1985 — 1990 |
Hanawalt, Philip C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Replication and Repair of Genetic Material
Differences in the processing of DNA damage in selected genomic domains may account for some of the profound differences seen in the carcinogenic response in different tissues or when different organisms are compared. Our long-term objective is to understand the "fine structure" of DNA replication and repair by using defined nucleotide sequences containing well-characterized DNA lesions. The T4 bacteriophage endonuclease V is used in a recently developed sensitive assay for ultraviolet (UV) induced pyrimidine dimers in restriction fragments of genes and other domains in the genomes of cultured rodent and human cells. Efforts focus upon studies to learn the rules governing accessibility of pyrimidine dimers, bulky adducts and interstrand crosslinks to repair. Repair will be assessed in the region surrounding the proficiently-repaired dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells to determine boundaries of one of the repairable domains in these generally repair deficient cells. Comparative analyses will be carried out in UV-sensitive CHO mutant cells expressing the cloned denV gene (ie.e., T4 endo V). Methylation levels in genomic regions will be assessed as possible signals for regulation of intragenomic heterogeneity in damage processing. The E. coli UvrABC complex will be used to analyze genomic distribution of bulky adducts produced by N-acetoxyacetylaminofluorene and the psoralens. A sensitive assay based upon renaturability of DNA will be used to monitor introduction and repair of psoralen interstrand cross-links in defined genomic fragments. Repair will be compared in genes that differ in their expression level and function in the cell type under examination. Repair and mutagenesis will be correlated in the same genes to determine whether differential repair accounts for rapid genomic evoluation in rodents. Replication of defined nucleotide sequences in the region around the DHFR gene will be studied to determine whether differential replication occurs in particular damaged genomic domains and whether daughter-strand discontinuities occur in those sequences. The studies should help to interpret the effects of DNA damage upon biological end points such as survival, mutagenesis, and transformation.
|
1 |
1985 — 1987 |
Hanawalt, Philip C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Basis of Dna Repair Deficiency in Xeroderma Pi
The long-term objective of this project is to understand the molecular basis of DNA repair deficiency in xeroderma pigmentosum (XP). The overall experimental strategy is to simplify the analysis by using agents such as UV that produce only a few types of lesions, using specific assays for each of these lesions, and using defined DNA sequences as substrates for repair so that the processing of damage can be analyzed at the molecular level. The following three experimental approaches and specific aims are planned: (1) We have discovered that XP cells from complementation group C exhibit an intracellular heterogeneity in the repair of DNA containing pyrimidine dimers. Some regions of the genome are repaired normally while others are relatively inaccessible to repair. We plan to analyze this response to determine what distinguishes repairable from non-repairable regions, using the methodology we have developed to isolate the repaired "domains." We will determine whether such heterogeneity is also a characteristic of other XP complementation groups. (2) Having obtained the first monoclonal antibody specific for thymine glycol in DNA we will use it to study the repair and relative biological significance of thymine glycol in normal and XP cells. We plan to prepare additional monoclonal antibodies and to use them as highly sensitive, specific assays for pyrimidine dimers and other well-defined photoproducts. Results obtained from immunological assays will be compared to those obtained using specific endonucleases to detect lesions and their distribution in the genome. (3) We will use defined chimeric plasmids to probe specific features of DNA repair in XP cells. We have found that 254 nm UV doses below 200 J/m squared enhance the transformation efficiency of pSV2-gpt while doses above 1000 J/m squared cause a decrease which is more pronounced in XP-A than in normal cells. We will determine the nature of the inactivating lesions and the cellular events that underly reactivation in normal cells. This research should contribute substantially to our understanding of the basis for DNA repair deficiency in XP and will also result in availability of new, sensitive probes for the analysis of damage and repair in human cells.
|
1 |
1987 — 1993 |
Hanawalt, Philip C |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Cellular Processing of Damaged Dna: Role in Oncogenesis
Our ultimate objective is to understand how the processing of damaged DNA in mammalian cells relates to carcinogenesis. Using relatively short, defined DNA sequences containing well characterized lesions, we have begun to analyze the intragenomic "fine structure" of DNA repair in cultured cells. Having discovered that certain regions of the nuclear genome repaired more efficiently than others, we hypothesize that the efficiency of repair of damage in mammalian chromatin depends upon the type of lesion, its location in the genome and the functional state of the DNA at the site of the lesion. Such specificity may account for some of the profound differences soon in the carcinogenic responses of different tissues and of the same tissue in different organisms. Having developed assays sensitive enough to detect repair of several different lesions, including pyrimidine dimers and interstrand cross-links, in restriction fragments from specific regions of the genome, we will compare the rate and extent of repair in genes that differ levels of expression, time of replication, genomic location and function. Examples include protooncogenes and other inducible or developmentally activated genes such as those for metallothioneins, alpha fetoprotein, fetal and adult beta-globin and myosin heavy chain in differentiating myoblasts. Chromatin conformation and methylation levels will be assessed as possible determinants of proficient repair. Repair and mutagenesis will be correlated in the same genes to determine whether differential repair might account for mutagenic changes related to carcinogenesis. Replication of defined nucleotide sequences containing damage will be studied to determine whether differential levels of replication occur in particular genomic domains and whether daughter-strand discontinuities occur in those sequences. Defined chimeric plasmids containing lesions at unique sites will be used to introduce genes into different genomic domains and to probe the specific features of damage processing the increase the frequency of stable transformation of human cells. This research should contribute substantially to our understanding of the basis for DNA damage processing deficiencies in certain cancer-prone hereditary diseases and it should also result in new, sensitive probes for the analysis of damage and repair in human cells. In addition, our studies should help to interpret the role of DNA damage in biologic end points such as survival, mutagenesis, and carcinogenesis.
