A5020926425- DNA Repair Mechanisms
- DNA and Nucleic Acid Chemistry
- CRISPR and Genetic Engineering
- Genomics and Chromatin Dynamics
- Bacterial Genetics and Biotechnology
- Carcinogens and Genotoxicity Assessment
- RNA Research and Splicing
- RNA and protein synthesis mechanisms
- RNA Interference and Gene Delivery
- Diffusion and Search Dynamics
- Molecular Biology Techniques and Applications
- Epigenetics and DNA Methylation
- Telomeres, Telomerase, and Senescence
- Microtubule and mitosis dynamics
- Bacteriophages and microbial interactions
- Genetic Neurodegenerative Diseases
- Peptidase Inhibition and Analysis
- Cancer-related Molecular Pathways
- Plant Genetic and Mutation Studies
- History and advancements in chemistry
- Cancer Genomics and Diagnostics
- PARP inhibition in cancer therapy
- Receptor Mechanisms and Signaling
- Genetics and Neurodevelopmental Disorders
- Histone Deacetylase Inhibitors Research
Pennsylvania State University
2013-2025
University of California, Berkeley
2016
University of Michigan
2008-2014
Ithaca College
2007
DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for polymerases and other proteins. The clamp needs to be opened installed onto by loader ATPase of AAA+ family. human factor C (RFC) proliferating cell nuclear antigen (PCNA) are both play critical roles in several diseases. Despite decades study, no structure RFC has been resolved. Here, we report bound PCNA cryogenic electron microscopy overall resolution...
Abstract Many types of damage, including abasic sites, block replicative DNA polymerases causing replication fork uncoupling and generating ssDNA. AP-Endonuclease 1 (APE1) has been shown to cleave sites in Importantly, APE1 cleavage ssDNA at a significant biological implications by double strand breaks that could collapse the fork. Despite this, molecular basis efficiency processing forks remain elusive. Here, we investigate substrates mimic interactions stalled or gaps. We determine robust...
Spontaneous DNA damage occurs throughout the genome, requiring that repair enzymes search each nucleotide every cell cycle. This is postulated to be more efficient if enzyme can diffuse along DNA, but our understanding of this process incomplete. A key distinction between mechanisms diffusion whether protein maintains continuous contact (sliding) or it undergoes microscopic dissociation (hopping). We describe a simple chemical assay detect ability modifying hop and have applied human...
In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to proliferating cell nuclear antigen (PCNA) sliding clamp form a holoenzyme. The stability of this complex integral every aspect replication. Most our understanding comes from Saccharomyces cerevisae where extreme pol holoenzyme ensures that nucleobase within an Okazaki fragment faithfully duplicated before dissociation but also necessitates active displacement mechanism recycling...
DNA repair proteins conduct a genome-wide search to detect and sites of damage wherever they occur. Human alkyladenine glycosylase (AAG) is responsible for recognizing variety base lesions, including alkylated deaminated purines, initiating their via the excision pathway. We have investigated mechanism by which AAG locates using an oligonucleotide substrate containing two damage. This was designed so that randomly binds either lesions. AAG-catalyzed creates intermediate, subsequent...
Translesion DNA synthesis (TLS) during S-phase uses specialized TLS polymerases to replicate a lesion, allowing stringent resume beyond the offending damage. Human involves conjugation of ubiquitin PCNA clamps encircling damaged and role this post-translational modification is under scrutiny. A widely-accepted model purports that ubiquitinated recruits such as pol η sites damage where they may also displace blocked replicative polymerase. We provide extensive quantitative evidence binding...
Abstract DNA polymerase δ (pol δ) holoenzymes, comprised of pol and the processivity sliding clamp, PCNA, carry out synthesis during lagging strand replication, initiation leading major damage repair tolerance pathways. Pol holoenzymes are assembled at primer/template (P/T) junctions initiate in a stepwise process involving single (ssDNA)-binding protein complex, RPA, clamp loader, RFC, PCNA δ. During this process, interactions RFC with P/T junction all significantly overlap. A burning issue...
