Neil Hunter - US grants

DESCRIPTION (provided by applicant): Recombination plays an essential mechanical role in meiotic chromosome segregation and defective recombination is linked to infertility, miscarriage and genetic disease in humans. The long term goal is to understand the molecular mechanism of recombination. Meiotic recombination occurs by the programmed formation and processing of DNA double-strand breaks (DSBs). DSB-ends interact sequentially with an homologous chromosome forming first a Single-End Invasion (SEI) and then a double-Holliday Junction (dHJ). The specific hypothesis is that the DSB-to-SEI and SEI-to-dHJ transitions occur via biochemically distinct processes, with unique contributions being made by the Dmc1, Rad51 and Rad52 proteins. DNA physical assays to directly monitor the chemical steps of meiotic recombination in Saccharomyces cerevisiae cells will form the cornerstone of this investigation. The Specific Aims are: 1. To characterize the in vivo roles of Dmc1 and Rad51. Indirect effects of null mutations and lack of in vivo assays to detect relevant strand-invasion products have previously limited our understanding of Dmc1 and Rad51 function. Preliminary experiments have identified conditions in which meiosis progresses efficiently when only Dmc1 or Rad51 is present. The following aspects of Dmc1-only and Rad51-only recombination will be characterized using physical, genetic and cytological assays: (a) the DNA events of recombination;(b) the gene products involved;(c) the distribution of crossovers formed;(d) the ability to promote chromosome pairing and formation of synaptonemal complex. 2. To characterize the mechanism of the SEI-to-dHJ transition. Events following SEI formation are uncharacterized but must include interaction of the second DSB-end, DNA synthesis and ligation. Preliminary evidence suggests the second DSB-end can interact by a process of single-strand-annealing. Physical assays will be used to characterize the following two aspects of the SEI-to-dHJ transition: (a) the roles of proteins implicated in the process of single-strand-annealing will be determined by analyzing a series of mutant strains;(b) the role of DNA synthesis and the involved factors will be established using chemical inhibitors of DNA synthesis and conditional alleles of replication proteins.

[unreadable] DESCRIPTION: Extension of the carious lesion into the matrix of dentine undermines the physical and functional integrity of the tooth and provides an increasingly noxious insult to the underlying pulp tissue. The bacteria associated with this process are not well understood. Currently, there are no reliable means of identifying the status of the pulp tissue underlying a carious lesion and no enhanced therapeutic modalities are available. This seriously impairs the efficacy of current diagnostic and treatment regimes for this clinically important disease. [unreadable] [unreadable] Preliminary studies have used a universal bacterial amplicon based on 16S rDNA to define the major bacterial populations of carious dentine. This work was complemented by real-time polymerase chain reaction quantification of bacterial DMA to determine levels of bacterial species identified by phylogenetic analysis. A complex array of predominantly anaerobic bacteria was demonstrated with characteristic Lactobacillus and Prevotella spp. dominant. Further dissection of the microbial composition of the developing lesion will be enhanced by development of methods for enriching the display in population analysis of low abundance species of interest. The sequence information obtained will be used to design specific oligonucleotide probes for the determination of the spatial localization of organisms within the lesion by fluorescence in situ hybridization combined with confocal microscopy. [unreadable] [unreadable] Experimental analysis will extend to provide information on the mechanisms of bacterial invasion of the Dental pulp at high resolution using both extractive and in situ approaches and relate this to the histopathological responses of the pulps. Data obtained from this study will provide an objective basis for an understanding of the extension of the carious process with potential diagnostic value for predicting pulp response including pulpal death and endodontic infection. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Crossing-over is essential for accurate chromosome segregation during meiosis. In Saccharomyces cerevisiae, crossing-over is specifically promoted by ScZip3, a putative E3-ligase for SUMO. Allelic variants of the human ScZip3 homolog, RNF212, have been linked to changes in genome-wide meiotic recombination rates. To examine the role of mammalian ZIP3/RNF212, we have constructed a Zip3-/- knock-out mouse. Our long-term goal is to understand the roles of post-translational protein modification in meiotic crossing-over. The hypothesis is that ZIP3-promoted SUMOylation of recombination and/or chromosomal proteins promotes meiotic crossing-over. We will test this hypothesis using a combination of genetic, cytological and molecular approaches in both mouse and yeast. The Specific Aims are: 1. To analyze the function of mouse ZIP3. Preliminary immunofluorescence cytology shows that mouse ZIP3 (MmZIP3) localizes specifically to regions of chromosome synapsis. Localization can occur independently of recombination and is deregulated in the absence of synaptonemal complex protein, SYCP1. Immunofluorescence co-staining experiments will be used to test the idea that MmZIP3 normally localizes to sites of recombination. Additional mutant lines will be analyzed to further define the genetic requirements for MmZIP3 localization. Zip3-/- mutant mice will be analyzed in detail using histological and immunofluorescence cytology approaches. 2. To identify meiotic SUMO-conjugates and substrates of yeast and mouse ZIP3 proteins. The spectrum of SUMO-protein conjugates is dramatically altered in yeast zip3 mutants. ScZip3-dependent SUMO- conjugates will be identified by mass spectrometry. In parallel, candidate targets will be examined for ScZip3- dependent SUMOylation. In cases where yeast targets are conserved, these experiments will inform the identification of partners and potential substrates of MmZIP3. We will also utilize yeast 2-hybrid screening and co-immunoprecipitation from testis extracts to identify MmZIP3 partners and substrates. ScZip3- and MmZIP3- dependent in vitro SUMOylation assays will be reconstituted using purified components and used to confirm candidate substrates. 3. To analyze the role of SUMOylation for proteins identified in Specific Aim 2. Site-directed mutagenesis will be used to diminish SUMOylation of identified substrates in yeast and the effects on meiotic recombination and chromosome behavior will be examined using genetic analysis, specialized DNA physical assays, and immunofluorescence cytology. Relevance: Defects in recombination and post-translational modification have been linked to human infertility, miscarriage and genetic diseases, particularly cancer. An understanding of the mechanism and regulation of recombination will therefore help us better understand the etiology of these diseases. PUBLIC HEALTH RELEVANCE: Chromosome pairing and homologous recombination are required for sexual reproduction and chromosome repair. Defects in these processes are linked to human infertility, miscarriage and genetic diseases, particularly cancer. A greater understanding of their mechanism and regulation will help us better understand the etiology of these diseases.

