A5001664399- DNA Repair Mechanisms
- Genomics and Chromatin Dynamics
- DNA and Nucleic Acid Chemistry
- CRISPR and Genetic Engineering
- Bacterial Genetics and Biotechnology
- Microtubule and mitosis dynamics
- Epigenetics and DNA Methylation
- Advanced biosensing and bioanalysis techniques
- Cancer-related Molecular Pathways
- RNA Research and Splicing
- PARP inhibition in cancer therapy
- Carcinogens and Genotoxicity Assessment
- Plant Genetic and Mutation Studies
- Fungal and yeast genetics research
- RNA and protein synthesis mechanisms
- Renal and related cancers
- RNA modifications and cancer
- Chromosomal and Genetic Variations
- Genetic diversity and population structure
- Genetic Syndromes and Imprinting
- Plant Disease Resistance and Genetics
- Cancer therapeutics and mechanisms
- Pluripotent Stem Cells Research
- Molecular Biology Techniques and Applications
- Genetics and Neurodevelopmental Disorders
Memorial Sloan Kettering Cancer Center
2015-2025
Cornell University
2020
Cancer Research UK
2009-2013
University of California, Berkeley
1999-2005
Cold Spring Harbor Laboratory
2001
Abstract ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that interacts with MCM subunits Mcm2, 4 6, which both destabilizes Mcm2–5 interface inhibits ATPase activity. Using X-ray crystallography, contains two winged-helix domains in C-terminal half protein a catalytically inactive dioxygenase-related N-terminal domain, is important for loading, but not subsequent replication. We...
R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how inherently impact fork progression is not understood. Here, we characterize R-loop-replisome collisions using fully reconstituted eukaryotic DNA system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional head-on can by inducing stalling, uncoupling leading strand synthesis from replisome progression, nascent gaps. RNase H1 Pif1 suppress defects resolving...
DNA G-quadruplexes (G4s) are non-B-form secondary structures that threaten genome stability by impeding replication. To elucidate how G4s induce replication fork arrest, we characterized collisions with preformed in the parental using reconstituted yeast and human replisomes. We demonstrate a single G4 leading strand template is sufficient to stall replisomes arresting CMG helicase. Cryo-electron microscopy of stalled complexes reveal folded lodged inside central channel, translocation. The...
The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of wild-type several mutant variants. an ATPase, our studies show that ORC1 subunit essential for ATP hydrolysis ATP-dependent binding. Moreover, binding by reduces its activity. In vitro , binds to chromatin manner, this process depends on AAA + nucleotide-binding domain ORC1. Mutations ATP-binding are unable...
The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic metabolic enzymes. PCNA loaded around by the ATP-dependent loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded (dsDNA) junctions harboring a recessed 3’ end (3’ ss/dsDNA junctions) and nicks. To illuminate loading mechanism we have investigated structure of RFC:PCNA bound to ATPγS or nicked using cryogenic electron microscopy. Unexpectedly, observe...
In eukaryotes the sites for initiation of chromosomal DNA replication are believed to be determined in part by binding a heteromeric origin recognition complex (ORC) DNA. We have cloned genes encoding subunits Drosophila ORC. Each is unique and can mapped discrete locations implying that pattern developmental regulation usage not regulated solely large family different ORC proteins. The six-subunit reconstituted with recombinant proteins into restores ORC-depleted or Xenopus egg extracts.
Fundamental to our understanding of chromosome duplication is the idea that replication origins function both as sites where MCM helicases are loaded during G1 phase and synthesis begins in S phase. However, temporal delay between phases exposes replisome assembly pathway potential disruption prior replication. Using multicolor, single-molecule imaging, we systematically study consequences encounters actively transcribing RNA polymerases (RNAPs) initiation intermediates context chromatin. We...
Sumoylation is emerging as a posttranslation modification important for regulating chromosome duplication and stability. The origin recognition complex (ORC) that directs DNA replication initiation by loading the MCM replicative helicase onto origins sumoylated in both yeast human cells. However, biological consequences of ORC sumoylation are unclear. Here we report effects hypersumoylation hyposumoylation on activity function using multiple approaches. preferentially reduced subset early...
Abstract DNA polymerase epsilon (Pol ε) is required for genome duplication and tumor suppression. It supports both replisome assembly leading strand synthesis; however, the underlying mechanisms remain to be elucidated. Here we report that a conserved domain within Pol ε catalytic core influences of these replication steps in budding yeast. Modeling cancer-associated mutations this reveals its unexpected effect on incorporating into four-member pre-loading complex during assembly. In...
Faithful propagation of eukaryotic chromosomes usually requires that no DNA segment be replicated more than once during one cell cycle. Cyclin-dependent kinases (Cdks) are critical for the re-replication controls inhibit activities components pre-replication complexes (pre-RCs) following origin activation. The recognition complex (ORC) initiates assembly pre-RCs at origins replication and Cdk phosphorylation ORC is important prevention re-initiation. Here we show Drosophila melanogaster...
Eukaryotic replication origins are licensed by the loading of replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control multiple protein kinases that either promote or inhibit activation, which important for genome maintenance. Using reconstituted budding yeast system, we find flexible N-terminal extension (NTE) Mcm2 promotes stable recruitment Dbf4-dependent kinase (DDK) to Mcm2-7 hexamers, turn DDK phosphorylation Mcm4 and...
Cdc6, a subunit of the pre-replicative complex (pre-RC), contains multiple regulatory cyclin-dependent kinase (Cdk1) consensus sites, SP or TP motifs. In Saccharomyces cerevisiae, Cdk1 phosphorylates Cdc6-T7 to recruit Cks1, phospho-adaptor in S phase, for subsequent multisite phosphorylation and protein degradation. Cdc6 accumulates mitosis is tightly bound by Clb2 through N-terminal order prevent premature origin licensing It has been extensively studied how regulated cyclin-Cdk1 complex....