|
1 |
1988 — 1991 |
Jones, Patricia Yanofsky, Charles (co-PI) [⬀] Hanawalt, Philip Simoni, Robert [⬀] Campbell, Allan (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Biological Facilities Center
This award will fund the purchase of varied items of equipment to be housed in a central shared instrumentation facility for members of the Department of Biology at Stanford University. The items include a scintillation counter, ultracentrifuges and rotors, an ELISA plate reader and a protein sequencer. These items are of general use; all 20 members of the department will share in their use. Recently, progress in many, if not all, areas of biological research has depended on instruments and techniques originally developed for molecular biology and immunology. These equipment items will augment the supply at Stanford of instruments needed for a number of such techniques and thus facillitate research by members of a department which is one of the best in the country.
|
0.915 |
1989 — 1990 |
Hanawalt, Philip C |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Celular Processing of Damaged Dna--Role in Oncogenesis
Our ultimate objective is to understand how the processing of damaged DNA in mammalian cells relates to carcinogenesis. Using relatively short, defined DNA sequences containing well characterized lesions, we have begun to analyze the intragenomic "fine structure" of DNA repair in cultured cells. Having discovered that certain regions of the nuclear genome repaired more efficiently than others, we hypothesize that the efficiency of repair of damage in mammalian chromatin depends upon the type of lesion, its location in the genome and the functional state of the DNA at the site of the lesion. Such specificity may account for some of the profound differences soon in the carcinogenic responses of different tissues and of the same tissue in different organisms. Having developed assays sensitive enough to detect repair of several different lesions, including pyrimidine dimers and interstrand cross-links, in restriction fragments from specific regions of the genome, we will compare the rate and extent of repair in genes that differ levels of expression, time of replication, genomic location and function. Examples include protooncogenes and other inducible or developmentally activated genes such as those for metallothioneins, alpha fetoprotein, fetal and adult beta-globin and myosin heavy chain in differentiating myoblasts. Chromatin conformation and methylation levels will be assessed as possible determinants of proficient repair. Repair and mutagenesis will be correlated in the same genes to determine whether differential repair might account for mutagenic changes related to carcinogenesis. Replication of defined nucleotide sequences containing damage will be studied to determine whether differential levels of replication occur in particular genomic domains and whether daughter-strand discontinuities occur in those sequences. Defined chimeric plasmids containing lesions at unique sites will be used to introduce genes into different genomic domains and to probe the specific features of damage processing the increase the frequency of stable transformation of human cells. This research should contribute substantially to our understanding of the basis for DNA damage processing deficiencies in certain cancer-prone hereditary diseases and it should also result in new, sensitive probes for the analysis of damage and repair in human cells. In addition, our studies should help to interpret the role of DNA damage in biologic end points such as survival, mutagenesis, and carcinogenesis.
|
1 |
1992 — 2000 |
Hanawalt, Philip C |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Cellular Processing of Damaged Dna--Role in Oncogenesis
This project is concerned with the molecular epidemiology of cancer, beginning with the realization that as many as half a dozen independent genetic and epigenetic events may be involved in the transformation from a normal cell to malignancy. The changes in genomic DNA occur at specific sites and can lead to activation of protooncogenes or inactivation of tumor suppressor genes through mutation, recombination, gene amplification, translocation, or other chromosomal abnormalities. In some human hereditary diseases an increased incidence of neoplasia is correlated with a defect in the repair and/or replication of damaged DNA. Our ultimate objective is to understand how the processing of damaged DNA in mammalian cells relates to carcinogenesis. Having pioneered in the development of sensitive techniques for quantifying particular DNA lesions in restriction fragments from specific regions of the genome we will extend our analysis of intragenomic fine structure of DNA repair, to learn the factors that control the efficiency of the process in chromatin and in different functional domains of the genome, such as replication origins and expressed genes transcribed by different RNA polymerases. Having discovered preferential repair of the transcribed DNA strand in expressed genes, we will test a model for transcription-coupled repair based upon factors that enhance transcript shortening by the 5'- 3' exonuclease activity of RNA polymerase II. We will critically test the possibility that strand-specific DNA repair can be used as a sensitive assay for transcription. Nuclear matrix associated DNA will be characterized to determine whether that is the site of transcription-coupled repair. Domain limited repair in xeroderma pigmentosum, complementation group C, will be assessed to learn the basis for the cancer prone phenotype, and the deficiency in repair of expressed genes in Cockayne's syndrome will be studied to understand the basis for the defect and the absence of cancer proneness. Differences in the repair of particular genes at risk may account for some of the profound differences seen in the carcinogenic responses of different tissues and of the same tissue in different organisms. Plasmid probes carrying lesions at defined sites will be used in the analysis of specific sequence repair in cells of different genetic background. The defined chimeric plasmids will also be used to introduce genes into different genomic domains to study the specific features of damage processing that result in the enhanced integration of damaged DNA in human cells. We will also explore the possible role of localized DNA turnover in non-proliferating cells in the fine structure of mutagenesis to test our hypothesis that transcription- associated DNA turnover may result in anomalous high mutation frequencies in some domains. This research should contribute substantially to our understanding of the basis for DNA damage processing deficiencies in certain cancer-prone hereditary diseases and it should also result in new, sensitive probes for the analysis of damage and repair in human cells. In addition, our studies should help to interpret the role of DNA damage and DNA turnover in biological end points such as survival, mutagenesis, and carcinogenesis.