  Summary/Abstract    Homologous recombination is a chromosome repair process that plays essential roles in meiosis, the  specialized cell division that produces gametes. Defects in meiotic recombination are a leading cause of  infertility, pregnancy loss and congenital disease in humans. A major gap in our understanding of meiotic  recombination is the mechanism of the recombination-­associated DNA synthesis (RADS) that is essential to  restore chromosome integrity. This knowledge gap persists because of inherent challenges to studying meiotic  RADS in vivo, in particular the essential nature of DNA replication factors, the need to study RADS in isolation  from chromosomal replication, and the requirement for special assays to measure RADS. These hurdles have  now been overcome using an innovative combination of chemical, real-­time and molecular genetics tools in  budding yeast that enable acute inactivation of essential replication factors specifically during recombination,  and measurement of de novo DNA synthesis. This system utilizes an ATP-­analog sensitive allele of the Cdc7  kinase (cdc7-­as3) to synchronize cells after S-­phase, but before recombination is initiated. Real-­time  inactivation of essential replication factors is achieved using the auxin-­inducible degron (AID) system, which  has been rewired and optimized for use in meiotic cells. To monitor RADS, newly synthesized DNA is labeled,  isolated and quantified using 5-­ethynyl-­2?-­deoxyuridine (Edu) incorporation, biotin-­azide click chemistry,  streptavidin purification and quantitative PCR (qPCR). Exploiting these tools, the long-­term objectives of this  project are to understand the nature, function, mechanism and regulation of RADS. These objectives will be  pursued through three aims. Aim 1 will determine the role of RADS for both the DNA events of meiotic  recombination and the chromosomal events of meiotic prophase using the comprehensive battery of  molecular, genetic and cytological assays uniquely available in budding yeast. Aim 2 will test models of RADS  by delineating the replication factors involved and systematically analyzing their roles. Complementary studies  in mouse will analyze the localization and dynamics of replication factors at sites of recombination. Aim 3 will  identify and characterize factors involved in the recruitment of replication factors to recombination sites and the  regulation of RADS. Immunofluorescence cytology will be used to monitor chromosomal dynamics of  replication factors and determine the genetic requirements for their localization. The timing and extent of RADS  will be analyzed in strains mutant for factors predicted to modulate RADS, including meiosis-­specific  recombination proteins, DNA helicases and topoisomerases. The results of these aims will provide  unprecedented insights into the mechanism and regulation of RADS, filling a major gap in our understanding of  meiotic recombination. These findings will be germane to understanding pathologies associated with human  meiosis, and are expected to define paradigms that are broadly relevant for chromosome repair.            

This award is for support of the conference Genetic Recombination and Genome Rearrangements, to be held August 14-19, 2022 in Steamboat Springs, Colorado. This is a well-established conference that has occurred biennially since 1985. The meeting will include 8 plenary sessions, each of which will have 6 to 8 20 minute presentations. Two thirds of the speakers are by invitation, and the remainder will be from contributed abstracts, with an emphasis on early career scientists. This meeting will have over 200 participants representing all career stages, from trainees to established PI’s. The committee will make a special effort to provide plenary speaking opportunities and short poster talks to diverse junior scientists as well. Notably, all NSF funds will be used to support participation by trainees and young investigators, with an emphasis on those from under-represented groups as well as those with financial need. <br/><br/>The conference will focus on the topic of recombination and its implications for chromosome segregation, DNA repair, genome rearrangement, and mutation. These topics will be explored from multiple perspectives,including structural and biophysical analyses of the proteins involved in recombination, and genomic analyses of the consequences of recombination for genome structure. The meeting will emphasize breakthroughs spurred by cutting-edge approaches from structural biology, molecular genetics, and biochemistry as applied to DNA damage responses, chromatin remodeling, and the general processes of replication and recombination.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.