HMO2, a yeast HMGB protein that preferentially binds to DNA ends

DNA damage is a common hazard that all cells have to combat. Saccharomyces cerevisiae HMO2 is a high mobility group protein (HMGB) that is a component of the chromatin remodeling complex INO80, which is involved in double strand break repair. I show here using DNA end-joining and exonuclease protection assays that HMO2 binds preferentially to DNA ends. While HMO2 binds DNA with both blunt and cohesive ends, the sequence of a single stranded overhang significantly affects binding, supporting the conclusion that HMO2 recognizes features at DNA ends. Analysis of the effect of duplex length on the ability of HMO2 to protect DNA from exonucleolytic cleavage suggests that more than one HMO2 must assemble at each DNA end. HMO2 binds supercoiled DNA with higher affinity than linear DNA and has a preference for DNA with lesions such as pairs of tandem mismatches; however, comparison of DNA constructs of increasing length suggests that HMO2 may not bind stably as a monomer to distorted DNA. The remarkable ability of HMO2 to protect DNA from exonucleolytic cleavage, combined with reports that HMO2 arrives early at DNA double strand breaks, suggests that HMO2 may play a role in double strand break repair beyond INO80 recruitment. I also found that HMO2 has the ability to mediate both 3Œ and 5Œ DNA strand invasion, which is an essential step in homologous recombination. Also hmo2∆ and hmo2∆rad52∆ have slower growth phenotype in presence of hydroxyurea thus indicating that HMO2 might play important role in recovery of stalled DNA replication forks

Cellular Roles of DNA Polymerase Beta

Since its discovery and purification in 1971, DNA polymerase ß (Pol ߆) is one of the most well-studied DNA polymerases. Pol ß is a key enzyme in the base excision repair (BER) pathway that functions in gap filling DNA synthesis subsequent to the excision of damaged DNA bases. A major focus of our studies is on the cellular roles of Pol ß. We have shown that germline and tumor-associated variants of Pol ß catalyze aberrant BER that leads to genomic instability and cellular transformation. Our studies suggest that Pol ß is critical for the maintenance of genomic stability and that it is a tumor suppressor. We have also shown that Pol ß functions during Prophase i of meiosis. Pol ß localizes to the synaptonemal complex and is critical for removal of the Spo11 complex from the 5’ ends of double-strand breaks. Studies with Pol ß mutant mice are currently being undertaken to more clearly understand the function of Pol ß during meiosis. in this review, we will highlight our contributions from our studies of Pol ß germline and cancer-associated variants

The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

DNA damage is a common hazard that all cells have to combat. Saccharomyces cerevisiae HMO2 is a high mobility group protein (HMGB) that is a component of the chromatin-remodeling complex INO80, which is involved in double strand break (DSB) repair. We show here using DNA end-joining and exonuclease protection assays that HMO2 binds preferentially to DNA ends. While HMO2 binds DNA with both blunt and cohesive ends, the sequence of a single stranded overhang significantly affects binding, supporting the conclusion that HMO2 recognizes features at DNA ends. Analysis of the effect of duplex length on the ability of HMO2 to protect DNA from exonucleolytic cleavage suggests that more than one HMO2 must assemble at each DNA end. HMO2 binds supercoiled DNA with higher affinity than linear DNA and has a preference for DNA with lesions such as pairs of tandem mismatches; however, comparison of DNA constructs of increasing length suggests that HMO2 may not bind stably as a monomer to distorted DNA. The remarkable ability of HMO2 to protect DNA from exonucleolytic cleavage, combined with reports that HMO2 arrives early at DNA DSBs, suggests that HMO2 may play a role in DSB repair beyond INO80 recruitment

Student-Faculty Collaborative Research Grant Report

The project focused on elucidating the mechanism by which cancer-associated variants of Mre11 alter alternative non-homologous end-joining (aNHEJ) repair activity. This project seeks to understand the potential role of cancer-associated variants of DNA repair genes (such as Mre11) in inducing genomic instability. Mutations in Mre11 genes can have a significant impact on cancer prognosis and treatment. Our data from the summer suggests that the three variants of Mre11 are sensitive to bleomycin (BLM) and etoposide and have accumulated double strands breaks (DSBs). This indicates Mre11 variants induce genomic instability. Importantly, our results suggest that the status of Mre11 variants can influence treatment of tumors with BLM and etoposide. Work is ongoing to characterize how the Mre11 variants alter DSB repair mechanisms. Our study will enhance the basic mechanistic understanding of cancer biology and how the DNA repair status of germlines and somatic cells of individuals influences tumorigenesis and therapy

