Satya Prakash
University Of Texas Medical Br Galveston
Project start date: 2012-01-27
Project end date: 2016-11-30
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Satya Prakash
ROLE OF HUMAN DNA POLYMERASE KAPPA IN REPLICATIVE BYPASS OF DNA LESIONS
Satya Prakash, Professor
University Of Texas Medical Br Galveston, 301 University Blvd, Galveston, Tx 77555
Grant 5R01CA141209-02 from National Cancer Institute
Abstract: Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions. Humans possess four TLS Pols that belong to the Y-family, Pols ?, ?, ?, and Rev1, and another Pol, Pol?, that belongs to the B-family. These TLS Pols employ highly specialized mechanisms for replicating through DNA lesions. Of these, Pol? is adept at extending opposite from bulky N2-dG minor groove lesions. Pol? can also carry out TLS opposite other types of DNA lesions; in that case, however, the lesion bypass ability is not limited to Pol?, as other TLS Pols can also function in their bypass. To elucidate the role of Pol? in lesion bypass in human cells, we will use a combined biochemical, genetic, and structural approach. In Aim 1, the role of two unique structural features of Pol?, (i) the N-clasp which allows Pol? to encircle DNA, and (ii) the openness of its active site towards the minor groove at the template-primer junction, will be analyzed by determining the effects of mutations on the extension reaction from N2-dG minor groove adducts and other types of DNA lesions as well. In Aim 2, the role of Pol? in promoting replication through different types of DNA lesions in human cells will be analyzed using a newly devised SV40-based plasmid system. Among the DNA lesions to be studied are 8- oxoguanine (8-oxoG) and thymine glycol (TG) that result from cellular oxidative DNA damage; the ring-opened N2-(3-hydroxyl propyl-2´-deoxygunaosine [(r)-3HOPdG] and the bulky trans-4-hydroxy-2-non-enal- deoxyguanosine (HNE-dG) adducts that result from the reaction of N2-dG with aldehydes or enals generated from free radical attack on lipids in membranes; and the multi-cyclic benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE) N2-dG adduct that results from exposure to environmental pollutants and carcinogens. To gain a deeper understanding of how Pol? actually performs TLS opposite these DNA lesions, in Aim 3, we will determine crystal structures of Pol? with 8-oxoG, TG, and cis-syn TT dimer, as well as with the N2-dG minor groove adducts of (r)-3HOPdG, HNE, and BPDE. The proficient ability of Pol? for extending from the C inserted opposite the N2-dG adducts by another DNA Pol, such as ? or Rev1, would ensure error-free replication through such minor groove DNA adducts. Since a large variety of N2-dG adducts are formed in human cells from cellular oxidative reactions and from exposure to chemical and environmental carcinogens, Pol? will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. The proposed studies are highly relevant for cancer biology and etiology as they will reveal how human cells minimize the mutagenic and carcinogenic potential of DNA lesions. DNA lesions are generated in human cells from cellular oxidative damage and from exposure to environmental pollutants and carcinogens. By promoting error-free replication through DNA lesions, translesion synthesis DNA polymerases help to maintain genomic stability by keeping the rate of mutations low, and thereby reducing the incidence of cancers. The proposed studies will examine the role of human DNA polymerase ? in promoting error-free lesion bypass
Keywords: 2-Amino-6-Hydroxypurine; 3, 4-Benzopyrene; 3, 4-Benzpyrene; 5, 6-dihydroxy-5, 6-dihydrothymine; 5, 6-dihydroxydihydrothymine; 6H-Purin-6-one, 2-amino-1, 7-dihydro-; 7, 8-BaP-9, 10-Diol Epoxide; 7, 8-Dihydro-7, 8-dihydroxybenzo(a)pyrene 9, 10-oxide; 7, 8-Dihydroxy-9, 10-Epoxy-7, 8, 9, 10-Tetrahydrobenzo(a)pyrene; 7, 8-dihydro-8-oxoguanine; 8-OG; Active Sites; Address; Adverse effects; Affect; Aldehydes; Anti-BaPDE; Aromatic Polycyclic Hydrocarbons; Automobiles; BPDE; BPDE-N2-dG; Benzo(10, 11)chryseno(3, 4-b)oxirene-7, 8-diol, 7, 8, 8a, 9a-tetrahydro-; Benzo(a)pyrene; Benzo(a)pyrene 7, 8-Dihydrodiol 9, 10-Epoxide; Benzo(a)pyrene-7, 8-diol 9, 10-Epoxide; Binding; Binding (Molecular Function); Biochemical; Biochemical Genetics; Bypass; Cancer Biology; Cancer Causing Agents; Cancer Induction; Cancers; Carcinogen-DNA Adducts; Carcinogens; Causality; Cells; Chemicals; Complex; DNA; DNA Adducts; DNA Polymerases; DNA Replication; DNA Synthesis; DNA biosynthesis; DNA lesion; DNA-Dependent DNA Polymerases; DNA-Directed DNA Polymerase; Deoxyguanosine; Deoxynucleoside-triphosphate[{..}]DNA deoxynucleotidyltransferase (DNA-directed); Deoxyribonucleic Acid; EC 2.7.7.7; Ensure; Environmental Carcinogens; Environmental Pollutants; Epoxides; Epoxy Compounds; Etiology; Exposure to; Family; Free Radicals; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Genetic, Biochemical; Genome Stability; Glycols; Guanine; Guanosine, 2`-deoxy-; Human; Human, General; Hydroxyl; Hydroxyl Radical; Incidence; Lesion; Lipid Peroxidation; Lipids; Malignant Neoplasms; Malignant Tumor; Mammalian Cell; Man (Taxonomy); Man, Modern; Mediating; Membrane; Minor Groove; Molecular; Molecular Interaction; Mutation; Nucleotides; Oncogens; PAH; Plasmids; Polycyclic Hydrocarbons, Aromatic; Polymerase; Polynuclear Aromatic Hydrocarbons; Process; Reaction; Role; Solvents; Specificity; Stability, Genomic; Structure; System; System, LOINC Axis 4; Testing; Time; Treatment Side Effects; UV induced; adduct; base; benzo(a)pyrene 7, 8-diol-9, 10-epoxide-N2-deoxyguanosine; carcinogenesis; dimer; disease causation; disease etiology; disease/disorder etiology; disorder etiology; genome mutation; glycolthymine; human DNA; malignancy; membrane structure; neoplasm/cancer; oxidative DNA damage; oxidative damage; polynuclear aromatic hydrocarbon; public health relevance; side effect; social role; therapy adverse effect; thymine glycol; treatment adverse effect
Relevance: DNA lesions are generated in human cells from cellular oxidative damage and from exposure to environmental pollutants and carcinogens. By promoting error-free replication through DNA lesions, translesion synthesis DNA polymerases help to maintain genomic stability by keeping the rate of mutations low, and thereby reducing the incidence of cancers. The proposed studies will examine the role of human DNA polymerase ¿ in promoting error-free lesion bypass
