STABILITY OF GAA/TTC AND CTG/CAG TRINUCLEOTIDE REPEATS DURING BREAK-INDUCED REPLICATION IN YEAST

poster abstractSeveral human neurodegenerative disorders are caused by the expansion of trinucleotide repeats within or near the region of genes. To study the sta-bility of trinucleotide repeats in eukaryotic cells, we insert different number of GAA and CTG repeats in both orientations separately into the genome of Saccharomyces cerevisiae. In addition to S-phase replications, DNA can also be repaired by break-induced replication (BIR), an important process of DNA repair system that has been implicated in various chromosomal instabilities. In this study, we want to analyze the stability of trinucleotide repeats asso-ciated with BIR

PIF1 HELICASE AND POLYMERASE ZETA (ζ) CHARACTERIZE TWO PATHWAYS OF MUTAGENESIS ASSOCIATED WITH BREAK INDUCED REPLICATION.

poster abstractThe fidelity of DNA synthesis differs among the various processes in which it is involved. While normal S-phase DNA replication is highly accu-rate, DNA synthesis associated with DNA repair is often error-prone. Recent-ly, we have analyzed the accuracy of Break-induced replication, which is a unique cellular process that mimics normal DNA replication in its processivity and rate, but is initiated at double-strand breaks (DSBs) rather than at repli-cation origins. We have demonstrated that BIR is associated with approxi-mately a thousand-fold increase of the rate of frameshift mutations as com-pared to spontaneous events. Here we have identified 5’ – 3’ helicase Pif1p and translesion polymerase Pol ζ as the two major components in promoting frameshift mutations associated with BIR. We have also employed a rever-sion assay using base substitution reporter ura 3-29 to demonstrate that BIR elevates base substitution mutations by a fold of 400 over normal DNA repli-cation. This mutagenic character led us to explore the mode of repair synthesis associated with BIR.Our data suggests that BIR maybe following an unusual, conservative mode of synthesis very different from the usual semiconserva-tive mode of synthesis followed by normal S-phase DNA replication

ROLE OF CHECKPOINT PROTEINS IN THE SUCCESS OF BIR

poster abstractBreak-induced replication (BIR) is an important homologous recombina-tion (HR) pathway employed to repair DNA lesions and has been implicated in various chromosomal instabilities, including loss of heterozygosity, trans-locations, and alternative telomere lengthening. Here, we study the role of checkpoint proteins in DNA repair in yeast Saccharomyces cerevisiae. Cell cycle checkpoints are required for the proper progression of the cell cycle. These checkpoint proteins sense problems during the cell cycle and halt pro-gression to allow mistakes to be corrected and the loss of checkpoint con-trols leads to major defects. RAD9 and RAD24, two important checkpoint proteins play a vital role in arresting the cell cycle upon DNA damage and are also responsible for bringing together the DNA repair machinery. We ob-served that mutations made in the genes encoding RAD9 and RAD24 result-ed in the formation of multiple sectors in individual colonies where, every in-dividual sector repaired differently. We analyze the frequency of different re-pair outcomes associated with BIR in these multi-sectored events. We also report that defective BIR in these checkpoint mutants lead to formation of half-crossovers similar to NRTs reported in mammals, which are implicated in the initiation of cascades of genomic instability characteristic of human cancer cells. 1Department of Environmental and Radiological Health Sciences, College of Veterinary Medi-cine & Biomedical Sciences, Colorado State University, Fort Collins, CO 80523

CHARACTERIZATION OF MOLECULAR MODE OF DNA SYNTHESIS DURING BREAK INDUCED REPLICATION

poster abstractAbnormal repair of DNA double strand breaks can lead to Gross Chromosomal Rearrangements (GCRs) that are the root cause for abnormal genetic and cellular functions that lead to cancer. DSBs left with only one of the two broken DNA ends must be repaired by Break Induced Replication (BIR). BIR requires extensive DNA replication which is very mutagenic. BIR model proposes that the junction made between the invading broken chromosome and the donor molecule substitutes for the origin of DNA replication. This initiates the assembly of a replication fork, which copies the donor sequence till the end of the donor chromosome. The as-sembly of a replication fork and the mode of DNA replication during BIR remains untested. Our study using 2-Dimensional electrophoresis demonstrates that BIR follows an unusual type of DNA synthesis forming “bubble” like replication inter-mediates. Also our result using molecular combing experiments demonstrates that BIR follows a “conservative mode” of DNA synthesis. This unusual kind of DNA replication could explain the highly mutagenic nature of BIR