|
1 |
1992 |
Hanawalt, Philip C |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Mtg On Cellular Responses to Environmental Dna Damage @ American Association For Cancer Research
Support is requested for a scientific meeting organized by the American Association for Cancer Research, Inc. (AACR). The conference, entitled "Cellular Responses to Environmental DNA Damage," will be held at the Banff Springs Hotel in Banff, Alberta, Canada, on December 1-5, 1991. The Co-Chairpersons for this conference are Drs. Philip C. Hanawalt and Malcolm C. Paterson. Dr. Hanawalt is Principal Investigator. This confer- ence will bring to the attention of both laboratory and clinical scientists the most recent findings in the area of how living cells respond to environmentally inflicted DNA damage, and the application of these findings to our understanding and control of mutation, cancer, and aging. The invited speakers are well-known investigators in this field. They will make 30- to 60-minute presentations. Approximately 200-225 other conference registrants will have the opportunity to present their own novel work during poster sessions. The deleterious consequences of DNA damage in mammalian systems include mutation, cancer lethality, and some aspects of aging. Most physical and chemical carcinogens interact primarily with DNA and are consequently mutagenic. Comprehensive international meetings in the field of DNA repair have been held in North America every 3-5 years. In the past year the pace of activity and discovery has quickened with the isolation and cloning of a number of human DNA repair genes, with new information on the intragenomic heterogeneity in DNA damage processing and mutagenesis, and with new insights regarding the processing of environmental DNA damage in humans, both in vitro and in vivo. It is important to place these new discoveries in the context of current cancer research, since they affect the direction in which this field now develops. We plan to convene most of the individuals who have made major recent advances in this field, along with a large group of cancer researchers, genetic toxicologists, and others working on basic mechanisms of how living cells respond to environmentally inflicted DNA damage. Participants will be drawn from a variety of scientific areas and all levels of training. from graduate students to senior basic scientists or clinical investigators. It is therefore anticipated that the conference will result in a fruitful exchange of information that will enrich current research and suggest new conceptual insights into the genesis and propagation of the neoplastic state.
|
0.901 |
1993 |
Hanawalt, Philip C |
F06Activity Code Description: Undocumented code - click on the grant title for more information. |
Dna Turnover in Active Genes: Role in Carcinogenesis? |
1 |
1993 |
Hanawalt, Philip C |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Female Germ Cell Development and Toxicology @ Environmental Mutagen Society
Support is requested for a symposium at the 24th Annual Meeting of the Environmental Mutagen Society of America (EMS) to be held at the Norfolk Waterside Marriott, Norfolk, Virginia, April 17-23, 1992. The symposium, entitled "Female Germ Cell Development and Toxicology" is organized by Dr. Philip C. Hanawalt and Dr. Walderico M. Generoso and it will be co-chaired by Dr. Jack Bishop and Dr. Richard Tasca. This symposium will bring to the attention of a major segment of environmental genetic toxicologists: Issues of concern regarding women's reproductive health, such as infertility and birth defects, that may be caused by genetic and environmental factors; the many recent, dramatic advances in molecular genetics and in cellular physiology which now make possible the application of high resolution technologies to better understand the detrimental effects of genetic and environmental factors upon female germ cell development; and applications of the technologies in research on female germ cell toxicities directed toward understanding the causes and prevention of infertility and birth defects. The invited speakers are well-known investigators in the fields of mammalian germ cell biology and toxicology. They will make 30 to 40-minute presentations with adequate time for questions and discussion in the 3-hour symposium session.
|
0.907 |
1996 |
Hanawalt, Philip C |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Gordon Conference On Mutagenesis--1996 @ Gordon Research Conferences |
0.903 |
1997 — 2002 |
Hanawalt, Philip C |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Postgraduate Training Program in Cutaneous Biology
DESCRIPTION (from the application): The Stanford Postgraduate Training Program in Cutaneous Biology provides synergistic research interactions between cutting-edge basic science and important clinical problems in dermatology. The PI, Dr. Hanawalt, with 17 years experience directing a graduate program and training grant in biophysics/molecular and cell biology, and his collaborations over many years within the Dermatology Department have exemplified this synergism. A major difference in the program from previous years are the new laboratories in the Department of Dermatology under the direction of an expanded list of faculty members (Herron, Hoeffler, Khavari, Kim, Lane, Marinkovich, McGuire, and Swetter), thereby strengthening the core interest in cutaneous biology. Our program continues the tradition of including senior faculty with international reputations in their respective fields including the former Chairman of Biological Sciences (Hanawalt), and the Chairman of Molecular and Cellular Physiology (Nelson), Pathology (Furthmayr, Smoller), Microbiology and Immunology (McDevitt, Davis), and Pediatncs (Lane). Research training opportunities include the molecular biology of DNA repair; photobiology of DNA; biochemistry and molecular biology of the basement membrane, matrix and matrix metalloproteases; growth and cell biologic phenotypes of keratinocytes, endothelial cells and fibroblasts; stem cell biology; cytokines; basic immunology, including T-cell molecular biology and immune response mechanisms; angiogenesis and carcinogenesis; experimental pathology; hereditary and autoimmune blistering disorders of the skin; and basic developmental biology of human fetal skin. M.D. candidates, who plan careers in investigative dermatology requiring extensive backgrounds in basic laboratory science, or Ph.D. candidates pursuing research in skin biology in an integrative environment including clinical aspects, are encouraged to apply. We propose support for 5 trainees, two first year and three third year. Recruitment of trainees is highly selective, based on academic record, letters of evaluation, a strong foundation in clinical dermatology and/or basic science, and publication record. We will draw on the experience of the previous PI and current co-PI, Dr. Eugene Bauer, and upon the current Advisory Board: Hanawalt, Ph.D., Nelson, Ph.D, Furthmayer, M.D., and Lane, M.D., who are also responsible for trainee selection. Trainees present research seminars in the weekly training program meeting and also to the Residency Program, facilitating monitoring of research progress, and enriching the experience of all trainees. Our trainees have access to the best available research facilities in well-equipped laboratories.