Most cancers upregulate the telomere lengthening enzyme telomerase to achieve unlimited cell division. How chemotherapeutic nucleoside 6-thio-2-deoxyguanosine (6-thio-dG) targets inhibit maintenance in cancer cells and tumors was unclear. Here, we demonstrate that insertion of 6-thio-dGTP prevents synthesis additional telomeric repeats but does not disrupt binding telomeres. Specifically, 6-thio-dG inhibits extension after translocates along its product DNA reposition template, inducing a...
The DNA genome is constantly exposed to agents, such as ultraviolet radiation (UVR), that can alter or eliminate its coding properties through covalent modifications of the template bases. Many these damaging (i.e., lesions) persist into S-phase cell cycle where they may stall canonical replication machinery. In humans, stalling events are circumvented by one at least three interconnected damage tolerance (DDT) pathways; translesion synthesis (TLS), Template Switching (TS), and...
G-quadruplexes (G4s) are functional elements of the human genome, some which inhibit DNA replication. We investigated replication G4s within highly abundant microsatellite (GGGA, GGGT) and transposable element (L1 SVA) sequences. found that genome-wide, numerous motifs located preferentially on leading strand transcribed templates. directly tested replicative polymerase ϵ δ holoenzyme inhibition at these G4s, compared to low motifs. For all synthesis was higher G-rich than C-rich or control...
In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined human holoenzyme. The results indicate that proceeds in a stepwise manner. loader (RFC) loads (PCNA) primer/template junction but transiently bound DNA. Unable slide away, PCNA re-engages with RFC is unloaded. presence...
Abstract PARP2 is a DNA-dependent ADP-ribosyl transferase (ARTs) enzyme with Poly(ADP-ribosyl)ation activity that triggered by DNA breaks. It plays role in the Base Excision Repair pathway, where it has overlapping functions PARP1. However, additional roles for have emerged response of cells to replication stress. In this study, we demonstrate promotes stress-induced telomere fragility and prevents loss following chronic induction oxidative lesions BLM helicase depletion. Telomere results...
Large genomes pose a challenge to DNA repair pathways because rare sites of damage must be efficiently located from among vast excess undamaged sites. Human alkyladenine glycosylase (AAG) employs nonspecific binding interactions and facilitated diffusion conduct highly redundant search adjacent This ensures that every site is searched, but could detriment if the protein trapped in local segment DNA. Intersegmental transfer between segments are transiently close proximity provides an elegant...
The replicative polymerases cannot accommodate distortions to the native DNA sequence such as modifications (lesions) template bases from exposure reactive metabolites and environmental mutagens. Consequently, synthesis on an afflicted abruptly stops upon encountering these lesions, but replication fork progresses onward, exposing long stretches of damaged before eventually stalling. Such arrests may be overcome by translesion (TLS) in which specialized TLS bind resident proliferating cell...
In humans, proliferating cell nuclear antigen (PCNA) sliding clamps encircling DNA coordinate various aspects of metabolism throughout the cycle. A critical aspect this is restricting PCNA to vicinity its target site. For example, must be maintained at or near primer/template (P/T) junctions during synthesis. With a diverse array cellular factors implicated, many which interact with PCNA, DNA, both, it unknown how feat achieved. Furthermore, current biochemical assays that examine retention...
Translesion DNA synthesis (TLS) enables replication through damaging modifications to template and requires monoubiquitination of the proliferating cell nuclear antigen (PCNA) sliding clamp by Rad6/Rad18 complex. This posttranslational modification is critical survival following exposure DNA-damaging agents tightly regulated restrict TLS damaged DNA. Replication protein A (RPA), major single-strand (ssDNA) binding complex, forms filaments on ssDNA exposed at sites plays yet undefined roles...
DNA glycosylases perform a genome-wide search to locate damaged nucleotides among great excess of undamaged nucleotides. Many are capable facilitated diffusion, whereby multiple sites along the sampled during single binding encounter. Electrostatic interactions between positively charged amino acids and negatively phosphate backbone crucial for but extent which diffusing proteins rely on double-helical structure is not known. Kinetic assays were used probe searching mechanism human...