Xeroderma pigmentosum (XP) is a disease in which a genetic deficiency predisposes patients to sunlight induced cancers of the skin, including squamous and basal cell cancers and melanomas. XP is a multigenic disease involving at least 8 genes: XP groups A through G represent deficiencies in nucleotide excision repair (NER) of sunlight (UVB) damage to the DNA of the skin, and XPV represents a deficiency in replication of damaged DNA. XP cells all show elevated UV-induced mutagenesis, which correlates with the increased risk for sunlight induced cancer. The XPV gene encodes a DNA polymerase, hRad3O, po1 n, homologous to the UMUC, D' class of polymerases in E.coli, and functions in an error-free pathway for UV damage. The enzyme is a distributive polymerase with a high error-rate on even undamaged DNA. We have mapped the gene to 6p2l, identified 10 coding exons, and an untranslated exon I. Exon II is deleted in some normal transcripts and in an XP patient. We observe that the promoter has multiple AP1 and SP1 sites, suggesting a damage-responsive regulation. We found expression from a constitutive promoter to be toxic, but have achieved functional correction of several XPV phenotypes (UV survival, apoptosis) using expression of a fusion protein. We have also found that DNA replication after UV damage in XPV cells involves a double strand repair/recombination system involving hMre1 1/hRad50/Nbs1 and depends on p53. Understanding how a polymerase with such a high error-rate contributes to error-free replication of UV damaged DNA, and how it is regulated to avoid rampant error generation and toxicity is of major importance and the emphasis of this project.
Our specific aims are:
Aim I : To understand the mechanisms by which the low fidelity hRad3O polymerase is regulated to maintain genetic stability in normal and transformed cells. We will design expression vectors that complement XPV phenotypes in vitro, and use these to identify binding partners in control and UV damaged cells.
Aim II : To understand the role of recombination in the S phase checkpoint(s) in cells deficient in hRad3O. We will determine the role of hMre 11 recombination in specific stages of the S phase and identify components of the S phase signal transduction pathways.
Aim III : To develop mouse strains defective in hRad3O functions. We will express hRad3O on the keratin 14 promoter for over-expression in the skin, and make targeted knockout of mRad3O in vivo to identify roles for hRad3O in promoting and preventing carcinogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
2R01ES008061-04
Application #
6327308
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Velazquez, Jose M
Project Start
1998-02-01
Project End
2006-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
4
Fiscal Year
2001
Total Cost
$331,875
Indirect Cost
Name
University of California San Francisco
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
de Feraudy, Sebastien; Ridd, Katie; Richards, Lauren M et al. (2010) The DNA damage-binding protein XPC is a frequent target for inactivation in squamous cell carcinomas. Am J Pathol 177:555-62
Cleaver, James E; Feeney, Luzviminda; Tang, Jean Y et al. (2007) Xeroderma pigmentosum group C in an isolated region of Guatemala. J Invest Dermatol 127:493-6
Laposa, Rebecca R; Feeney, Luzviminda; Crowley, Eileen et al. (2007) p53 suppression overwhelms DNA polymerase eta deficiency in determining the cellular UV DNA damage response. DNA Repair (Amst) 6:1794-804
Cleaver, J E; Hefner, E; Laposa, R R et al. (2007) Cockayne syndrome exhibits dysregulation of p21 and other gene products that may be independent of transcription-coupled repair. Neuroscience 145:1300-8
de Feraudy, S; Limoli, C L; Giedzinski, E et al. (2007) Pol eta is required for DNA replication during nucleotide deprivation by hydroxyurea. Oncogene 26:5713-21
Laposa, R R; Huang, E J; Cleaver, J E (2007) Increased apoptosis, p53 up-regulation, and cerebellar neuronal degeneration in repair-deficient Cockayne syndrome mice. Proc Natl Acad Sci U S A 104:1389-94
Marti, Thomas M; Hefner, Eli; Feeney, Luzviminda et al. (2006) H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks. Proc Natl Acad Sci U S A 103:9891-6
Cleaver, James E (2005) Splitting hairs--discovery of a new DNA repair and transcription factor for the human disease trichothiodystrophy. DNA Repair (Amst) 4:285-7
Limoli, Charles L; Giedzinski, E; Cleaver, J E (2005) Alternative recombination pathways in UV-irradiated XP variant cells. Oncogene 24:3708-14
Wechsler, Thomas; Chen, Benjamin P C; Harper, Ryan et al. (2004) DNA-PKcs function regulated specifically by protein phosphatase 5. Proc Natl Acad Sci U S A 101:1247-52

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