|
Overview
Our laboratory's major interest is in the mechanisms that maintain the stability of DNA in human and other mammalian cells. Our emphasis is on the excision-replacement systems of nucleotide (NER), base (BER) and mismatch (MMR) repair as well as touching on mechanisms of recombination and apoptosis that are triggered by DNA double strand breaks. We have developed mouse knockouts for one DNA repair gene and others are under construction. We presently work in several specific areas of NER, DNA replication arrest and base excision repair. The PI is involved in UCSF graduate training programs, which include the Program in Biological Sciences (PIBS), Biophysics, and programs for Dermatology residents.
Xeroderma Pigmentosum: A Hereditary Disease of DNA Repair and Carcinogenesis
Current research involves a study of human genetic diseases that show increased susceptibility to genotoxic agents including ultraviolet light and chemical carcinogens. Xeroderma pigmentosum (XP) and related diseases, which fail to repair damage from ultraviolet (UV) light and other DNA damaging agents, have played a pivotal role in relating DNA damage and repair to the mechanisms underlying genomic instability in environmentally-induced carcinogenesis. XP patients have a 1000-fold increase in susceptibility to solar-induced skin cancers, and this is due to an inability to process DNA damage (pyrimidine dimers) correctly by repair and replication. The genes involved in XP regulate both NER (XP groups A through G) and DNA replication arrest (XP group V).
We have established the spectrum of mutations carried by patients suffering from XP-A, a defect in the damage recognition mechanism, and related these to the functional regions of the gene product. We are using XP-A cells for study of gene delivery using adeno-associated virus into which the XPA gene and the green fluorescent protein (GFP) gene have been inserted. This is a tissue culture model for gene therapy. We are developing a database of all known XP mutations to be posted under the auspices of the Cancer Center and the Human Genome Organization (HUGO)
The XP variant represents about 25% of all XP patients: they have normal excision repair of UV damage, but are clinically indistinguishable from excision defective patients. XP variants, instead, suffer premature arrest of DNA replication forks during the S phase in UV damaged cells, and increased UV-induced mutagenesis, and represent a defect in a polymerase (hRAD30) associated with replication fidelity. Human cells also contain another polymerase (pol zeta) which is responsible for inserting incorrect bases during DNA replication in XP cells in the absence of NER or hRAD30. We have recently demonstrated that the phenotype of XP variant cells can be significantly altered by transformation using either SV40 or HPV16, indicating a pathway for interaction between the XP variant gene product and p53, which influences genomic stability after UV irradiation. We have recently discovered that arrested DNA replication forks in UV damaged XP variant cells develop into DNA double strand breaks that stimulate a recombination pathway and rapid apoptosis. We are isolating bacterial artificial chromosomes (BACs) for both polymerases in mouse and human in order to sequence structural gene and promoter regions to understand their regulation and develop knockout animal models for the XP variant.
Mouse Models of Defects in Base Excision Repair
Base excision repair (BER) is the primary mechanism by which cells correct naturally-occurring DNA damage such as deamination, oxidation, and methylation. Defects in this repair may be involved in neurodegenerative diseases such as Cockayne syndrome and some groups of XP. We are investigating the developmental and physiological role of BER by studying one of the central proteins in the pathway, Xrcc1. Xrcc1 interacts with DNA ligase III (LIG3), DNA polymerase beta, and poly[ADP-ribose] polymerase (PARP), and evidence indicates Xrcc1 regulates the activity of LIG3 and PARP. We constructed mice with a knockout mutation in Xrcc1 and found that Xrcc1-/- embryos were unable to develop beyond embryonic day 6.5 (E6.5). Prior to death Xrcc1-/- embryos contained an increased level of spontaneous DNA damage and a high number of apoptotic cells compared to wild type embryos. This indicates that BER is essential for mouse embryonic development. We are currently doing site-directed mutagenesis experiments to determine essential protein-protein interactions, and genetic studies to determine other genes linked to the pathway. We are also planning embryo culture experiments designed to rescue knockouts. Several non-conservative Xrcc1 mutations have been found in human breast cancer patients hypersensitive to radiation treatment. We plan to construct a mouse model of these human patients and test these animals to determine if Xrcc1 might be a variable in individual radiation sensitivity and cancer susceptibility. We will also be developing a conditional expression system for Xrcc1 in mice such that the role of BER can be investigated over the life-span of the animals and establish whether repair of endogenously-generated oxidative damage is related to aging.
Additional Resource
See also http://xpmutations.org for a searchable website providing extra background and summarizing the mutations in XP, CS, and TTD diseases.
Cutaneous Oncology Program
The PI, J.E.Cleaver, is currently group leader of the Cancer Center's Cutaneous Oncology Program, together with Dr. M. Kashani-Sabet, who is clinical leader. This program draws together many faculty interested in all forms of skin cancer: squamous carcinoma, basal cell carcinoma, cutaneous and uveal melanoma, and cutaneous T-cell lymphoma. This program aims at a comprehensive understanding of molecular mechanisms underlying skin cancers and development of translational activities in prevention and treatment. The program has access to an archive of skin cancer tissues for investigational uses, and has patient contacts and clinical trials through the UCSF Dermatology Department and the Melanoma Center, and patient support groups such as the XP Society.
|
