A5089715804- DNA Repair Mechanisms
- DNA and Nucleic Acid Chemistry
- Carcinogens and Genotoxicity Assessment
- PARP inhibition in cancer therapy
- High Altitude and Hypoxia
- Molecular Biology Techniques and Applications
- Bacterial Genetics and Biotechnology
- HIV/AIDS drug development and treatment
- Cancer therapeutics and mechanisms
- Mitochondrial Function and Pathology
- Genomics and Chromatin Dynamics
- RNA and protein synthesis mechanisms
- Salmonella and Campylobacter epidemiology
- HIV Research and Treatment
- Cancer-related Molecular Pathways
- CRISPR and Genetic Engineering
- Bacteriophages and microbial interactions
- Epigenetics and DNA Methylation
- Plant Genetic and Mutation Studies
- Integrated Circuits and Semiconductor Failure Analysis
- Advanced biosensing and bioanalysis techniques
- Genetic factors in colorectal cancer
- Acute Lymphoblastic Leukemia research
- Polyomavirus and related diseases
- Spaceflight effects on biology
University of Agricultural Sciences, Bangalore
2024
National Institute of Environmental Health Sciences
2012-2021
National Institutes of Health
2010-2020
Government of the United States of America
2018
St. John's University
2016
Defence Institute of Physiology and Allied Sciences
1995-2015
Triangle
1997-2014
Research Triangle Park Foundation
1998-2011
University of Pittsburgh
2011
Los Alamos National Laboratory
2006
DNA polymerase beta (pol beta) fills single nucleotide (nt) gaps in produced by the base excision repair pathway of mammalian cells. Crystal structures have been determined representing intermediates 1 nt gap-filling reaction pol beta: binary complex with a gapped substrate (2.4 A resolution), ternary including ddCTP (2.2 A), and product containing only nicked (2.6 A). Upon binding to gap complex, thumb subdomain rotates into closed conformation contact otherwise solvent-exposed...
Base excision repair (BER) is one of the cellular defense mechanisms repairing damage to nucleoside 5′-monophosphate residues in genomic DNA. This pathway initiated by spontaneous or enzymatic N-glycosidic bond cleavage creating an abasic apurinic-apyrimidinic (AP) site double-stranded Class II AP endonuclease, deoxyribonucleotide phosphate (dRP) lyase, DNA synthesis, and ligase activities complete site. In mammalian cell nuclear extract, BER can be mediated a macromolecular complex...
The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated vitro. A 51-base pair synthetic substrate containing single was used, incorporation dCMP during exclusively to replace uracil. neutralizing polyclonal antibody against polymerase β (β-pol) inhibited reaction. ddCTP also reaction, whereas aphidicolin had no significant effect,...
Base excision repair (BER) is a cellular defense mechanism repairing modified bases in DNA. Recently, G:U reaction has been reconstituted with several purified enzymes from Escherichia coli (Dianov, G., and Lindahl, T. (1994) Curr. Biol. 4, 1069-1076). Using bovine testis crude nuclear extract, we have shown that repaired efficiently vitro, DNA polymerase β (β-pol) responsible for the single nucleotide gap-filling synthesis (Singhal, R. K., Prasad, R., Wilson, S. H. (1995) J. Chem. 270,...
Mitochondria have been proposed to possess base excision repair processes correct oxidative damage the mitochondrial genome. As only DNA polymerase (pol) present in mitochondria, pol gamma is necessarily implicated such processes. Therefore, we tested ability of catalytic subunit human participate uracil-provoked reconstituted vitro with purified components. Subsequent actions uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease, was able fill a single nucleotide gap presence 5'...
The two base excision repair (BER) subpathways in mammalian cells are characterized by the number of nucleotides synthesized into patch. They "single-nucleotide" BER pathway and "long patch" (several incorporated) pathway. Both these involve a damaged and/or nearby DNA synthesis to fill gap. Whereas polymerase β (pol β) is known participate single-nucleotide pathway, identity polymerases involved long patch has remained unclear. By analyzing products generated during uracil-containing...
