A5060041272- DNA Repair Mechanisms
- Genetic factors in colorectal cancer
- RNA and protein synthesis mechanisms
- DNA and Nucleic Acid Chemistry
- Bacterial Genetics and Biotechnology
- Cancer Genomics and Diagnostics
- RNA modifications and cancer
- RNA Research and Splicing
- Bacteriophages and microbial interactions
- Epigenetics and DNA Methylation
- Genomics and Chromatin Dynamics
- Carcinogens and Genotoxicity Assessment
- Diffusion and Search Dynamics
- CRISPR and Genetic Engineering
- Advanced biosensing and bioanalysis techniques
- Enzyme Structure and Function
- PARP inhibition in cancer therapy
- Digestive system and related health
- Genomic variations and chromosomal abnormalities
- Amino Acid Enzymes and Metabolism
- Colorectal Cancer Screening and Detection
- Microbial Metabolic Engineering and Bioproduction
- Heat shock proteins research
- Cancer therapeutics and mechanisms
- Colorectal Cancer Treatments and Studies
Duke University Hospital
2009-2020
Duke Medical Center
2009-2020
Howard Hughes Medical Institute
2009-2020
Duke University
1996-2016
Institute of Cytochemistry and Molecular Pharmacology
2004-2008
Laboratoire de Biochimie
2008
Durham Technical Community College
2005-2007
Brandeis University
2001
National Cancer Center
1999
University Hospitals of Cleveland
1998
A subset of sporadic colorectal tumors and most developing in hereditary nonpolyposis cancer patients display frequent alterations microsatellite sequences. Such have been thought to manifest replication errors (RER+), but the basis for has remained conjectural. We demonstrate that mutation rate (CA)n repeats RER+ tumor cells is at least 100-fold RER- show by vitro assay increased mutability associated with a profound defect strand-specific mismatch repair. This deficiency was observed...
Repair of dsDNA breaks requires processing to produce 3'-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); Replication protein A (RPA). Resection occurs via two routes. In one, BLM physically specifically interact resect in a process that is ATP-dependent nuclease functions. RPA essential for both unwinding by enforcing 5' →...
Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective is evidenced by associated instability microsatellite sequences (MSI). Germ-line mutations are causally with inherited MSI cancer, somatic sporadic cancer. Previously however, we demonstrated that many all genes wild type. To investigate this class tumors further, examined a group cancer cell lines, most were documented as established from antecedent...
A mismatch-binding heterodimer of hMSH2 and a 160-kilodalton polypeptide has been isolated from HeLa cells by virtue its ability to restore mismatch repair nuclear extracts hMSH2-deficient LoVo colorectal tumor cells. This heterodimer, designated hMutSα, also restores alkylation-tolerant MT1 lymphoblastoid HCT-15 cells, which are selectively defective in the base-base single-nucleotide insertion-deletion mismatches. Because appear be free mutations, this selective defect is likely result...
DNA mismatch correction is a strand-specific process involving recognition of noncomplementary Watson-Crick nucleotide pairs and participation widely separated sites. The Escherichia coli methyl-directed reaction has been reconstituted in purified system consisting MutH, MutL, MutS proteins, helicase II, single-strand binding protein, polymerase III holoenzyme, exonuclease I, ligase, along with ATP (adenosine triphosphate), the four deoxynucleoside triphosphates. This set proteins can seven...
The human lymphoblastoid MT1 B-cell line was previously isolated as one of a series mutant cells able to survive the cytotoxic effects N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). nevertheless remain sensitive mutagenesis by MNNG and display mutator phenotype. These phenotypes have been attributed single genetic alteration postulated confer defect in strand-specific mismatch repair, proposal that attributes effect DNA alkylation wild-type futile attempts correct mispairs arise during...
Bacterial and mammalian mismatch repair systems have been implicated in the cellular response to certain types of DNA damage, genetic defects this pathway are known confer resistance cytotoxic effects DNA-methylating agents. Such observations suggest that addition their ability recognize base-pairing errors, members MutS family may also respond lesions produced by damage. We show human recognition activity MutSalpha recognizes several lesion including 1,2-intrastrand d(GpG) crosslink...