|
1 |
1998 — 2002 |
Hanawalt, Philip C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Rna Polymerase Ii and Dna Repair
DESCRIPTION: The nucleotide excision DNA repair (NER) pathway exists as a widely distributed biological mechanisms for removing various structurally unrelated DNA adducts. Preferential NER occurs for some DNA adducts due to a transcription-coupled repair (TCR) mechanisms that is proposed to initiate as consequence of arrested RNA polymerase II (RNA pol II) at transcription blocking lesions. The objective of this proposal is aimed at understanding how DNA repair enzymes recognize the arrested transcription complex and facilitate transcription-coupled repair. Taking advantage of an in vitro transcription assay that utilizes purified rat liver RNA pol II and initiation factors, the P.I.s propose to determine the structural properties of transcription complexe arrested at cyclobutane pyrimidine dimer (CPD) or other lesions. A comparison will be made to the properties of complexes arrested at naturally occurring arrest sites. To achieve the objective, three specific aims were described. First, three parameters of the RNA pol II complex arrested at CPD sites will b examined. The P.I.s have proposed 1) to determine the site of the front and rear edge of the RNA pol II arrested complex on a site-specific CPD-containing DNA ligand; 2) to locate the position of the catalytic site of RNA pol II with respect to the 3'-end of the incomplete transcript following transcription arrest; and 3) to determine the effect of nucleotide sequence context on the footprint of RNA pol II complexes in bent DNA. The second aim centers on examining the unique character of RNA pol II complexes arrested at CPD sites compared to complexes arrested at other types of pausing sites (e.g. sequence dependent arrest sites, DNA binding protein, DNA binding drug and nucleotide depletion). The third aim is proposed to extend the analysis described in Specific Aims 1 and 2 for CPDs to other types of DNA lesions (psoralen crosslinks, psoralen monoadducts, thymine glycol, abasic sites, and single-strand breaks). It is hoped that an analysis of different types of arrested RNA pol II complexes will provide an understanding of the recognition process used to attract DNA repair enzymes and initiate TCR.
|
1 |
1999 |
Hanawalt, Philip C |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Gordon Conference On Mammalian Dna Repair, 1999 @ Gordon Research Conferences
Funds are requested to enable scientists to participate in the seventh bi-annual Gordon Research Conference on Mammalian DNA Repair, to be held in Ventura, CA, Feb. 7- Feb. 12, 1999. The selected speakers are current leaders in the fields of DNA repair and related cellular processes. Besides providing a forum for these experts to present their most recent results and ideas, the conference will facilitate wide- ranging interactions among all attendees through Plenary Discussion Sessions and at posters. Selected poster presenters (generally students and younger scientists) will be given the opportunity to highlight their research results in short presentations, to stimulate debate following the plenary talks. (Several overhead transparencies, but no slides will be permitted for these "Discussion Points"). The Conference is normally oversubscribed with attendance limited to 135 participants. The attendees will be chosen globally from universities, research institutes, and government and industrial research labs. Every effort will be made to select a well-balanced mix of individuals ranging from leads in mammalian DNA repair to younger scientists new to the filed. A concerted attempt will be made to identify and attract qualified minority scientists to the conference. The 1999 Conference differs from the previous one, in that it will focus more narrowly on mammalian systems. This reflects the explosive growth of the DNA repair field and the exciting, new data linking DNA repair to human genetic disease and cancer (e.g. BRCA gene mutations that predispose to breast cancer appear to have roles in DNA repair). The first session will feature a keynote address by Dr. Richard Setlow and two other special lectures, by Prof. Jacqueline Barton and by Dr. Thomas Lindahl. The following eight sessions will define areas in which exciting new information is emerging but in which experts may disagree. The session topics include: Repair of endogenous damage, in nuclei and mitochondria; Translesion synthesis and repair of mismatches; DNA repair enzyme structure and substrate interactions; Nucleotide excision repair- role of transcription; Base excision repair- overlap with other pathways; Inducible responses and cell cycle checkpoints; Cellular localization of repair and effects of bound proteins; and DNA repair deficiency in human genetic disease. In summary, this Conference will examine DNA repair as a key component in the genomic surveillance that is so crucial to the overall integrity and function of mammalian cells. Recent discoveries have catapulted DNA repair into a pivotal position in fundamental research in the fields of oncology, aging, environmental health, and developmental biology. We hope to highlight the most promising and exciting avenues of research in robust discussions at this conference.