Project start date: 2009-07-01
Project end date: 2014-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-07-070
5R01CA141209-02 (2010): $471484
1R01CA141209-01 (2009): $475682
TRANSCRIPTION-COUPLED DNA REPAIR IN YEAST
Satya Prakash, Professor
University Of Texas Medical Br Galveston, 301 University Blvd, Galveston, Tx 77555
Grant 5R01CA035035-24 from National Cancer Institute
Abstract: The long term objectives are to identify the mechanisms eukaryotic cells employ to overcome blocks to RNA polymerase II (Pol II) transcription conferred by DNA lesions in the template strand. The specific aims of this proposal are to determine the roles of the yeast Rad26, Rad28, and Rad2 proteins, and of TFIIH, particularly its Rad3 and Rad25 DNA helicase subunits, in transcription elongation on undamaged and damaged DNAs, and in transcription-coupled DNA repair. In Specific Aim 1, the involvement of the RAD26, RAD28, RAD2, RAD3, and RAD25 genes in transcription elongation in undamaged cells will be examined by determining the effects of mutations in these genes on Pol II transcription in yeast cells grown in the presence of 6-azauracil (6AU), and by determining the in vivo occupancy of promoters and open reading frames (ORFs) of various genes by the Rad26, Rad28, Rad2, Rad3, and Rad25 proteins by chromatin immunoprecipitation (CHIP) assays in yeast cells treated with 6AU. In Specific Aim 2, biochemical studies will be done to examine the effects of purified Rad26, Rad28, Rad2, and TFIIH on Pol II transcription elongation through intrinsic arrest sites on undamaged DNA templates. In Specific Aim 3, the interactions of the Rad2, Rad26, and Rad28 proteins with Pol II will be examined. In Specific Aim 4, the involvement of Rad26, Rad28, Rad2, and TFIIH in promoting Pol II transcription through damaged bases and through abasic sites will be studied by genetic and biochemical means. To provide evidence for the in vivo roles of these genes in the transcriptional bypass of DNA lesions, Pol II transcription will be examined in the various rad mutant strains that are also deleted for the genes that function in the removal of damaged bases or abasic sites, and treated with the alkylating agent MMS. The presence of the Rad26, Rad28, Rad2, Rad3, and Rad25 proteins with elongating Pol II will be analyzed by ChIP analyses in cells treated with MMS. Biochemical studies will be done with purified Rad26, Rad28, Rad2, and TFIIH to examine their role in promoting Pol II transcription through a thymine glycol and an abasic site. In Specific Aim 5, the hypothesis that after promoting the forward translocation of Pol II through certain DNA lesions, the Rad26, Rad28, Rad2 and TFIIH proteins remain bound at the lesion site and provide the nucleation site for the subsequent assembly of other repair factors, thereby enabling the preferential repair of DNA lesions from the transcribed strand, will be tested. Mutations in the human counterparts of yeast genes being studied here can cause Cockayne syndrome (CS); the proposed studies in yeast should yield a better understanding of the underlying basis of the severe growth and developmental defects in CS
Keywords: 5, 6-dihydroxy-5, 6-dihydrothymine; 5, 6-dihydroxydihydrothymine; 6-azauracil; Abscission; Alkylating Agents; Alkylators; Assay; BTF2; BTF2 transcription factor; BTF2-p89 Gene; Base Excision Repairs; Basic Transcription Factor 2 89 kDa Subunit Gene; Binding; Binding (Molecular Function); Bioassay; Biochemical; Biochemical Genetics; Biologic Assays; Biological Assay; Bypass; CHIP assay; Cells; ChIP (chromatin immunoprecipitation); Cockayne Syndrome; DNA; DNA Base Excision Repair; DNA Damage; DNA Damage Repair; DNA Helicases; DNA Injury; DNA Polymerase I; DNA Polymerase alpha; DNA Repair; DNA Unwinding Proteins; DNA lesion; DNA unwinding enzyme; DNA-Dependent DNA Polymerase I; DNA-Dependent RNA Polymerase II; Defect; Deoxyribonucleic Acid; Development; ERCC3; ERCC3 gene; Eukaryote; Eukaryotic Cell; Excision; Extirpation; GTF2H; Gene Transcription; Generalized Growth; Genes; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Genetic, Biochemical; Goals; Growth; Histone H3.3; Human; Human, General; In Vitro; Klenow Fragment; Lesion; Man (Taxonomy); Man, Modern; Molecular Interaction; Mutation; Nucleotide Excision Repair; ORFs; Open Reading Frames; Pol I; Polymerase; Progeria-Like Syndrome; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Protein Coding Region; Proteins; RAD25; RNA Expression; RNA Polymerase B; RNA Polymerase II; Removal; Repairs, Base Excision; Role; S cerevisiae; SII transcription elongation factor; Saccharomyces cerevisiae; Site; Surgical Removal; TFIIH; TFIIH 89 kDa Subunit Gene; TFIIH Basal Transcription Factor Complex Helicase XPB Subunit Gene; TFIIS; TFIIS elongation factor; Tail; Testing; Tissue Growth; Transcription; Transcription Elongation; Transcription, Genetic; Unscheduled DNA Synthesis; XPB; Yeast, Baker`s; Yeast, Brewer`s; Yeasts; azauracil; base; basic transcription factor 2; chromatin immunoprecipitation; elongation factor SII; eukaryotida; experiment; experimental research; experimental study; gene function; gene product; genome mutation; glycolthymine; helicase; in vivo; mutant; oligo(dC); oligodeoxycytidylic acid; ontogeny; repair; repaired; research study; resection; social role; strand transfer protein alpha; thymine glycol; transcription elongation factor A; transcription elongation factor S-II; transcription factor IIH; transcription factor IIS; transcription factor S-II; transcription factor TFIIH
Project start date: 1983-07-01
Project end date: 2010-04-30
Budget start date: 1-MAY-2008
Budget end date: 30-APR-2010
5R01CA035035-24 (2008): $0
5R01CA035035-23 (2007): $330390
5R01CA035035-22 (2006): $340257
5R01CA035035-21 (2005): $348446
2R01CA035035-20 (2004): $348446
HOOGSTEEN BASE PAIRING IN HUMAN DNA POLYMERASE IOTA
Satya Prakash, Professor
University Of Texas Medical Br Galveston, 301 University Blvd, Galveston, Tx 77555
Grant 5R01CA115856-06 from National Cancer Institute