PURPOSE We evaluated the response to Temodal (Schering-Plough Research Institute, Kenilworth, NJ) of patients with newly diagnosed malignant glioma, as well predictive value quantifying tumor DNA mismatch repair activity and O6-alkylguanine-DNA alkyltransferase (AGT). PATIENTS AND METHODS Thirty-three glioblastoma multiforme (GBM) five anaplastic astrocytoma (AA) were treated at a starting dose 200 mg/m2 daily for 5 consecutive days repeat dosing every 28 after first dose. Immunochemistry...
A human MSH2-human MSH3 (hMSH2·hMSH3) complex of approximately 1:1 stoichiometry (human MutSβ (hMutSβ)) has been demonstrated in several tumor cell lines and purified to near homogeneity. In vitro, hMutSβ supports the efficient repair insertion/deletion (I/D) heterologies 2–8 nucleotides, is weakly active on a single-nucleotide I/D mispair, not detectably eight base-base mismatches. Human MutSα (hMutSα), heterodimer hMSH2 hMSH6, efficiently mismatches, mispairs, all substrates tested that...
Nuclear extracts derived from HeLa and Drosophila melanogaster KC cell lines have been found to correct single base-base mispairs within open circular DNA heteroduplexes containing a strand-specific, site-specific incision located 808 base pairs the mismatch. Correction in both extract systems is strand specific, being highly biased incised strand. Different homologous set of were processed with different efficiencies (G.T greater than G.G approximately equal A.C C.C), correction was...
Hypermutable H6 colorectal tumor cells are defective in strand-specific mismatch repair and bear defects both alleles of the hMLH1 gene. We have purified to near homogeneity an activity from HeLa that complements nuclear extracts restore proficiency on a set heteroduplex DNAs representing eight base-base mismatches as well number slipped-strand, insertion/deletion mispairs. This behaves single species during fractionation copurifies with proteins 85 110 kDa. Microsequence analysis...
The Escherichia coli mutS gene product is involved in mismatch correction this organism. We have purified a biologically active form of the 97,000 Mr protein to near homogeneity from an overproducing strain. Enzymatic and chemical protection ("footprinting") experiments demonstrated that mutS-encoded specifically binds DNA regions containing single base-pair mismatch. displayed variable affinity for limited set mismatches tested (G-T greater than G-A approximately equal A-C T-C).
An assay has been developed that permits analysis of DNA mismatch repair in cell-free extracts Escherichia coli. The method relies on heteroduplex molecules f1 R229 DNA, which contain a base-pair within the single EcoRI site molecule. As observed with heteroduplexes lambda [Pukkila, P. J., Peterson, Herman, G., Modrich, & Meselson, M. (1983) Genetics, press], vivo correction is directed by state dam methylation d(G-A-T-C) sequences duplex. Thus, (formula: see book) repaired to an...
To evaluate the substrate specificity of methyl-directed mismatch repair in Escherichia coli extracts, we have constructed a set DNA heteroduplexes, each which contains one eight possible single base pair mismatches and hemimethylated d(GATC) site. Although all were located at same position within heteroduplex molecules embedded sequence environment, they not corrected with equal efficiencies vitro. G-T was most efficiently, A-C, C-T, A-A, T-T, G-G being repaired rates 40-80% that mispair....
The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use in vivo methylated ColE1 DNA or vitro with purified enzyme. recognizes symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site duplex product methylation being 6-methylaminopurine. This work also demonstrated that Dpn I restriction endonuclease cleaves on 3' side modified adenine within to yield fragments possessing fully base-paired termini. All sequences enzyme...
The error-free repair of double-stranded DNA breaks by homologous recombination requires processing broken ends. These processed ends are substrates for assembly strand exchange proteins that mediate invasion. Here, we establish human BLM helicase, a member the RecQ family, stimulates nucleolytic activity exonuclease 1 (hExo1), 5'-->3' exonuclease. stimulation is specific because other homologs fail to stimulate hExo1. Stimulation resection hExo1 independent helicase and is, instead,...