119.010594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956524PMC
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2019.102749DOI Listing
January 2020

DNA polymerase beta and other gap-filling enzymes in mammalian base excision repair.

Enzymes 2019 ;45:1-26

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Science, National Institutes of Health, Durham, NC, United States. Electronic address:

DNA polymerase β plays a central role in the base excision DNA repair pathway that cleanses the genome of apurinic/apyrimidinic (AP) sites. AP sites arise in DNA from spontaneous base loss and DNA damage-specific glycosylases that hydrolyze the N-glycosidic bond between the deoxyribose and damaged base. AP sites are deleterious lesions because they can be mutagenic and/or cytotoxic. DNA polymerase β contributes two enzymatic activities, DNA synthesis and lyase, during the repair of AP sites; these activities reside on carboxyl- and amino-terminal domains, respectively. Accordingly, its cellular, structural, and kinetic attributes have been extensively characterized and it serves as model enzyme for the nucleotidyl transferase reaction utilized by other replicative, repair, and trans-lesion DNA polymerases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.enz.2019.08.002DOI Listing
October 2019

Cutting-edge perspectives in genomic maintenance VI.

DNA Repair (Amst) 2019 09 5;81:102677. Epub 2019 Aug 5.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2019.102677DOI Listing
September 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.9b00433DOI Listing
September 2019

Damage sensor role of UV-DDB during base excision repair.

Nat Struct Mol Biol 2019 08 22;26(8):695-703. Epub 2019 Jul 22.

Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

UV-DDB, a key protein in human global nucleotide excision repair (NER), binds avidly to abasic sites and 8-oxo-guanine (8-oxoG), suggesting a noncanonical role in base excision repair (BER). We investigated whether UV-DDB can stimulate BER for these two common forms of DNA damage, 8-oxoG and abasic sites, which are repaired by 8-oxoguanine glycosylase (OGG1) and apurinic/apyrimidinic endonuclease (APE1), respectively. UV-DDB increased both OGG1 and APE1 strand cleavage and stimulated subsequent DNA polymerase β-gap filling activity by 30-fold. Single-molecule real-time imaging revealed that UV-DDB forms transient complexes with OGG1 or APE1, facilitating their dissociation from DNA. Furthermore, UV-DDB moves to sites of 8-oxoG repair in cells, and UV-DDB depletion sensitizes cells to oxidative DNA damage. We propose that UV-DDB is a general sensor of DNA damage in both NER and BER pathways, facilitating damage recognition in the context of chromatin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-019-0261-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684372PMC
August 2019

The Pol β variant containing exon α is deficient in DNA polymerase but has full dRP lyase activity.

Sci Rep 2019 07 9;9(1):9928. Epub 2019 Jul 9.

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

DNA polymerase (Pol) β is a key enzyme in base excision repair (BER), an important repair system for maintaining genomic integrity. We previously reported the presence of a Pol β transcript containing exon α (105-nucleotide) in normal and colon cancer cell lines. The transcript carried an insertion between exons VI and VII and was predicted to encode a ~42 kDa variant of the wild-type 39 kDa enzyme. However, little is known about the biochemical properties of the exon α-containing Pol β (exon α Pol β) variant. Here, we first obtained evidence indicating expression of the 42 kDa exon α Pol β variant in mouse embryonic fibroblasts. The exon α Pol β variant was then overexpressed in E. coli, purified, and characterized for its biochemical properties. Kinetic studies of exon α Pol β revealed that it is deficient in DNA binding to gapped DNA, has strongly reduced polymerase activity and higher Km for dNTP during gap-filling. On the other hand, the 5'-dRP lyase activity of the exon α Pol β variant is similar to that of wild-type Pol β. These results indicate the exon α Pol β variant is base excision repair deficient, but does conduct 5'-trimming of a dRP group at the gap margin. Understanding the biological implications of this Pol β variant warrants further investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-45846-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616571PMC
July 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-biochem-013118-111315DOI Listing
June 2019

Molecular basis for the faithful replication of 5-methylcytosine and its oxidized forms by DNA polymerase β.

J Biol Chem 2019 05 18;294(18):7194-7201. Epub 2019 Mar 18.

From the Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

DNA methylation is an epigenetic mark that regulates gene expression in mammals. One method of methylation removal is through ten-eleven translocation-catalyzed oxidation and the base excision repair pathway. The iterative oxidation of 5-methylcytosine catalyzed by ten-eleven translocation enzymes produces three oxidized forms of cytosine: 5-hydroxmethylcytosine, 5-formylcytosine, and 5-carboxycytosine. The effect these modifications have on the efficiency and fidelity of the base excision repair pathway during the repair of opposing base damage, and in particular DNA polymerization, remains to be elucidated. Using kinetic assays, we show that the catalytic efficiency for the incorporation of dGTP catalyzed by human DNA polymerase β is not affected when 5-methylcytosine, 5-hydroxmethylcytosine, and 5-formylcytosine are in the DNA template. In contrast, the catalytic efficiency of dGTP insertion decreases ∼20-fold when 5-carboxycytosine is in the templating position, as compared with unmodified cytosine. However, DNA polymerase fidelity is unaltered when these modifications are in the templating position. Structural analysis reveals that the methyl, hydroxymethyl, and formyl modifications are easily accommodated within the polymerase active site. However, to accommodate the carboxyl modification, the phosphate backbone on the templating nucleotide shifts ∼2.5 Å to avoid a potential steric/repulsive clash. This altered conformation is stabilized by lysine 280, which makes a direct interaction with the carboxyl modification and the phosphate backbone of the templating strand. This work provides the molecular basis for the accommodation of epigenetic base modifications in a polymerase active site and suggests that these modifications are not mutagenically copied during base excision repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.006809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509498PMC
May 2019

A Transition-State Perspective on Y-Family DNA Polymerase η Fidelity in Comparison with X-Family DNA Polymerases λ and β.

Biochemistry 2019 04 14;58(13):1764-1773. Epub 2019 Mar 14.

Department of Biological Sciences, Dana and David Dornsife College of Letters, Arts, and Sciences , University of Southern California , University Park Campus , Los Angeles , California 90089 , United States.

Deoxynucleotide misincorporation efficiencies can span a wide 10-fold range, from ∼10 to ∼10, depending principally on polymerase (pol) identity and DNA sequence context. We have addressed DNA pol fidelity mechanisms from a transition-state (TS) perspective using our "tool-kit" of dATP- and dGTP-β,γ substrate analogues in which the pyrophosphate leaving group (p K = 8.9) has been replaced by a series of bisphosphonates covering a broad acidity range spanning p K values from 7.8 (CF) to 12.3 [C(CH)]. Here, we have used a linear free energy relationship (LFER) analysis, in the form of a Brønsted plot of log( k) versus p K, for Y-family error-prone pol η and X-family pols λ and β to determine the extent to which different electrostatic active site environments alter k values. The apparent chemical rate constant ( k) is the rate-determining step for the three pols. The pols each exhibit a distinct catalytic signature that differs for formation of right (A·T) and wrong (G·T) incorporations observed as changes in slopes and displacements of the Brønsted lines, in relation to a reference LFER. Common to this signature among all three pols is a split linear pattern in which the analogues containing two halogens show k values that are systematically lower than would be predicted from their p K values measured in aqueous solution. We discuss how metal ions and active site amino acids are responsible for causing "effective" p K values that differ for dihalo and non-dihalo substrates as well as for individual R and S stereoisomers for CHF and CHCl.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.9b00087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681824PMC
April 2019

A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition.

Nucleic Acids Res 2019 04;47(6):3197-3207

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, P.O. Box 12233, Research Triangle Park, NC 27709-2233, USA.

4,6-Diamino-5-formamidopyrimidine (Fapy•dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapy•dG is in its nucleotide triphosphate form, Fapy•dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapy•dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymerases. Here, we evaluated insertion kinetics and high-resolution ternary complex crystal structures of a configurationally stable Fapy•dGTP analog, β-C-Fapy•dGTP, with DNA polymerase β. The crystallographic snapshots and kinetic data indicate that binding of β-C-Fapy•dGTP impedes enzyme closure, thus hindering insertion. The structures reveal that an active site residue, Asp276, positions β-C-Fapy•dGTP so that it distorts the geometry of critical catalytic atoms. Removal of this guardian side chain permits enzyme closure and increases the efficiency of β-C-Fapy•dG insertion opposite dC. These results highlight the stringent requirements necessary to achieve a closed DNA polymerase active site poised for efficient nucleotide incorporation and illustrate how DNA polymerase β has evolved to hinder Fapy•dGTP insertion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451102PMC
April 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2018.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312470PMC
January 2019

Cutting-edge perspectives in genomic maintenance V.

DNA Repair (Amst) 2018 11 25;71:1-2. Epub 2018 Sep 25.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2018.09.012DOI Listing
November 2018
-->