|
0.903 |
2000 — 2002 |
Hanawalt, Philip C |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Domain Organization of Dna Repair in Human Cells
Through nucleotide excision repair (NER), an organism removes the damaged nucleotides e.g. UV-induced cyclobutane pyrimidine dimers from its genome. There are two sub-pathways of NER; global genome repair (GGR) and transcription coupled repair (TCR). There is some evidence that TCR is more efficient and occurs early. Apparently, RNA polymerase stops at the damaged DNA and the transcription blocking lesion from the transcribed strands of the expressed gene is removed. While much is known about the genetic control of GGR and TCR, very little is known about their cellular localization. The investigators will use 5-iododeoxyuridine (IdU) labeling of repair patches in human fibroblasts with deficiencies either in GGR (Xeroderma Pigmentosa-C) or in TCR (Cockayne's syndrome) to determine the focal sites of DNA synthesis. Pulse-labeling experiments will be done to identify and enumerate the early and late foci of repair synthesis. Using different thymidine analogs, the investigators expect to map the early and late domains of repair and to relate these to chromosomal regions of high and low gene density. They will also analyze the relative distribution and co-localization in interphase nuclei of repair foci and proteins diagnostic of GGR and TCR. It is anticipated that these cytological approaches will complement the basic biochemical understanding of NER and provide new insights into the regulation of GGR and TCR in human cells.
|
1 |
2002 — 2006 |
Hanawalt, Philip Courtland |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transcription Coupled Dna Repair and Human Disease
DESCRIPTION: (PROVIDED BY APPLICANT) This project is concerned with the molecular epidemiology of cancer. Many independent genetic events occur in the transformation from a normal cell to malignancy. Changes in genomic DNA occur at specific sites and can lead to activation of proto-oncogenes or inactivation of tumor suppressor genes through mutation, recombination, gene amplification, translocation, or other chromosomal abnormalities. In some human hereditary diseases, an increased incidence of neoplasia is correlated with a defect in the repair and/or replication of damaged DNA. Our ultimate objective is to understand how the processing of damaged DNA in mammalian cells relates to carcinogenesis. We are particularly interested in how human cells process DNA lesions through the respective pathways of global genomic excision repair (GGR) and transcription-coupled repair (TCR). While a deficiency in GGR is well-known to predispose to cancer, a defect in TCR, as in the hereditary disease Cockayne syndrome (CS), does not. The characteristic developmental and neurological problems in CS are thought to be a consequence of defective TCR of endogenous oxidative DNA damage. We have documented a TCR deficiency in "UV Sensitive syndrome" (UVSS), a hereditary disease that does not present the developmental/neurological features of CS. We propose to test our hypothesis that the UVSS gene product is essential for TCR through the nucleotide excision repair pathway but not through the base excision repair pathway that deals with oxidative DNA lesions. UVSS could be a key gene in the link between DNA repair and transcription. Our proposal includes the following sub-projects: (1) The role of TCR in repair of oxidative DNA lesions will be assessed in UVSS cells, using established methods for gene-specific repair. (2) Repair of other classes of DNA damage (e.g., Benzo[a]pyrene diol-epoxide) will be assessed, using monoclonal antibodies, 32P post-labeling, and gene specific repair assays, to further characterize the repair deficiency in UVSS cells. (3) Mutagenesis studies will be performed in UV-irradiated UVSS cells for comparison with those in CS. (4) The phenomenon of inhibited GGR in active or inactive genes in TCR-deficient cells will be further characterized. (5) A complementation assay will be used to characterize cells from photosensitive patients of unknown genotype, obtained from existing collections, for assignment to UVSS, CS, or other known or unknown syndromes. The results should enhance our understanding of the role of TCR in relation to human tumorigenesis and development. New genes implicated in TCR may be discovered. Novel interactions may be revealed that will clarify relationships between cellular DNA transactions.
|
1 |
2002 — 2006 |
Hanawalt, Philip Courtland |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transcription Coupled Dna Repair in E. Coli
DNA damage and DNA repair have many important consequences for human health. Cancer and developmental defects have been associated with congenital deficiencies in DNA repair. Cancer treatment is often based upon damaging DNA or inhibiting DNA repair in the diseased tissue. In part because of its implications for human health, nucleotide excision repair (NER) has been the subject of intense investigation and a major focus of our research for nearly four decades. Based largely upon pioneering work in our laboratory, a close relationship between DNA repair and transcription has been documented in mammalian, yeast, and bacterial cells. RNA polymerase (RNAP) is a prime candidate for an essential role in this relationship, but we still do not understand exactly how it participates. Results of numerous investigations indicate that RNAP interacts with other proteins involved in DNA repair, but current ideas about the details of the interactions are sometimes contradictory. This is particularly true for transcription coupled repair in human cells in which both NER and base excision repair have been implicated. Although our ultimate goal is to understand the mechanism of transcription-coupled NER (TC-NER) in human cells, on the basis of past experience we believe that important general principles may be revealed by studying the process in the simplest systems in which it can be demonstrated. Therefore, we will focus upon the monomeric RNAP of bacteriophage T7 and the multisubunit RNAP of Escherichia coli. I. Having obtained evidence that transcription of a gene by the T7 RNAP results in enhanced repair of the transcribed strand relative to the non-transcribed strand (the hallmark of TC-NER) after UV-irradiation, we will study the biochemical basis of this effect, including the requirements for other proteins such as Mfd and mismatch proteins. II. We will identify properties of the E. coli RNAP subunits involved in TC-NER by testing well characterized mutants (rpoA, rpoB, rpoC, rpoD) for UV sensitivity. UV sensitive mutants will then be analyzed for global genomic NER and TC-NER. III. We will measure DNA turnover in the undamaged lac operon when it is expressed, or repressed, comparing the frequency of "gratuitous" repair synthesis in each strand using an approach developed in this laboratory. In addition, the nature of repair synthesis following thymine deprivation, its dependence upon transcription, and the possibility that it may reflect "gratuitous" TC-NER will be assessed. Gene expression profiles during thymine deprivation will be assessed by microarrays. The results of these experiments are relevant to an understanding of the adverse consequences of folate deprivation in humans.
