Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.
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2019-01-07
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Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.
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Klein, Hannah L, Giedrė Bačinskaja, Jun Che, Anais Cheblal, Rajula Elango, Anastasiya Epshtein, Devon M Fitzgerald, Belén Gómez-González, et al. (2019). Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways. Microbial cell (Graz, Austria), 6(1). pp. 1–64. 10.15698/mic2019.01.664 Retrieved from https://hdl.handle.net/10161/18039.
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Scholars@Duke
Sue Jinks-Robertson
My research focuses on the regulation of genetic stability and primarily uses budding yeast (Saccharomyces cerevisiae) as a model genetic system. The two primary research goals in the budding yeast system are (1) defining molecular structures and mechanisms of mitotic recombination intermediates and (2) understanding how and why transcription destabilizes the underlying DNA template. We also have initiated studies of mutagenesis in the pathogenic fungus Cryptococcus neoformans. We have found that a shift to the human body temperature mobilizes transposable elements, and suggest that this promotes rapid adaptation to the harsh host environment.
Thomas Douglas Petes
My lab is active in three somewhat related research areas: 1) the mechanism of mitotic recombination, 2) the genetic regulation of genome stability, and 3) genetic instability associated with interstitial telomeric sequences. Almost all of our studies are done using the yeast Saccharomyces cerevisiae.
Mechanism of mitotic recombination
Mitotic recombination, an important mechanism for the repair of DNA damage, is less well characterized than meiotic recombination. One difficulty is that mitotic recombination events are 104-fold less frequent than meiotic recombination events. We developed a greatly improved system for identifying and mapping mitotic crossovers at 1-kb resolution throughout the genome. This system uses DNA microarrays to detect loss of heterozygosity (LOH) resulting from mitotic crossovers. We identified motifs associated with high levels of spontaneous mitotic recombination. In particular, we demonstrated that a “hotspot” for mitotic recombination was generated by a pair of inverted retrotransposons. We also used this system to make the first genome-wide map of UV-induced recombination events. Finally, and most importantly, we demonstrated that most spontaneous mitotic recombination events reflect the repair of two sister-chromatids broken at the same position. This result argues that the DNA lesions that initiate mitotic recombination are a consequence of chromosome breakage in unreplicated DNA, contrary to the common belief that most recombinogenic lesions reflect broken replication forks. We are currently analyzing recombination events that occur in the absence of DNA mismatch repair.
Genetic regulation of genome stability
In wild-type cells, the frequency of genomic alterations of any type (point mutations, deletions, insertions, and chromosome rearrangements) is very low. We are interested in the genes that regulate genome stability. One rationale for this interest is that the cells of most solid tumors have very high levels of chromosome rearrangements (deletions, duplications, and translocations) as well as high levels of aneuploidy. To understand this type of instability, we are examining the chromosome instability associated with various genome-destabilizing conditions in yeast. We are currently concentrating on mutations that affect DNA replication. We have mapped chromosome rearrangements in yeast strains with low levels of DNA polymerase alpha. This mapping indicated that DNA breaks occur in regions of the genome in which replication forks are slowed or stalled. This pattern of recombination events is quite different from that observed in cells with normal replication. In collaboration with Sue Jinks-Robertson’s lab, we have also characterized chromosome alterations in strains with mutations in Topoisomerase I and cells treated with Topoisomerase I inhibitors. Our analysis is currently being extended into strains with mutations affecting Topoisomerase II, and mutations in DNA damage repair checkpoint genes. Our preliminary study shows that loss of Topoisomerase II results in an interesting pattern of chromosome non-disjunction in which chromosomes segregate in a manner similar to the first division of meiosis.
Genetic regulation of genome stability
Although telomeric sequences are usually located at the ends of the chromosome, mammalian chromosomes also have interstitial telomeric repeats (ITSs), and these ITSs are often sites of chromosome rearrangements in tumor cells. In collaboration with Sergei Mirkin’s lab, we developed methods of detecting ITS-induced genome instability in yeast. We are currently examining the effects of mutations in recombination (RAD52, RAD51, MUS81, RAD50, MRE11, LIG4, RAD59), DNA repair (RAD1, MSH2), DNA replication (REV3), and telomere length maintenance (TEL1, RIF1) pathways on the rates and types of ITS-induced events. The goal of this project is to identify the proteins required to initiate DNA lesions at ITSs and the proteins required to catalyze the ITS-associated rearrangements.
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