In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: "single-nucleotide BER" requiring DNA synthesis of just one nucleotide "long patch multi-nucleotide synthesis. single-nucleotide BER, polymerase β (β-pol) accounts for both gap filling removal the 5′-deoxyribose phosphate (dRP) site, whereas involvement various polymerases in long BER is less well understood. Recently, we found that β-pol...
Recently, photoaffinity labeling experiments with mouse cell extracts suggested that PARP-1 functions as a surveillance protein for stalled BER intermediate. To further understand the role of in BER, we examined DNA synthesis and flap excision steps long patch using reconstituted system containing 34-base pair substrate five purified human enzymes: uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, polymerase β, endonuclease-1 (FEN-1), PARP-1. stimulates strand displacement by β...
The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction the PARP-1/AP site complex resulted covalent cross-linking PARP-1 DNA; identity cross-linked was confirmed by mass spectrometry. Using purified PARP-1, specificity binding AP site-containing competition experiments. only weakly activated conduct synthesis upon DNA, but strongly for strand incision...
The current model for base excision repair (BER) involves two general sub-pathways termed single-nucleotide BER and long patch that are distinguished by their sizes the enzymes/co-factors involved. Both involve a series of sequential steps from initiation to completion repair. designed sequester various intermediates, passing them along one step next without allowing these toxic molecules trigger cell cycle arrest, necrotic death, or apoptosis. Although variety DNA-protein protein-protein...
PARP1-dependent poly-ADP-ribosylation (PARylation) participates in the repair of many forms DNA damage. Here, we used atomic force microscopy (AFM) and single molecule fluorescence to examine interactions PARP1 with common intermediates. AFM volume analysis indicates that binds at nicks, abasic (AP) sites, ends as a monomer. Single tightrope assays were follow real-time dynamic behavior absence presence AP endonuclease (APE1) on damage arrays. These experiments revealed conducted search...
DNA polymerase iota (pol iota) is one of several recently discovered polymerases in mammalian cells whose function unknown. We report here that human pol has an intrinsic 5'-deoxyribose phosphate (dRP) lyase activity. In reactions reconstituted with uracil-DNA glycosylase (UDG), apurinic/apyrimidinic (AP) endonuclease and ligase I, can use its dRP activities to repair G*U A*U pairs DNA. These data three distinct catalytic properties implicate it specialized forms base excision (BER).
DNA polymerase β (β-pol) cleaves the sugar-phosphate bond 3′ to an intact apurinic/apyrimidinic (AP) site (<i>i.e.</i> AP lyase activity). The same is cleaved even if has been previously 5′-incised by endonuclease, resulting in a 5′ 2-deoxyribose 5-phosphate dRP We characterized these reactions steady-state kinetics with amino-terminal 8-kDa domain of β-pol and entire 39-kDa polymerase. Steady-state kinetic analyses show that Michaelis constants for both activities are similar. However,...
To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The was in situ, using an end-labeled oligonucleotide containing synthetic abasic site; this site incised by apurinic/apyrimidinic endonuclease creating nick 3′-hydroxyl 5′-reduced sugar phosphate groups at margins, then dNMP carrying photoreactive adduct added to group. With near-UV...
DNA alkylation damage is primarily repaired by the base excision repair (BER) machinery in mammalian cells. In of N-alkylated purine lesion, for example, alkyl adenine glycosylase (Aag) recognizes and removes base, polymerase beta (beta-pol) contributes gap tailoring synthesis steps. It loss beta-pol-mediated 5'-deoxyribose phosphate removal that renders mouse fibroblasts alkylation-hypersensitive. Here we report hypersensitivity beta-pol-deficient cells after methyl methanesulfonate-induced...
In the crystal structure of a substrate complex, side chains residues Asn279, Tyr271, and Arg283 DNA polymerase β are within hydrogen bonding distance to bases incoming deoxynucleoside 5′-triphosphate (dNTP), terminal primer nucleotide, templating respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. Kraut, J.(1994) Science 264, 1891-1903). We have altered these through individual site-directed mutagenesis. Each mutant protein was expressed in Escherichia coli soluble. The...