|
1 |
2003 — 2007 |
Hanawalt, Philip C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Rna Polymerase Ll in Dna Repair
DESCRIPTION (provided by applicant): Multiple strategies have evolved to minimize the genotoxic consequences of endogenous and environmental agents that damage DNA. The ubiquitous process of excision repair removes a variety of structurally unrelated DNA lesions that are mutagenic and that may result in malignancy, faulty differentiation patterns and cell death. The efficiency of excision repair varies throughout the genome and a dedicated pathway of transcription-coupled repair (TCR) deals with lesions in the transcribed strands of expressed genes. Our favored model for the mechanism of TCR implicates an arrested RNA polymerase as a signal to recruit excision repair enzymes to the transcription blocking lesion. To understand how repair proteins recognize an arrested RNA polymerase to initiate a repair event, we intend to continue characterizing the unique features of the transcription complex when it encounters different impediments during the normal course of elongation. We propose to extend this analysis to elucidate the protein-interactions of the RNA polymerase complex when it is arrested at a cyclobutane pyrimidine dimer (CPD). Utilizing an in vitro transcription assay with purified T7 RNA polymerase or rat liver RNA polymerase II (RNAP II) and unique DNA substrates we will analyze the behavior of RNA polymerase at site-specific lesions, focusing upon lesions that impose different types of constraints, including an abasic site or oxidized abasic site; a DNA bubble structure (intermediate in TCR); psoralen monoadducts and interstrand crosslinks; and mismatch repair complexes bound to 8-oxoG (a lesion that does not in itself arrest RNAP II). Protein-interactions and protein-modifications of RNAP II transcription complexes when arrested at a DNA lesion will be identified and characterized. Isolated RNAP II ternary complexes will be incubated with relevant purified proteins (e.g. XAB2, CSA, CSB, TFIIH, XPG, MSH2-MSH6, MSH2-MSH3) in the presence or absence of the elongation factor SII to determine by gel mobility shift assays how coupling factors interact with an arrested polymerase. The position of the transcription "bubble" following RNAP II arrest at a DNA lesion will be mapped with KMnO4, before or after addition of putative coupling factors; and after SII-mediated reverse translocation of RNAP I1. To study the role of ubiquitination upon transcription arrest at a lesion, the isolated RNAP II ternary complexes will be incubated with HeLa cell extracts and His-tagged ubiquitin, followed by detection of the reaction products by Western blotting. The unique features of a single translocating RNAP II complex as it is arrested at a CPD, and reversed by SII will be visualized using an optical trap. Ultimately our goal is to reconstitute TCR in vitro, building upon and benefiting from results obtained as we achieve the foregoing aims. This research is relevant to an understanding of the relationship between DNA repair and oncogenesis, as well as the possible application of such understanding to more effective cancer therapy.
|
1 |
2005 |
Hanawalt, Philip Courtland |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
9th International Conference On Environmental Mutagens @ Environmental Mutagen Society
DESCRIPTION (provided by applicant): We request support for invited speakers participating in the 9th International Conference on Environmental Mutagens (9th ICEM) hosted by the Environmental Mutagen Society (EMS). The meeting will be held September 3-8, 2005 at the Hyatt Regency Hotel at Embarcadero in San Francisco, CA. The ICEM is held every 4 years and was last hosted in the U.S. 16 years ago. We anticipate attracting1200 participants worldwide, including the members of the EMS and corresponding environmental mutagen societies in many other countries. The EMS is the primary scientific society devoted to the understanding of the basic mechanisms of mutagenesis and their implications for human genetic disease, cancer, and aging. The EMS is unique in that it also is the relevant society for applied science in this field, including genotoxicity risk assessment and regulatory matters. It is the appropriate society for researchers interested in the development and application of transgenic technology for toxicological studies and for the development of methods for studying mutations, mutagenic mechanisms, environmental carcinogens, and DNA repair in model cellular systems as well as humans. The theme for the 9th ICEM is "Global Issues in Genetic Toxicology and Environmental Mutagenesis." To highlight these issues and to explore solutions to these global threats to our collective health, the 9th ICEM will offer 46 symposia, 2 keynote lectures, and 12 plenary lectures from the world leaders in environmental mutagenesis and related fields. Broadly, speakers will address how environmental contaminants can damage the genome, how such damage may be repaired or else processed into mutations, and how the resulting mutations may lead to disease. Symposia at the cutting-edge of basic and applied research will present details of new technologies in molecular biology and biomedical research as they apply to the global problem of maintaining genomic integrity and human health. There will be opportunities for researchers from all countries to present their most recent research in poster and platform sessions. We will also include relevant workshops and special programs for students. Emerging issues in environmental health science, and ways of identifying, characterizing, and solving some of these problems, will be presented. The new scientific information and insights from this Conference will be of importance to wise policy decisions to protect the public from significant environmental health hazards.