Abstract: The long term objectives are to delineate the action mechanisms of human DNA polymerase i (Poll) on undamaged and damaged DNAs. The specific aims of this project are to examine the role of Hoogsteen base pairing in the replication of undamaged and damaged DNAs by Poll, and the proposed studies will use a combined biochemical and structural approach, hi Aim 1, biochemical studies will be done with base analogs of adenine and guanine to analyze the contributions of different hydrogen bond acceptors/donors in template purines to their Hoogsteen pairing with the incoming pyrimidine nucleotide (nt). Aim 2 will test the hypothesis that Hoogsteen base pairing enables Poll to incorporate nts opposite DNA lesions that severely impinge upon the minor groove of the template base or which impair its Watson-Crick base pairing ability. The proficiency of Poll for incorporating nts opposite such lesions and to extend there from will be determined by steady-state kinetic methods. Aim 3 will determine the structural bases of Hoogsteen base pairing in Poll´s active site by analyzing the effects that mutational changes in the residues of the fingers and palm domains, that are seen to interact with the templating nt or with the incoming nt in Poll ternary structure, have on the efficiency and fidelity of nt incorporation, hi Aim 4, the X-ray structures of ternary complexes of Poll with undamaged templates G, T, and C, in the presence of different incoming nts will be determined, and in Aim 5, the ternary structures of Poll with DNA containing lesions at the templating site that severely impinge upon the minor groove, or which affect Watson-Crick base pairing, and others, in the presence of different incoming nts will be determined. The results will be highly relevant for cancer biology, as the manner in which DNA lesions are bypassed during replication has a major impact on genome stability; and, in fact, the inactivation of Polr in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum
Keywords: 1H-Purin-6-amine; 2-Amino-6-Hydroxypurine; 6H-Purin-6-one, 2-amino-1, 7-dihydro-; Active Sites; Adenine; Adopted; Affect; Amino Acids; Angioma Pigmentosum Atrophicum; Atrophoderma Pigmentosum; Automobile Driving; Base Pairing; Biochemical; Bypass; Cancer Biology; Cancer Cause; Cancer Etiology; Complex; DNA; DNA Damage; DNA Injury; DNA Polymerases; DNA Replicating Damage; DNA Replication; DNA Replication Damage; DNA Synthesis; DNA biosynthesis; DNA lesion; DNA polymerase iota; DNA-Dependent DNA Polymerases; DNA-Directed DNA Polymerase; Deoxynucleoside-triphosphate[{..}]DNA deoxynucleotidyltransferase (DNA-directed); Deoxyribonucleic Acid; Drivings, Automobile; EC 2.7.7.7; Fingers; Genetic Alteration; Genetic Change; Genetic defect; Genome Stability; Goals; Guanine; Human; Human, General; Hydrogen Bonding; Kaposi Dermatosis; Kaposi Disease; Kinetic; Kinetics; Lesion; Man (Taxonomy); Man, Modern; Melanosis Lenticularis Progressiva; Methods; Minor Groove; Molecular Configuration; Molecular Conformation; Molecular Stereochemistry; Mutation; NAC precursor; Nucleotides; PARK1 protein; PARK4 protein; Pigmented Epitheliomatosis; Position; Positioning Attribute; Purines; Pyrimidine Nucleotides; RAD30B; Radiation, X-Rays; Radiation, X-Rays, Gamma-Rays; Relative; Relative (related person); Roentgen Rays; Role; SNCA; SNCA protein; Site; Stability, Genomic; Structure; Syndrome; Testing; Variant; Variation; Vitamin B4; X-Radiation; X-Rays; Xeroderma Pigmentosum; Xeroderma Pigmentosum Syndrome; Xeroderma of Kaposi; Xrays; adduct; adenine analog; alpha synuclein; alphaSP22; aminoacid; base; conformation; conformational state; driving; genome mutation; human DNA; non A-beta component of AD amyloid; non A4 component of amyloid precursor; nucleotide analog; poliota; purine; social role
Project start date: 2005-09-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
5R01CA115856-06 (2010): $354888
5R01CA115856-05 (2009): $339737
5R01CA115856-04 (2008): $329839
Sponsored Links Excellgen http://Excellgen.com
5R01CA115856-03 (2007): $326573
5R01CA115856-02 (2006): $326533
1R01CA115856-01 (2005): $337402
Repair Of UV Irradiated DNA: Excision Genes Of Yeast
Satya Prakash, Professor
University Of Texas Medical Br Galveston 301 University Blvd Galveston, Tx 77555
Grant 5R37CA041261-21 from National Cancer Institute IRG: RAD
Abstract: The long term objectives are to define, in Saccharomyces cerevisiae, the roles of various nucleotide excision repair (NER) protein factors in damage recognition and in the assembly of the NER machinery at the damage site. The proposed studies will determine the role of the newly identified gene RFI1 in the repair of non-transcribed DNA and they will examine the damage binding properties of the purified Rfi1 protein. Genetic and biochemical studies will be performed to determine the role of the C4 and C3HC4 sequence motifs present in the Rfi1 protein and in the Rad16 protein. The physical and functional interaction of Rfi1 with the Rad7-Rad16 protein complex and with the other NER factors will be studied, and the role of the C3HC4 sequence present in Rfi1 and Rad16 on promoting association between these two proteins will be examined. The Abf1 protein is a component of the Rad7-Rad16 complex. The role of ABF1 in the repair of non-transcribed DNA will be examined and the damage binding properties of Abf1 protein will be studied. The Mms19 protein complex, which is comprised of four proteins of 100 (Mms19), 80, 55, and 43 kDa will be studied further. The identity of these Mmsl9 associated proteins will be determined by mass spectrometry, and genetic and biochemical studies will examine the role of these subunits and of the Mmsl9 complex in NER and in Pol II transcription. The order by which different NER factors assemble on damaged DNA will be analyzed by gel retardation and by DNase I footprinting. The role of the Rad7-Rad16 complex and of associated proteins in chromatin remodeling will be analyzed, and the effect of these proteins on the removal of UV lesions from nucleosomal DNA examined in the reconstituted system. Xeroderma pigmentosum (XP) patients are defective in NER of UV damaged DNA, and as a consequence, they suffer from a high incidence of skin cancers. Our studies in yeast should continue to provide important insights on the role of various protein factors in damage recognition and other steps of the NER process in eukaryotes, including in humans.