MMBIRFinder: A Tool to Detect Microhomology-Mediated Break-Induced Replication

The introduction of next-generation sequencing technologies has radically changed the way we view structural genetic events. Microhomology-mediated break-induced replication (MMBIR) is just one of the many mechanisms that can cause genomic destabilization that may lead to cancer. Although the mechanism for MMBIR remains unclear, it has been shown that MMBIR is typically associated with template-switching events. Currently, to our knowledge, there is no existing bioinformatics tool to detect these template-switching events. We have developed MMBIRFinder, a method that detects template-switching events associated with MMBIR from whole-genome sequenced data. MMBIRFinder uses a half-read alignment approach to identify potential regions of interest. Clustering of these potential regions helps narrow the search space to regions with strong evidence. Subsequent local alignments identify the template-switching events with single-nucleotide accuracy. Using simulated data, MMBIRFinder identified 83 percent of the MMBIR regions within a five nucleotide tolerance. Using real data, MMBIRFinder identified 16 MMBIR regions on a normal breast tissue data sample and 51 MMBIR regions on a triple-negative breast cancer tumor sample resulting in detection of 37 novel template-switching events. Finally, we identified template-switching events residing in the promoter region of seven genes that have been implicated in breast cancer

Molecular and Cellular Mechanisms of M. tuberculosis and SARS-CoV-2 Infections-Unexpected Similarities of Pathogenesis and What to Expect from Co-Infection

Tuberculosis is still an important medical and social problem. In recent years, great strides have been made in the fight against M. tuberculosis, especially in the Russian Federation. However, the emergence of a new coronavirus infection (COVID-19) has led to the long-term isolation of the population on the one hand and to the relevance of using personal protective equipment on the other. Our knowledge regarding SARS-CoV-2-induced inflammation and tissue destruction is rapidly expanding, while our understanding of the pathology of human pulmonary tuberculosis gained through more the 100 years of research is still limited. This paper reviews the main molecular and cellular differences and similarities caused by M. tuberculosis and SARS-CoV-2 infections, as well as their critical immunological and pathomorphological features. Immune suppression caused by the SARS-CoV-2 virus may result in certain difficulties in the diagnosis and treatment of tuberculosis. Furthermore, long-term lymphopenia, hyperinflammation, lung tissue injury and imbalance in CD4+ T cell subsets associated with COVID-19 could propagate M. tuberculosis infection and disease progression

Identification of autoimmune markers in pulmonary tuberculosis

IntroductionPathogenesis of many autoimmune diseases is mainly promoted by poorly regulated and/or wrong targeted immune response to pathogens including M. tuberculosis. Autoimmunity is one of the processes with are characteristics of tuberculosis (Tbc). The aim was to determine the autoimmune clinical and immunological features in patients with pulmonary Tbc.Materials and methodsA prospective comparative study was performed in 2017 – 2019 with the inclusion of 46 patients with Tbc. The trigger factors and clinical manifestations, autoantibodies, peripheral blood B cell subsets were stained with fluorochrome-conjugated monoclonal antibodies. 40 healthy volunteers in the control group, were matched for age with no chronic diseases, contacts with TB patients and changes in their laboratory parameters. A statistical analysis was done with GraphPad Prism 6, Statistica 10 (Statsoft) and MedCalc – version 18.2.1 values.ResultsThere were no significant ASIA triggers in Tbc patients and control group. 21.1% of Tbc patients had a high level of a rheumatoid factor and in 47.4% complement system factor C3 was high; anti-MCV was detected in 60.7% of Tbc patients. Relative and absolute frequencies of “naïve” Bm1 cells and eBm5 were significantly decreased and activated pre-germinal-center Bm2’ cells were significantly increased in Tbc patients. The CD24++CD38++ B cells were increased in Tbc vs control group (10.25% vs 5.42%), p < 0.001, and 19 cell/1μL (10; 290 vs 11 cell/1μL (6; 20), p = 0.029, respectively). The frequency of CXCR3+CCR4– Tfh1 cells was significantly lower in Tbc vs control one (26.52% vs. 31.00%, p = 0.004), while CXCR3–CCR4+ Tfh2 cells were increased in Tbc (20.31% vs. controls (16.56%, p = 0.030). The absolute numbers of Tfh1 cells were decreased in the Tbc vs. control (24 cell/1μL vs. 37 cell/1μL p = 0.005).ConclusionThe results of our study showed that the detection of a rheumatoid factor, the components of complement system and anti-MCV in complex with alterations in B cells and follicular Th cell subsets may indicate a presence of autoimmunity in the pathogenesis of tuberculosis, but they are not specific. The indicators of autoimmune-related provide new opportunities in the Tbc treatment

RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends

Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent

Defective Resection at DNA Double-Strand Breaks Leads to De Novo Telomere Formation and Enhances Gene Targeting

The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2–4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Δ exo1Δ mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Δ exo1Δ, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Δ exo1Δ cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair