A5069424550- DNA Repair Mechanisms
- Cancer-related Molecular Pathways
- DNA and Nucleic Acid Chemistry
- Genomics and Chromatin Dynamics
- CRISPR and Genetic Engineering
- PARP inhibition in cancer therapy
- Molecular Biology Techniques and Applications
- RNA Research and Splicing
- Carcinogens and Genotoxicity Assessment
- RNA modifications and cancer
- RNA and protein synthesis mechanisms
- HIV/AIDS drug development and treatment
- Bacteriophages and microbial interactions
- Epigenetics and DNA Methylation
- Plant Virus Research Studies
- Nuclear Structure and Function
- Polyomavirus and related diseases
- Microtubule and mitosis dynamics
- Bacterial Genetics and Biotechnology
- Mitochondrial Function and Pathology
- Enzyme Structure and Function
- Cancer therapeutics and mechanisms
- Animal Genetics and Reproduction
- Genetics and Neurodevelopmental Disorders
- Herpesvirus Infections and Treatments
New York Medical College
2013-2025
Catalyst Foundation
2012-2018
Weatherford College
1994-2013
Leonardo (United Kingdom)
2013
Evonik (Germany)
2013
Ospedale L. Bonomo
2007
University of Miami
1985-1998
The Ohio State University
1995
University of California System
1992
DNA polymerase delta from calf thymus has been purified to apparent homogeneity by a new procedure which utilizes hydrophobic interaction chromatography with phenyl-Sepharose at an early step separate most of the calcium-dependent protease activity and alpha. The enzyme migrates as single protein band on polyacrylamide gel electrophoresis under nondenaturing conditions. sedimentation coefficient is 7.9 S, Stokes radius 53 A. A molecular weight 173K calculated for native enzyme. Sodium...
Human DNA polymerase η (Pol η) is best known for its role in responding to UV irradiation-induced genome damage. We have recently observed that Pol also required the stability of common fragile sites (CFSs), whose rearrangements are considered a driving force oncogenesis. Here, we explored molecular mechanisms underlying this newly identified role. demonstrated accumulated at CFSs upon partial replication stress and could efficiently replicate non-B sequences within CFSs. deficiency led...
Cancer cells rely on the activation of telomerase or alternative lengthening telomeres (ALT) pathways for telomere maintenance and survival. ALT involves homologous recombination (HR)-dependent exchange and/or HR-associated synthesis telomeric DNA. Utilizing proximity-dependent biotinylation (BioID), we sought to determine proteome in cancer that employ these distinct elongation mechanisms. Our analysis reveals multiple DNA repair networks converge at telomeres. These include specialized...
In 1975, a Greek letter nomenclature system was adopted for the DNA polymerases from higher eukaryotes [I].This defined a, and y .The recent isolation characterization of at least two new classes polymerases, collectively called polymerase 6, has
Homologous recombination (HR) is essential for maintaining genomic integrity, which challenged by a wide variety of potentially lethal DNA lesions. Regardless the damage type, known to proceed RAD51-mediated D-loop formation, followed repair synthesis. Nevertheless, participating polymerases and extension mechanism are not well characterized. Here, we present reconstitution this step using purified human proteins. In addition Pol δ, TLS polymerases, including η κ, also can extend D-loops....
Mammalian DNA polymerase (Pol) delta is essential for replication. It consists of four subunits, p125, p50, p68, and p12. We report the discovery that p12 subunit rapidly degraded in cultured human cells by damage or replication stress brought about treatments with UV, methyl methanesulfonate, hydroxyurea, aphidicolin. The degradation due to an accelerated rate proteolysis inhibited proteasome inhibitors, MG132 lactacystin. UV treatment converts Pol vivo three-subunit form lacking This was...
DNA polymerase delta was isolated from human placenta and identified as such on the basis of its association with a 3'- to 5'-exonuclease activity. The exonuclease activities maintained throughout purification attempted separations by physical or electrophoretic methods. Moreover, ratios two remained constant during steps, both were inhibited aphidicolin, oxidized glutathione, N-ethylmaleimide. purified enzyme had an estimated molecular weight 172,000, Stokes radius 53.6 A sedimentation...
AbstractThe ubiquitination of PCNA is an essential event in the operation DNA Damage Tolerance (DDT) pathway that activated after damage caused by UV or chemical agents during S-phase. This allows bypass translesion synthesis would otherwise cause replication fork stalling. mono-ubiquitinated Rad18-Rad6, and polyubiquitinated Rad5-Ubc13/Uev1 DDT pathway. Mono-and polyubiquitination are key processes template switching sub-pathways DDT. IR causes DSBs, which trigger Response (DDR). The...
The p-n-butylphenyl- and p-n-butylanilino- substituted analogs of dGTP dATP, respectively, were tested as inhibitors purified human placental DNA polymerases alpha delta. It was observed that polymerase activity potently inhibited by these with I0.5 values low the nanomolar range, whereas delta poorly inhibited, ca. 100 micromolar. These results argue for a distinct identity two enzymes, demonstrate usefulness probes structures. In addition, provide rapid method discrimination enzyme...
Common fragile sites (CFS) are chromosomal regions that exhibit instability during DNA replication stress. Although the mechanism of CFS expression has not been fully elucidated, one known feature is a severely delayed S-phase. We used an in vitro primer extension assay to examine progression synthesis through various sequences within FRA16D by replicative human polymerases δ and α, with cell-free extracts. found specific cis-acting sequence elements perturb elongation, causing inconsistent...
Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays crucial and versatile role in replication repair processes. We have reconstituted human Pol complexes insect cells infected with single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of tetrameric holoenzyme, p125/p50 core dimer, core+p68 trimer core+p12 trimer, as well p125 catalytic subunit. These isolated milligram amounts reproducible purity specific activities...
A 12-kDa and two 25-kDa polypeptides were isolated with highly purified calf thymus DNA polymerase δ by conventional chromatography. 16-mer peptide sequence was obtained from the polypeptide which matched a new open reading frame human EST (AA402118) encoding hypothetical protein of unknown function. The designated as p12. Human AA402118 identified putative homologue Schizosaccharomyces pombe Cdm1 tBlastn search data base usingS. Cdm1. AA402118encoded 107 amino acids predicted molecular mass...
Proliferating cell nuclear antigen (PCNA) has recently been identified as a target for the binding of proteins involved in DNA replication, repair, and cycle control. The interactions between PCNA number these are known to be mediated by conserved peptide motif. In this study, random library which sequences displayed on E. coli bacterial flagellin protein was screened PCNA-binding sequences. Analysis retrieved verified presence addition, second, larger group peptides containing different...
The cDNA of human DNA polymerase delta was cloned. had a length 3.5 kb and encoded protein 1107 amino acid residues with calculated molecular mass 124 kDa. Northern blot analysis showed that the hybridized to mRNA 3.4 kb. Monoclonal polyclonal antibodies C-terminal 20 specifically immunoblotted pol catalytic polypeptide. A multiple sequence alignment constructed. This is closely related yeast herpes virus polymerases. levels message were found be induced concomitantly activity synthesis in...
Mammalian DNA polymerase delta (pol delta), a key enzyme of chromosomal replication, consists four subunits as follows: the catalytic subunit; p125, which is tightly associated with p50 p68, proliferating cell nuclear antigen (PCNA)-binding protein; and fourth subunit, p12. In this study, functional roles p12 subunit pol were studied. The inter-subunit interactions determined by yeast two-hybrid assays pulldown assays. These revealed that interacts p125 well p50. This dual interaction...
This study examines the role of p12 subunit in function human DNA polymerase δ (Pol δ) holoenzyme by comparing kinetics synthesis and degradation catalyzed four-subunit complex, three-subunit complex lacking p12, site-directed mutants each proofreading exonuclease activity. Results show that modulates rate fidelity Pol δ. All four complexes synthesize a rapid burst phase slower, more linear phase. In presence rates are ∼5 times faster, while affinity enzyme for its dNTP substrates appears...
Ubiquitination of proliferating cell nuclear antigen (PCNA) to ub-PCNA is essential for DNA replication across bulky template lesions caused by UV radiation and alkylating agents, as orchestrates the recruitment switching translesion synthesis (TLS) polymerases with polymerases. This allows proceed, leaving be repaired subsequently. Defects in a TLS polymerase, Pol η, lead form Xeroderma pigmentosum, disease characterized severe skin sensitivity sunlight damage an increased incidence cancer....
We have shown before that constitutive DNA damage signaling represented by H2AX-Ser139 phosphorylation and ATM activation in untreated normal tumor cells is a reporter of the persistent replication stress induced endogenous oxidants, by-products aerobic respiration. In present study we observed exposure mitogenically stimulated lymphocytes or cell lines A549, TK6 A431 to metformin, specific activator 5'AMP-activated protein kinase (AMPK) an inhibitor mTOR signaling, resulted attenuation H2AX...