Keywords: DNA binding protein, DNA damage, DNA repair, fungal protein, ultraviolet radiation, fungal genetics, molecular genetics, radiation genetics, DNA footprinting, Saccharomyces cerevisiae, gel mobility shift assay, laboratory rabbit, mass spectrometry
Project start date: 1985-12-01
Project end date: 2006-12-31
5R37CA041261-21 (2006): $363746
5R37CA041261-20 (2005): $372500
5R37CA041261-19 (2004): $372500
5R37CA041261-18 (2003): $372500
EXCISION REPAIR OF UV IRRADIATED DNA IN YEAST
Satya Prakash, Professor
University Of Texas Medical Br Galveston 301 University Blvd Galveston, Tx 77555
Grant 5R37CA035035-19 from National Cancer Institute IRG: NSS
Abstract: The long term objectives are to define the different biological functions of genes required for excision repair of ultraviolet (UV) damaged DNA in the eukaryote, Saccharomyces cerevisiae, determine the biochemical activities of their protein products, and reconstitute the damage specific incision activity. This proposal focuses on five excision repair genes RAD1, RAD2, RAD3, RAD4, and RAD10, and their encoded proteins. Studies will be undertaken to define the role of RAD3 in excision repair and viability. The effect of superhelicity, ATP binding vs. its hydrolysis, and other factors on specific binding of RAD3 protein to UV damaged DNA will be determined, and the role of RAD3 in stimulating strand displacement synthesis by yeast DNA polymerase examined. The possible involvement of RAD3 in coupling excision repair and transcription will be investigated by examining whether the rad3 Arg48 mutation defective in helicase activities, and cs rad3 mutations (identical to mutations in the human XPDC gene, the RAD3 homolog, that cause Cockayne syndrome, CS), are defective in preferential repair of the transcribed DNA strand. The rad3 ts14 mutation that stops growth rapidly at 37oC will be further analyzed for its effects on transcription and DNA replication. The possibility that the severe transcriptional defect observed in the rad3 ts14 mutant at 37oC arises from a defect in RNA chain elongation or in promoter specific transcription initiation by the three RNA polymerases will be tested. The interaction of RAD3 with RNA polymerase II and the TATA binding factor will be examined by co- immunoprecipitation and protein-affinity chromatography. Genes encoding proteins that interact with RAD3 will be identified using the genetic strategy of the two hybrid system. Interactions among purified RAD proteins will be examined by hydrodynamic studies, protein affinity chromatography, and other methods. The stoichiometry and other biophysical properties of the RAD1/RAD10 complex will be determined. The role of the leucine zipper motif in RAD1, and of two contiguous helix motifs in RAD10, in complex formation between these two proteins will be ascertained. Purified RAD1 protein will be examined for its biochemical activities alone and in combination with purified RAD10 protein. The RAD2 and RAD4 proteins will be purified and their biochemical activities characterized. Biochemical activities of combinations of RAD proteins will be determined and the enzyme activity for incision of UV damaged DNA reconstituted in vitro. Xeroderma pigmentosum (XP) patients are defective in excision repair of UV damaged DNA, and as a consequence, they suffer from a high incidence of skin cancers. Because remarkable evolutionary conservation exists among the excision repair genes in eukaryotes from yeast to human, information from the proposed studies with the yeast genes and proteins should serve as a useful model for delineating the biological roles of human excision repair genes, in characterizing the biochemical activities of their protein products, and in defining the incision mechanism in humans.