|
0.907 |
2008 — 2016 |
Hanawalt, Philip Courtland |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Role of Transcription in Genomic Stability
DESCRIPTION (provided by applicant): Role of transcription in genomic stability RNA polymerase (RNAP) sensitively detects damage as it performs one-dimensional scanning of template DNA, thus initiating the dedicated pathway of transcription-coupled repair (TCR). However, transcription can also increase mutagenesis. Hot spots for genomic instability implicated in human genetic disease and carcinogenesis have been localized in DNA sequences that can adopt non- canonical DNA structures (e.g. Z-DNA, H-DNA, G4-DNA, triplet repeats and palindromes forming hairpins/cruciforms). We wish to gain a mechanistic perspective on possible outcomes when RNAP encounters such structures. We hypothesize that TCR may be mutagenic when it occurs at or near non-canonical DNA structures, certain lesion types, or bound complex ligands. To test this hypothesis we will determine the precise signals that can arrest RNAP and elicit TCR, to learn whether TCR might be error-prone under some circumstances. An in vitro transcription assay using purified T7RNAP and mammalian RNAPII with required factors on defined DNA substrates will be utilized to: (1) Characterize RNAP arrested at site-specific non-canonical DNA structures and lesions, including psoralen monoadducts vs. interstrand crosslinks, and adducts of the acylfulvenes, which are reportedly subject to TCR but not global excision repair. The arrested RNAP, transcription bubble, and RNA/DNA hybrid will be mapped. Effects of added factors such as TFIIS, CSB and TFIIH, which may modulate transcription arrest, as well as effects of mismatch repair proteins and RecQ, which modulate some non B-form DNA structures will be determined. Recognition of non-canonical structures by repair enzymes in human cell extracts will be assessed (2) Evaluate cooperative effects on transcription of abasic sites or 8oxoGuanine introduced into Z-DNA, and effects of complex ligands, such as the Z-DNA binding protein ADAR1, and topoisomerase 1 trapped at abasic sites. (3) Utilize stable complexes of peptide nucleic acid (PNA) to explore novel aspects of these unique ligands for targeted gene alterations, while revealing mechanistic details of transcriptional processing. PNA binding will also be explored as an alternative to promoter-driven transcription, to possibly achieve higher efficiency of substrate usage, toward improved assays for transcription behavior at lesions and cell-free TCR assays. PUBLIC HEALTH RELEVANCE: The results from this project will enhance our understanding of the roles of transcription and TCR in processing lesions and other abnormalities in DNA that have been implicated in human disease. Since prolonged transcription arrest generates a strong signal for apoptosis, the research may lead to novel modes of chemotherapy, involving selective inhibition of TCR in target cells combined with administration of transcription-blocking drugs.
|
1 |
2010 — 2011 |
Hanawalt, Philip Courtland |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Oxidative Dna Damage Processing;Role in Human Pathology and Aging
DESCRIPTION (provided by applicant): Our overall objective is to understand how processing of damaged DNA relates to human genetic disease, cancer and aging. Having pioneered the discovery of nucleotide excision repair (NER), we are elucidating the sub-pathways of global genomic repair (GGR) and transcription-coupled repair (TCR). The TCR-deficient diseases, Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS), present indistinguishable biochemical responses to UV;UVSS patients have only superficial consequences of sunburn while those with CS suffer severe neurological/developmental defects, segmental progeria and early death. Notably, no cancers of any type have been reported for patients with these syndromes. We hypothesize that the severe features of CS are due to apoptosis triggered by prolonged transcription arrest as a consequence of defective TCR of oxidative base damage or to defective transcriptional bypass of such damage generated by endogenous reactive oxygen species, while UVSS cells are normal with respect to processing base damage in expressed genes. In support of this model we find that CS cells are hypersensitive to oxidants, and that UVSS but not CS cells are proficient in host cell reactivation of plasmids containing oxidized bases. However, definitive biochemical evidence for TCR of oxidized bases is lacking. Our model for TCR postulates that an arrested RNA polymerase (RNAP) recruits repair enzymes to transcription- blocking lesions. Reported studies with an oxidized base positioned at a unique site in the DNA template strand indicate that RNAP can bypass, transiently pause or arrest at these lesions. We propose to use a novel transcription assay with multiple randomly-positioned lesions induced in the template, to let the transcription system tell us which lesions and which sequence contexts are most relevant for further analysis. After determining the types and positions of the lesions that cause arrest in vitro, we will construct single-lesion vectors for transfection into human cells to measure in vivo transcription rates upstream and downstream of the lesion;sequencing the transcripts will reveal transcriptional mutagenesis. We propose to develop the sensitive Comet-FISH approach with gene-specific probes to comparatively quantify low levels of particular oxidative lesions and their removal from transcribed or silent sequences and from the genome overall. Cells with missing or reduced base excision repair or NER activities will be employed to investigate processing of oxidative lesions. Specific enhancement of 8oxoG in DNA will be achieved by interference RNA-mediated MTH1 knockdown, to eliminate complications of other lesions and other oxidative effects. We will focus on differences between CS and UVSS as a model system to elucidate the role of processing of oxidative base damage in aging, disease and neurological degeneration, as well as the underlying cause of the cancer-resistance of these syndromes. PUBLIC HEALTH RELEVANCE: Free radicals from endogenous and environmental sources are a constant threat to genomic integrity. The induced damage can arrest DNA and RNA polymerases, events that can unleash irreversible apoptotic pathways or mutagenicity. We propose novel approaches for elucidation of the effects of oxidative DNA lesions on transcription, and for the analysis of repair of physiologically relevant levels of these lesions in transcriptionally active or silent genomic domains and in the genome overall, using the Comet- FISH assay. Results from the project will advance our understanding of cellular processes leading to carcinogenesis, aging, and other pathologies. They will also further the development of effective strategies for therapeutic intervention in human disease.
