Lab Projects
Lab Projects | ||
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UV Repair | ||
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The UV component of sunlight induces a variety of DNA damage products which have both cytotoxic and mutagenic effects. The pyrimidine dimers are the most prevalent and probably most significant of these UV-induced photoproducts. Pyrimidine dimers fall into two classes: the cyclobutane dimers (CPDs), which make up approximately 75% of UV-induced damage products, and the pyrimidine [6-4] pyrimidinone dimers (6-4 products), which make up most of the remaining 25% of UV-induced lesions. Both of these classes of dimers have been demonstrated to block the progress of both DNA and RNA polymerases. The removal of these lesions is essential not only for efficient DNA synthesis, but also for transcription. Almost all living things produce enzymes, termed "photolyases", whose single function is the reversal of pyrimidine dimers, via a process that requires a photon in the UV-A to blue range. Interestingly, however, while almost all organisms studied (with the notable exception of placental mammals) produce a CPD-specific photolyase, only a subclass of these organisms, including Arabidopsis, produce a second photolyase that can reverse 6-4 products. |  | |||
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A second repair mechanism for the excision of dimers and a wide variety of other DNA damage products also exists. The light-independent mechanism, nucleotide excision repair (NER), takes on the burden of UV-repair in placental mammals; its importance is demonstrated in the lethal effects of solar UV in individuals that carry homozygous genetic deficiencies in any of the dozen genes required for this process. The role of NER in the repair of UV-induced damage in organisms that produce photolyases is less clear. NER is generally a slower process than photolyase-driven photoreactivation, though it is possible that transcription-coupled NER plays an important role in rapidly clearing dimers from the path of transcriptionally active RNA polymerases. CS-B is a human gene required for transcription coupled repair. Arabidopsis has a large number of CS-B homologs, some of which (uvr4, uvr5) are required for UV-resistance. Arabidopsis possesses all of the UV-repair mechanisms described above, and mutants defective in these processes have been identified on the basis of their UV-sensitive phenotype (NER defective mutants can be identified by their UV-sensitivity in the absence of photoreactivating light). uvr1, uvh1, uvr5, and uvr7 mutants are all defective in NER, and the UVH1 gene has been shown to be a homolog of the yeast RAD1 NER excinuclease. Arabidopsis uvr2 and uvr3mutants are defective in the CPD- and 6-4-product-specific photolyases, respectively. Ongoing research in the lab currently focuses on the phenomenology of transcription-coupled repair. | ||
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Mechanisms of Recombination | ||
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Double strand breaks (DSBs) occur routinely during DNA replication and during meiosis. They can also be induced by DNA damaging agents and transposable elements. DSBs are repaired via two basic classes of pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). All organisms possess both pathways, but organisms with less compact genomes appear to repair most breaks generated in mitotic cells via NHEJ. We are interested in determining the genetic basis of NHEJ in plants, using Arabidopsis as a model system. We are taking both reverse genetic and classical genetic approaches, with the goal of determining a) whether Arabidopsis homologs of genes involved in NHEJ in yeast and mammals are also required for this process in plants, and b) whether novel mutants, representing previously undiscovered genes or genes unique to plants, are involved in the processing of DSBs. A better understanding of DSB repair, and the creation of mutants defective in NHEJ, may have direct applications to genetic engineering, as transgenes are integrated into the plant genome via this process. Our research suggests that the canonical DNA ligase IV/Ku-dependent NHEJ pathway, while functional and important in plants, is “backed up” by a second, relatively sloppy, but still effective, NHEJ pathway that requires neither Ku nor Lig4. Our research is focusing on identifying the genes involved in this noncannonical pathway.
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Cell Cycle Checkpoints | ||
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Plants, like other organisms, respond to DNA damage by arresting cell division, thus allowing time for the cellular machinery to repair the DNA. After the damage is repaired the cell then resumes division. Our lab is taking both functional genomic and classical approaches to determine whether the plant homologs of damage response genes actually function in response in plants, and to identify novel factors involved in cell cycle arrest in response to damage. Knockouts of genes that are critical for damage response and repair in mammals often exhibit a lethal in mammals. Fortunately, plants with similar null mutations are viable. This permits us to definitively answer many questions regarding the functions of these genes that cannot be addressed in mammals, including their roles in development, general DNA replication, and meiosis.
Aphidicolin-treated atr root
tips lose their nuclei. DNA is here stained blue with
DAPI. In plants and mammals, ATR is required to induce cell cycle
arrest in response to replication blocks. In wild-type cells,
the cell cycle is slowed so that replication can be accurately completed
before progression into mitosis. atr null plants lack
this safeguard, and dividing cells die when exposed to replication
blocking agents such as the DNA polymerase poison aphidicolin. | ||
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Repair and response to DNA damageinduced by space radiation |
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Both solar flares and galactic radiation contribute to the production of a radiation flux that includes both high energy photons (gamma and X-rays) and high energy, high charge relativistic nuclei which are very effective DNA damaging agents. While gamma rays will skim through tissues, interacting with matter via Compton scattering and depositing relatively diffuse “tracks” of ion pairs, heavier, relativistic, highly charged particles will deposit a dense “tunnel” of ionized matter as they rip through tissue. Fortunately, the Earth’s magnetic field currently shields us from these charged particles. This shielding is reduced at high altitudes and at the poles. This radiation is particularly hazardous to individuals participating in long term missions in outer space (i.e., in the space station, and in the planned manned mission to Mars). Thus far we have failed to develop shielding materials capable of absorbing these particles. Our lab is currently working on identifying the repair and damage response genes required for resistance to these particles, in the hope of developing genetic screens for particularly susceptible individuals. This project is funded by NASA.
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The Earth's magnetic field, and the solar wind, act to shield the Earth's surface from cosmic rays. |
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Section of Plant Biology - University of California at Davis - Davis, CA 95616 | ||
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