Keywords: DNA repair, genetic mapping, microorganism genetics, radiation genetics, ultraviolet radiation, xeroderma pigmentosum, DNA directed DNA polymerase, cell transformation, fungal genetics, gene mutation, genetic manipulation, genetic transcription, nucleic acid sequence, protein biosynthesis, transcription factor, Saccharomyces, disease /disorder model, radioimmunoassay, tissue /cell culture
Project start date: 1983-07-01
Project end date: 2003-04-30
5R37CA035035-19 (2001): $346139
5R37CA035035-18 (2000): $336057
5R37CA035035-17 (1999): $326268
Sponsored Links Excellgen http://Excellgen.com
5R37CA035035-15 (1997): $275448
5R37CA035035-13 (1995): $254586
5R37CA035035-12 (1994): $239175
2R37CA035035-11 (1993): $196304
5R01CA035035-10 (1992): $265344
DNA MISMATCH REPAIR IN EUKARYOTES
Satya Prakash, Professor
Internal Medicineuniversity Of Texas Medical Br Galveston
301 University Blvd
galveston, Tx 77555
Grant 5R01CA080882-03 from National Cancer Institute IRG: BIO
Abstract: The long term goals are to define the process of mismatch repair in eukaryotes. Because of the high degree of conservation of mismatch repair genes and proteins between yeast and humans, we will use the yeast Saccharomyces cerevisae as a model for these studies. We will examine the roles of the Msh2-Msh3, Msh2-Msh6, and Mlh1-Pms1 complexes in mismatch recognition and in subsequent steps of mismatch repair and will determine at which step(s) the ATP binding/hydrolysis activity of Msh2-Msh3 and Msh2-Msh6 is utilized in mismatch repair. Mismatch binding by the Msh2-Msh3 and Msh2-Msh6 complexes will be examined by Dnase I and hydroxyl radical footprinting, and the effect of Mlh1-Pms1 on mismatch binding by Msh complexes determined. Genetic and biochemical studies of mutant msh2, msh3, and msh6 genes and proteins harboring a lysine to alanine alteration in the GKS nucleotide binding motif will examine the role of ATP binding/hydrolysis by the Msh protein complexes in mismatch recognition, in ternary complex formation with Mlh1-Pms1 and mismatched DNA, and in the looping of mismatched DNA. The other components of mismatch repair, including the 5´ yields 3´ and 3´ yields 5´ exonucleases and DNA helicases, will be identified by genetic and biochemical studies; their biochemical activities will be further defined, and their manner of interaction with the other mismatch repair proteins determined. Mismatch repair will be examined in vitro in yeast extracts, and the involvement of different nucleases, helicases, and of other enzymes in mismatch repair will be determined. Mismatch repair will be reconstituted with the highly purified yeast proteins, setting the stage for in-depth analyses of the intricacies of this repair process. Mismatch repair maintains the integrity of genomic DNA by correcting replication errors. Defects in mismatch repair lead to enhanced mutability and microsatellite instability, and mutations in human mismatch repair genes result in hereditary non-polyposis colorectal cancer (HNPCC) and other types of cancers. Because of the remarkable conservation of the mismatch repair machinery between yeast and humans, these studies should provide important and novel insights into the mechanisms of this repair process in humans
Keywords: DNA binding protein, DNA repair, Saccharomyces cerevisiae, adenosine triphosphate, eukaryote, hydrolysis, protein binding DNA replication, adenosinetriphosphatase, biological model, dimer, enzyme activity, enzyme mechanism, exonuclease, fungal genetics, helicase, nucleic acid sequence, nucleic acid structure, phosphoester ligase DNA footprinting, fungal protein, protein purification
Project start date: 1999-04-01
Project end date: 2004-01-31
5R01CA080882-03 (2001): $282896
5R01CA080882-02 (2000): $276280
1R01CA080882-01 (1999): $232842
5R01CA080882-05 (2003): $296729
REPAIR OF UV IRRADIATED DNA EXCISION GENES OF YEAST
Satya Prakash, Professor
University Of Texas Medical Br Galveston 301 University Blvd Galveston, Tx 77555
Grant 5R01CA041261-11 from National Cancer Institute IRG: RAD
Abstract: Our long term goals are to elucidate the genetic and molecular mechanisms of excision repair of DNA damaged by ultraviolet (UV) light irradiation in the yeast Saccharomyces cerevisiae. The overall objective of this proposal is to study the structure, regulation, and function of the RAD7, RAD14, RAD16, RAD23, and MMS19 genes involved in excision of pyrimidine dimers. The RAD14, RAD16, and MMS19 genes will be isolated by complementation of UV or MMS sensitivity of the corresponding mutants. The complementing DNA fragments will be subcloned and the identity of the cloned gene confirmed by mapping it to its location in the yeast genome. The cloned genes will be used for making deletions of the corresponding genes in the yeast genome, and the effects of deletions studies. The size and direction of the RAD14, RAD16, and MMS19 transcripts will be determined by using M13mp18 and M13mp19 phages containing an internal fragment of the RAD or MMS gene. Radioactively labeled single-stranded DNA probes from these recombinant phages will be used in hybridizations to yeast RNA. The nucleotide sequence of the RAD14, RAD16, RAD23, and MMS19 genes will be determined by the Sanger dideoxy method. The 5 and 3 mRNA termini of the RAD7, RAD14, RAD16, RAD23, and MMS19 genes will be located by S1 nuclease mapping. To determine if the RAD7, RAD14, RAD16, RAD23, and MMS19 genes show regulated expression, transcript levels, and Beta-galactosidase levels in yeast strains containing in-frame fusions of these genes with the E. coli lacZ gene, will be examined following UV irradiation. RAD-lacZ fusions will be used for determining the intracellular location of the hybrid proteins by cell fractionation and immunofluorescence. The amino acid sequence determinants in these RAD and MMS proteins affecting their nuclear localization will be identified. The RAD7, RAD14, RAD16, RAD23, and MMS19 genes will be fused to a strong yeast promoter in a multicopy plasmid for overproducing their proteins, and the RAD or MMS gene encoded proteins purified from yeast strains containing these multicopy plasmids. The purified proteins will be examined for DNA binding, DNA unwinding, ATPase, UV endonuclease, exonuclease and nucleosome binding. Human xeroderma pigmentosum patients are defective in excision of UV induced pyrimidine dimers from DNA and show an increased frequency of cancers. The proposed studies should provide a model system for understanding the complex mechanisms of excision repair in eukaryotes, including humans.