|
1 |
2012 — 2014 |
Hanawalt, Philip Courtland |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Oxidative Dna Damage Processing; Role in Human Pathology and Aging
DESCRIPTION (provided by applicant): Our overall objective is to understand how processing of damaged DNA relates to human genetic disease, cancer and aging. Having pioneered the discovery of nucleotide excision repair (NER), we are elucidating the sub-pathways of global genomic repair (GGR) and transcription-coupled repair (TCR). The TCR-deficient diseases, Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS), present indistinguishable biochemical responses to UV; UVSS patients have only superficial consequences of sunburn while those with CS suffer severe neurological/developmental defects, segmental progeria and early death. Notably, no cancers of any type have been reported for patients with these syndromes. We hypothesize that the severe features of CS are due to apoptosis triggered by prolonged transcription arrest as a consequence of defective TCR of oxidative base damage or to defective transcriptional bypass of such damage generated by endogenous reactive oxygen species, while UVSS cells are normal with respect to processing base damage in expressed genes. In support of this model we find that CS cells are hypersensitive to oxidants, and that UVSS but not CS cells are proficient in host cell reactivation of plasmids containing oxidized bases. However, definitive biochemical evidence for TCR of oxidized bases is lacking. Our model for TCR postulates that an arrested RNA polymerase (RNAP) recruits repair enzymes to transcription- blocking lesions. Reported studies with an oxidized base positioned at a unique site in the DNA template strand indicate that RNAP can bypass, transiently pause or arrest at these lesions. We propose to use a novel transcription assay with multiple randomly-positioned lesions induced in the template, to let the transcription system tell us which lesions and which sequence contexts are most relevant for further analysis. After determining the types and positions of the lesions that cause arrest in vitro, we will construct single-lesion vectors for transfection into human cells to measure in vivo transcription rates upstream and downstream of the lesion; sequencing the transcripts will reveal transcriptional mutagenesis. We propose to develop the sensitive Comet-FISH approach with gene-specific probes to comparatively quantify low levels of particular oxidative lesions and their removal from transcribed or silent sequences and from the genome overall. Cells with missing or reduced base excision repair or NER activities will be employed to investigate processing of oxidative lesions. Specific enhancement of 8oxoG in DNA will be achieved by interference RNA-mediated MTH1 knockdown, to eliminate complications of other lesions and other oxidative effects. We will focus on differences between CS and UVSS as a model system to elucidate the role of processing of oxidative base damage in aging, disease and neurological degeneration, as well as the underlying cause of the cancer-resistance of these syndromes.
|
1 |
2014 |
Hanawalt, Philip Courtland |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
2014 Dna Damage, Mutation and Cancer Gordon Research Conference @ Gordon Research Conferences
Project Summary/Abstract The progression from damaged DNA and its cellular processing to mutagenesis during DNA replication is a complex process. Depending upon the nature of the mutations and the genes affected the further progression to a tumor can be even more complex. The initial damage may have occurred in unusual DNA sequences or structures that are refractory to repair, it may be embedded in chromatin structures that restrict access to repair enzymes and occasionally, the primary causal event may be an undamaged DNA sequence that has been processed incorrectly by transcription and/or replication. The 2014 Gordon Research Conference on DNA damage, mutation and cancer will focus on the manner in which DNA lesions and unusual DNA structures can be recognized and processed by cellular enzymes to modulate their mutagenic effects. Multiple proteins may recognize the same DNA alteration and impact the pathway choice for its resolution. Furthermore, each step in a repair pathway generates another lesion, often accessible to enzymes from other pathways. Crosstalk between competing or collaborating DNA repair pathways will be an important theme in this Conference. We will also explore chemotherapeutic interventions to take advantage of potential genotypic differences between normal cells and those in a designated tumor. The synthetic lethality approach is important, but there are other approaches to selectively inactivate tumor cells in the sea of normal cells. We will also address the evidence for the role of DNA damage in aging, and the importance of telomere processing in aging and cancer. The nine provisional sessions are titled: Mutations cause cancer, reveal etiology and provide clues for therapy; Sensing environmental genomic damage, finding needles in the haystack; Weak links in the genome, intrinsic challenges to replication and transcription; Cutting edges at the initiation of DNA repair; Oxidative DNA damage and base excision repair, roles of PARP; Mismatch repair and responses to arrested replication or transcription; Cell cycle checkpoints and strand-break repair; Designing chemotherapies based upon cancer genotypes; and Relationships of DNA damage to telomere maintenance and aging. Poster sessions and open afternoons will provide ample opportunities for engagement between investigators at all levels. We anticipate that scientific interactions during this conference will impact cancer research in significant ways and result in establishing productive multi-disciplinary research collaborations. The 2014 DNA Damage, Mutation & Cancer Gordon Research Conference will be held at the Beach Marriott, in Ventura, California March 16 - 21, on the 50th anniversary of the reported discovery of excision repair and the 40th anniversary of the first international workshop on DNA repair mechanisms.
|
0.903 |