Dr. Lawrence Grossman
Mechanisms for the Repair of Damaged DNA
University Distinguished Service Professor of Biochemistry and Molecular Biology,The Johns Hopkins Bloomberg School of Public Health
Joint Appointment in Environmental Health Services
Joint Appointment in Oncology
Ph.D., Johns Hopkins University, Baltimore, MD
Johns Hopkins University, McCollum-Prett Institute Biology Department, Baltimore, MD
The ability of cells to survive hostile environments is due, in part, to surveillance systems that recognize damaged sites in DNA and are capable of either reversing the damage or removing damaged bases or nucleotides, generating sites that lead to a cascade of events restoring DNA to its original structural and biological integrity. Both endogenous and exogenous environmental agents can damage DNA. A number of repair systems are regulated by the stressful effects of such damage, by affecting the levels of responsible enzymes or by modifying their specificity. Repair enzymes apparently are the most highly conserved proteins, showing their important role throughout evolution. The nucleotide excision repair pathway in Escherichia coli is comprised of at least six structural and two regulatory genes; the products of the former genes have been amplified and purified to homogeneity. The UvrA, UvrB and UvrC proteins in concert with ATP carry out dual incision of DNAs damaged by a wide variety of structurally unrelated damage in which an initial 5’-cut occurs 7 nucleotides from a damaged site and the second 3’-cut occurs 3 nucleotides from the same site regardless of the nature of the damage. The UvrAB protein complex serves to locally unwind DNA a single helical turn followed by a unidirectional (5’->3’) strand displacement, which is accompanied by waves of supercoiling in front of and behind the nucleoprotein complex. The energy needed for recognition is derived from the binding of ATP generating associations between DNAs with protein. Protein-protein and nucleoprotein dissociations and translocation are driven by ATP hydrolysis. The damaged oligonucleotide is excised by the UvrD gene product-helicase II, DNA polymerases I or III in the presence of dNTPs and ligase restores the integrity of the strands.
Dr. Grossman’s lab is studying the mechanism-architecture paradigm of these multiprotein systems by Ònearest neighborÓ analyses with monoclonal antibodies against the structural motifs of the UvrA, B and C proteins in vitro and in vivo. The UvrB protein(s) has been crystallized and its structure under study. A catalytically active ultiprotein-DNA-membrane complex from UV-damaged cells has been isolated consisting of 17 characterized proteins. From reversible cross-linking studies under SOS conditions (cells previously damaged with UV light), the lab has identified the b-subunit of RNA polymerase (RNAP) and UvrA in close proximity. The role of the RNAP is to provide a signal for the landing of the repair complex on the apparatus strands and sites. In collaboration with doctoral students from Epidemiology, the lab developed a DNA repair assay that can be applied to human cancer populations. It was found in a study of 450 subjects that reduced DNA repair of UV-induced damage contributes directly to basal cell carcinoma (BCC) in those individuals with a prior sunlight exposure. It was further discovered that a family history of skin cancer is a predictor of low DNA repair. Repair of UV-damaged DNA declines at a fixed rate as a function of age in normals.
The DNA repair differences between young skin cancer cases and their controls disappear as they age, hence BCC, in terms of DNA repair, is a premature aging disease. The group originally set out to examine whether an individual’s vulnerability to sunlight leading to basal cell carcinoma in the general population is related to DNA repair capacity. It soon became apparent that the xeroderma pigmentosum-DNA repair paradigm-UV exposure was applicable to the general Caucasian population. This disease is rare in Oriental and Black populations. It was found in Taiwanese populations endemic for Blackfoot disease that affected individuals exposed to arsenic in their drinking water have significantly lower repair than controls, suggesting that DNA repair is a risk factor in this disease study in a Blackfoot disease endemic area in Taiwan. Additionally, DNA repair declined at the rate of one percent per annum among controls. Subsequent studies with Dr. Ken Kraemer’s group of the NCI revealed that mutation rates increased also at the same rate, implying that DNA repair contributed to the aging process by affecting the persistence of DNA damage in repair defective cells and individuals.
Hildebrand EL, Grossman L. Introduction of a Tryptophan Reporter Group in the ATP Binding Motif of the Escherichia Coli UvrB Protein for the Study of Nucleotide Binding and Conformational Dynamics. Journ Biol Chem 273: 7818-7827, 1998. Lin G-L, Kovalsky O, Grossman L. Transcription Coupled Nucleotide Excision Repair by Isolated Escherichia Coli Membrane-associated Nucleoids. NAR 26: 1466-1472, 1998. Wei Q, Matanoski GM, Farmer ER, Hedayati MA, Grossman L. DNA Repair and Aging in Basal Cell Carcinoma: A Molecular Epidemiology Study. Proc Natl Acad Sci USA 90: 1614-1618, 1993. Van der Riet P, Karp D, Farmer E, Wei Q, Grossman L, Tokino K, Ruppert JM, Sidransky D. Progression of Basal Cell Carcinoma Through Loss of Chromosome 9q and Inactivation of a Single p53 Allele. Cancer Res 54: 25-27, 1994. Grossman L, Wei Q. DNA Repair Capacity (DRC) as a Biomarker of Human Variational Responses to the Environment in DNA Repair Mechanisms: Impact on Human Diseases and Cancer. (Vos J-M, ed). pp 327-345, 1994. Wu M-M. DNA Repair and Arsenical Skin Cancer: a Case Control Study in a Blackfoot Disease Endemic Area in Taiwan. PhD thesis in preparation for both Cancer Res and J Epidemiology. Hedayati M, Matanoski G, Grossman L. Moriwaki S-I, Ray S, Tarone RE, Kraemer KH, Grossman L. The Effects of Aging on the Processing of UV-Damaged DNA in Human Cells: Reduced DNA Repair Capacity and Increased DNA Mutability. Mut Res-DNA Repair, 364:117-123, 1996. Khan SG, Metter EJ, Tarone RE, Bohr VA, Grossman L, Hedayati M, Bale SJ, Kraemer KH. A New Xeroderma Pegmentosum GroupC Poly AT Insertion/Deletion Polymorphism. Carcinogenesis, 2000. Updated 7/17/01