Keywords: DNA repair, DNA replication, fungal genetics, genetic manipulation, genetic mapping, radiation genetics, radiation sensitivity, ultraviolet radiation, DNA binding protein, DNA topoisomerase, binding protein, gene deletion mutation, gene expression, gene mutation, genetic transcription, ionizing radiation, meiosis, methionine, nucleic acid sequence, protein biosynthesis, protein purification, protein sequence, ubiquitin, Escherichia coli, Saccharomyces cerevisiae, disease model, gel electrophoresis, molecular cloning, plasmid, xeroderma pigmentosum
Project start date: 1985-12-01
Project end date: 1996-03-31
5R01CA041261-11 (1995): $223967
Sponsored Links Excellgen http://Excellgen.com
5R01CA041261-10 (1994): $201418
7R01CA041261-09 (1993): $177905
REPAIR OF UV IRRADIATED DNA--EXCISION GENES OF YEAST
Satya Prakash, Professor
University Of Rochester 517 Hylan Bldg., Box 270140 Rochester, Ny 14627
Grant 5R01CA041261-07 from National Cancer Institute IRG: RAD
Abstract: Our long term goals are to elucidate the genetic and molecular mechanisms of excision repair of DNA damaged by ultraviolet (UV) light irradiation in the yeast Saccharomyces cerevisiae. The overall objective of this proposal is to study the structure, regulation, and function of the RAD7, RAD14, RAD16, RAD23, and MMS19 genes involved in excision of pyrimidine dimers. The RAD14, RAD16, and MMS19 genes will be isolated by complementation of UV or MMS sensitivity of the corresponding mutants. The complementing DNA fragments will be subcloned and the identity of the cloned gene confirmed by mapping it to its location in the yeast genome. The cloned genes will be used for making deletions of the corresponding genes in the yeast genome, and the effects of deletions studies. The size and direction of the RAD14, RAD16, and MMS19 transcripts will be determined by using M13mp18 and M13mp19 phages containing an internal fragment of the RAD or MMS gene. Radioactively labeled single-stranded DNA probes from these recombinant phages will be used in hybridizations to yeast RNA. The nucleotide sequence of the RAD14, RAD16, RAD23, and MMS19 genes will be determined by the Sanger dideoxy method. The 5 and 3 mRNA termini of the RAD7, RAD14, RAD16, RAD23, and MMS19 genes will be located by S1 nuclease mapping. To determine if the RAD7, RAD14, RAD16, RAD23, and MMS19 genes show regulated expression, transcript levels, and Beta-galactosidase levels in yeast strains containing in-frame fusions of these genes with the E. coli lacZ gene, will be examined following UV irradiation. RAD-lacZ fusions will be used for determining the intracellular location of the hybrid proteins by cell fractionation and immunofluorescence. The amino acid sequence determinants in these RAD and MMS proteins affecting their nuclear localization will be identified. The RAD7, RAD14, RAD16, RAD23, and MMS19 genes will be fused to a strong yeast promoter in a multicopy plasmid for overproducing their proteins, and the RAD or MMS gene encoded proteins purified from yeast strains containing these multicopy plasmids. The purified proteins will be examined for DNA binding, DNA unwinding, ATPase, UV endonuclease, exonuclease and nucleosome binding. Human xeroderma pigmentosum patients are defective in excision of UV induced pyrimidine dimers from DNA and show an increased frequency of cancers. The proposed studies should provide a model system for understanding the complex mechanisms of excision repair in eukaryotes, including humans.
Keywords: DNA repair, DNA replication, fungal genetics, genetic manipulation, genetic mapping, radiation genetics, radiation sensitivity, ultraviolet radiation, DNA binding protein, DNA topoisomerase, binding protein, gene deletion mutation, gene expression, gene mutation, genetic transcription, ionizing radiation, meiosis, methionine, nucleic acid sequence, protein biosynthesis, protein purification, protein sequence, ubiquitin, Escherichia coli, Saccharomyces cerevisiae, disease model, gel electrophoresis, molecular cloning, plasmid, xeroderma pigmentosum
Project start date: 1985-12-01
Project end date: 1996-03-31
5R01CA041261-07 (1992): $208397
REPAIR OF UV IRRADIATED DNA: EXCISION GENES OF YEAST
Satya Prakash, Professor
University Of Rochester 517 Hylan Bldg., Box 270140 Rochester, Ny 14627
Grant 5R01CA041261-02 from National Cancer Institute IRG: RAD
Abstract: Our long term goals are to elucidate the genetic and molecular mechanisms of excision repair of DNA damaged by ultraviolet (UV) light irradiation in the yeast Saccharomyces cerevisiae. The overall objective of this proposal is to study the structure, regulation, and function of the RAD7, RAD14, RAD16, RAD23, and MMS19 genes involved in excision of pyrimidine dimers. The RAD14, RAD16, and MMS19 genes will be isolated by complementation of UV or MMS sensitivity of the corresponding mutants. The complementing DNA fragments will be subcloned and the identity of the cloned gene confirmed by mapping it to its location in the yeast genome. The cloned genes will be used for making deletions of the corresponding genes in the yeast genome, and the effects of deletions studies. The size and direction of the RAD14, RAD16, and MMS19 transcripts will be determined by using M13mp18 and M13mp19 phages containing an internal fragment of the RAD or MMS gene. Radioactively labeled single-stranded DNA probes from these recombinant phages will be used in hybridizations to yeast RNA. The nucleotide sequence of the RAD14, RAD16, RAD23, and MMS19 genes will be determined by the Sanger dideoxy method. The 5 and 3 mRNA termini of the RAD7, RAD14, RAD16, RAD23, and MMS19 genes will be located by S1 nuclease mapping. To determine if the RAD7, RAD14, RAD16, RAD23, and MMS19 genes show regulated expression, transcript levels, and Beta-galactosidase levels in yeast strains containing in-frame fusions of these genes with the E. coli lacZ gene, will be examined following UV irradiation. RAD-lacZ fusions will be used for determining the intracellular location of the hybrid proteins by cell fractionation and immunofluorescence. The amino acid sequence determinants in these RAD and MMS proteins affecting their nuclear localization will be identified. The RAD7, RAD14, RAD16, RAD23, and MMS19 genes will be fused to a strong yeast promoter in a multicopy plasmid for overproducing their proteins, and the RAD or MMS gene encoded proteins purified from yeast strains containing these multicopy plasmids. The purified proteins will be examined for DNA binding, DNA unwinding, ATPase, UV endonuclease, exonuclease and nucleosome binding. Human xeroderma pigmentosum patients are defective in excision of UV induced pyrimidine dimers from DNA and show an increased frequency of cancers. The proposed studies should provide a model system for understanding the complex mechanisms of excision repair in eukaryotes, including humans.
Keywords: GENETICS, GENETIC REGULATION, GENETIC INDUCTION-REPRESSION-DEREPRESSION, TRANSCRIPTION, GENE SPLICING, GENETICS, MICROBIAL, FUNGAL, NUCLEIC ACIDS REPAIR, NUCLEIC ACIDS, COMPLEMENTARY DNA, RADIATION STUDY SECTION, genetic mapping, radiation sensitivity, CARBOHYDRASES, BETA-GALACTOSIDASE, GENETICS, BIOCHEMICAL GENETICS, GENETIC CODING, GENETICS, BIOCHEMICAL GENETICS, MOLECULAR CLONING, GENETICS, GENETIC REGULATION, GENETIC INDUCTION-REPRESSION-DEREPRESSION, TRANSCRIPTION, GENETICS, RADIATION GENETICS, IMMUNOLOGY, ANTIBODY SPECIFICITY, PROTEINS-PEPTIDES STRUCTURE, AMINO ACIDS SEQUENCE, RADIATION, ELECTROMAGNETIC WAVES, ULTRAVIOLET RAYS (290NM TO 380NM), RADIATION, IONIZING RADIATION, genetic manipulation, BACTERIA, ENTEROBACTERIACEAE, ESCHERICHIA COLI, FUNGI, YEASTS, SACCHAROMYCES
Project start date: 1985-12-01
Project end date: 1990-11-30
REPAIR OF UV IRRADIATED DNA--EXCISION GENES OF YEAST
Satya Prakash, Professor
University Of Texas Medical Br Galveston 301 University Blvd Galveston, Tx 77555
Grant 5R01CA041261-16 from National Cancer Institute IRG: RAD
Abstract: adapted from the investigator s ) The long term objectives are to define the roles of different protein assemblies in nucleotide excision repair (NER) in the eukaryote, Saccharomyces cerevisiae, to determine the sequence of events that underlie dual incision of UV damaged DNA, and to examine the role of protein phosphorylation in the modulation of activities of NER proteins and of subunits of Pol II transcriptional factor IIH (TFIIH). The proposed studies will identify the protein factors that modulate the proficiency of the reconstituted incision reaction, and will examine the biochemical properties of NER proteins individually and in protein assemblies. The Rad7 and Rad16 proteins will be purified and biochemical activities of Rad7, Rad16, and of Rad7-Rad16 complex defined. Interaction domains in Rad7 and Rad16 proteins will be identified and the role of ATP in Rad7- Rad16 complex formation examined. Whether Rad7, Rad16, and Rad7-Rad 16 proteins have affinity for UV damaged DNA will be determined. The role of PHR1-encoded photolyase in NER will be studied by genetic and biochemical means. Whether addition of the PHR1, Rad 7, and Rad16 proteins to the reconstituted system increases the efficiency of dual incision of a linear DNA fragment containing a site specific cyclobutane pyrimidine dimer (CPD) will be examined. The patterns of interactions among proteins in the Rad14-Rad1-Rad10 complex will be analyzed and DNA damage binding affinity of the complex determined. The involvement of Rad23 ubiquitin domain in Rad4-Rad23 complex formation will be examined, and the role of Rad4-Rad23 complex in assembling the NER machinery studied. The hierarchy by which different NER factors are assembled onto the DNA damage site will be analyzed by native gel electrophoresis DNA binding assay and by footprinting studies. The MMS19-associated protein complex will be purified, genes encoding MMS19-associated proteins will be cloned, and their role in NER and Pol II transcription determined. The role of MMS19 associated kinase in phosphorylation of NER proteins and of TFIIH components will be examined. Xeroderma pigmentosum (XP) patients are defective in NER of UV damaged DNA, and as a consequence, they suffer from a high incidence of skin cancers. Because of the remarkable evolutionary conservation of the NER machinery between yeast and humans, these studies should continue to provide insights into the mechanisms of this complex process in humans, and they are expected to reveal the roles of human XP genes in processes other than NER.
Keywords: DNA repair, fungal genetics, genetic mapping, radiation genetics, radiation sensitivity, ultraviolet radiation, DNA binding protein, gene expression, gene mutation, phosphorylation, protein biosynthesis, protein sequence, transcription factor, ubiquitin, DNA footprinting, Saccharomyces cerevisiae, gel electrophoresis, molecular cloning
Project start date: 1985-12-01
Project end date: 2001-12-31
5R01CA041261-16 (2000): $352448
5R01CA041261-15 (1999): $340674
5R01CA041261-14 (1998): $329351
Sponsored Links Excellgen http://Excellgen.com
5R01CA041261-13 (1997): $318465
Satya Prakash
University Of Texas Medical Br Galveston
Project start date: 2008-04-01
Project end date: 2013-01-31
ROLE OF REV1 IN ERROR-FREE REPLICATION OF DNA DAMAGE AND IN MUTATION PREVENTION
Satya Prakash, Professor
University Of Texas Medical Br Galveston, 301 University Blvd, Galveston, Tx 77555
Grant 5R01ES016666-03 from National Institute Of Environmental Health Sciences
Keywords: 2-Amino-6-Hydroxypurine; 2-Propenal; 3, 4-Benzopyrene; 3, 4-Benzpyrene; 6H-Purin-6-one, 2-amino-1, 7-dihydro-; Acraldehyde; Acrolein; Acrylaldehyde; Acrylic Aldehyde; Affect; Aldehydes; Allyl Aldehyde; Amino Acids; Apoenzymes; Arginine; Arginine, L-Isomer; Benzo(a)pyrene; Binding; Binding (Molecular Function); Biochemical; Biochemical Genetics; Butadiene; Bypass; Cancer Biology; Cancer Induction; Cancers; Carcinogen-DNA Adducts; Carcinogens, Environmental; Causality; Cells; Chemicals; Complement; Complement Proteins; Complex; DNA; DNA Adducts; DNA Binding; DNA Binding Interaction; DNA Damage; DNA Injury; DNA Polymerases; DNA Replicating Damage; DNA Replication; DNA Replication Damage; DNA Synthesis; DNA biosynthesis; DNA lesion; DNA repair protein; DNA-Dependent DNA Polymerases; DNA-Directed DNA Polymerase; Deoxynucleoside-triphosphate[{..}]DNA deoxynucleotidyltransferase (DNA-directed); Deoxyribonucleic Acid; EC 2.7.7.7; Environmental Carcinogens; Environmental Pollutants; Epoxides; Epoxy Compounds; Ethylene Aldehyde; Etiology; Eukaryota; Eukaryote; Exposure to; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Genetic, Biochemical; Genome Stability; Genomics; Glycols; Goals; Guanine; Human; Human, General; Hydrogen Bonding; Incidence; L-Arginine; Lesion; Lipid Peroxidation; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Minor Groove; Molecular Interaction; Mutate; Mutation; Nucleotides; Plasmids; Polymerase; Prevention; Proteins; Reaction; Role; Site; Specificity; Stability, Genomic; Structure; Testing; Yeasts; acryaldehyde; adduct; aminoacid; base; cancer prevention; carcinogenesis; disease causation; disease etiology; disease/disorder etiology; disorder etiology; eukaryotida; gene product; genome mutation; in vivo; insight; malignancy; neoplasm/cancer; nucleotide analog; oxidative damage; social role; synthetic DNA; synthetic construct
Project start date: 2008-04-01
Project end date: 2013-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
5R01ES016666-03 (2010): $436742
REPAIR OF UV IRRADIATED DNA: EXCISION GENES OF YEAST
Satya Prakash, Professor
University Of Texas Medical Br Galveston, 301 University Blvd, Galveston, Tx 77555
Grant 5R37CA041261-25 from National Cancer Institute
Keywords: 20S Catalytic Proteasome; 20S Core Proteasome; 20S Proteasome; 20S Proteosome; Abscission; Angioma Pigmentosum Atrophicum; Atrophoderma Pigmentosum; Biochemical; Cancer Cause; Cancer Etiology; Cells; Cockayne Syndrome; Complex; DNA; DNA Damage; DNA Injury; DNA lesion; DNA-Dependent RNA Polymerase II; Defect; Deoxyribonucleic Acid; E3 Ligase; E3 Ubiquitin Ligase; Enzymes; Event; Excision; Extirpation; Gene Transcription; Generalized Growth; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic analyses; Genetic defect; Growth; Histones; Human; Human, General; Kaposi Dermatosis; Kaposi Disease; Macropain; Macroxyproteinase; Man (Taxonomy); Man, Modern; Melanosis Lenticularis Progressiva; Monoubiquitination; Multicatalytic Proteinase; Mutation; Neurologic; Neurological; Nucleotide Excision Repair; Patients; Physiologic; Physiological; Pigmented Epitheliomatosis; Polymerase; Progeria-Like Syndrome; Prosome; Proteasome; Proteasome Endopeptidase Complex; Proteins; Proteosome; RNA Expression; RNA Polymerase B; RNA Polymerase II; Reaction; Removal; Role; S cerevisiae; Saccharomyces cerevisiae; Site; Surgical Removal; Syndrome; System; System, LOINC Axis 4; Tissue Growth; Transcription; Transcription, Genetic; UV damaged DNA; UV induced DNA damage; UV irradiated DNA; UV lesions; Ubiquitilation; Ubiquitin-Conjugating Enzyme E2; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligase E3; Ubiquitination; Ubiquitinoylation; Xeroderma Pigmentosum; Xeroderma Pigmentosum Syndrome; Xeroderma of Kaposi; Yeast, Baker`s; Yeast, Brewer`s; Yeasts; base; chromatin remodeling; experiment; experimental research; experimental study; gene product; genetic analysis; genome mutation; multicatalytic endopeptidase complex; ontogeny; protein complex; reconstitute; reconstitution; repair; repaired; research study; resection; response; social role; ubiquination; ubiquitin conjugation; ubiquitin ligase; ubiquitin-protein ligase; ultraviolet lesions
Project start date: 1985-12-01
Project end date: 2011-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2010
5R37CA041261-25 (2010): $364577
Role Of Rev1 In Error-free Replication Of DNA Damage And In Mutation Prevention
Satya Prakash, Professor
Biochemistry And Molecular Biologyuniversity Of Texas Medical Br Galveston
Grant 5R01ES016666-02 from National Institute Of Environmental Health Sciences IRG: CE
Project start date: 2008-04-01
Project end date: 2013-01-31
Repair Of UV Irradiated DNA: Excision Genes Of Yeast
Satya Prakash, Professor
University Of Texas Medical Br Galveston 301 University Blvd Galveston, Tx 77555
Grant 4R37CA041261-22 from National Cancer Institute IRG: NSS
Project start date: 1985-12-01
Project end date: 2011-12-31
4R37CA041261-22 (2007): $383374
REPAIR OF UV IRRADIATED DNA--EXCISION GENES OF YEAST
Satya Prakash, Professor
University Of Texas Medical Br Galveston
301 University Blvd
galveston, Tx 77555
Grant 2R01CA041261-12 from National Cancer Institute IRG: RAD
Project start date: 1985-12-01
Project end date: 2001-01-31
2R01CA041261-12 (1996): $307998
EXCISION REPAIR OF UV IRRADIATED DNA IN YEAST
Satya Prakash, Professor
Biochemistry And Molecular Biologyuniversity Of Texas Medical Br Galveston
301 University Blvd
galveston, Tx 77555
Grant 5R37CA035035-14 from National Cancer Institute IRG: RAD
Project start date: 1983-07-01
Project end date: 1998-04-30
5R37CA035035-14 (1996): $264853
REPAIR OF UV IRRADIATED DNA: EXCISION GENES OF YEAST
Satya Prakash, Professor
University Of Rochester
517 Hylan Bldg., Box 270140
rochester, Ny 14627
Grant 5R01CA041261-08 from National Cancer Institute IRG: RAD
Project start date: 1985-12-01
Project end date: 1993-06-30
5R01CA041261-08 (1993): $38827
Sponsored Links Excellgen http://Excellgen.com