Student-Faculty Collaborative Research Grant Report

This project focused on testing the hypothesis that DNA repair gene variants (GVs) contribute to elevated cancer risk. It seeks to understand the potential role of cancer-associated variants of DNA repair genes (such as Mre11) in inducing genomic instability. During summer 2018, I collaborated with 3 students (Ashley Headrick, Cristina Mateos, and Itzel Romero) and focused on the following aims: 1) to test the hypothesis that tumor-derived variants of Mre11 can drive genomic instability and carcinogenesis, and 2) if the variants of Mre11 have the potential to influence responses to DNA damaging agents which are important for cancer treatment. Our preliminary data from the summer suggests that the three variants of Mre11 are sensitive to DNA damaging agent bleomycin (BLM) and have accumulated double-strand DNA breaks (DSBs). This indicates Mre11 variants induce genomic instability. Importantly, our results suggest that the status of Mre11 variants can impact treatment of tumors with BLM. Likewise, other DNA damaging agents will be tested

Interaction of <i>Saccharomyces cerevisiae</i> HMO2 Domains with Distorted DNA

The <i>Saccharomyces cerevisiae</i> high mobility group protein HMO2 is a component of the chromatin remodeling complex INO80. In this capacity, it has been shown to direct INO80 to DNA double-strand breaks, thereby contributing to double-strand break repair. Consistent with such function, HMO2 binds DNA ends, protecting them from exonucleolytic degradation. We show here that both domains of HMO2, HMO2-BoxA and HMO2-BoxB, bind preferentially to distorted DNA, with HMO2-BoxA binding preferentially to four-way DNA junctions and DNA with tandem mismatches and HMO2-BoxB binding four-way junctions as well as DNA with stem–loop structures, tandem mismatches, and abasic sites. As previously reported for mammalian high mobility group proteins, the acidic C-terminal extension significantly attenuates DNA binding. Notably, the unique ability of HMO2 to protect DNA ends is conferred by the Box A domain. Considering the reported roles for INO80 in other events such as recovery of stalled replication forks and nucleotide excision repair, we assessed the effect of DNA damaging agents on an <i>hmo2</i>Δ strain; while modest growth inhibition is seen upon exposure to UV light, exposure to hydroxyurea, which causes replication fork arrest, induces severe growth deficiency. These data suggest that HMO2 may also participate in directing the INO80 complex to sites such as stalled replication forks; the preferred binding of HMO2 domains to damaged DNA and intermediates in homologous recombination is consistent with such function

Mutations in Mre11 Induce Genomic Instability

Exogenous agents such as ionizing radiation that challenge the DNA and produces various DNA lesions lead to genome instability. These lesions at the DNA level led to changes in genetic information, leading to mutagenesis, which can propagate in subsequent rounds of replication, eventually resulting in the disruption of normal cell function, and uncontrolled cell growth thus forming tumors. DNA double strand breaks (DSBs) are the most lethal type of DNA damage that cells combat. One of the pathways to repair DSBs is homology-dependent repair (HDR). The 3′-to-5′ exonuclease activity of Mre11 generate protruding 3′ ssDNA at DSBs. Further, Rad51, a factor that performs homology search and strand invasion, then binds the ssDNA. Mre11 is responsible for the initial short-range resection which is followed by the long resection by Exo1 before strand invasion in HDR. The purpose of this study is to test the hypothesis that MRE11 gene variants contribute to genomic instability by aberrant DNA repair. This project seeks to understand the potential role of disease-associated variants of Mre11 in defective resection function thus inducing genomic instability

Characterization of Mre11 Gene Variants in Cancer

Among the different damages that a cell can undergo, DNA double-strand break (DSB) is the most detrimental one. DNA contains all the necessary information for a cell\u27s proper functioning and replication; therefore, a break that damages both of its strands completely destabilizes the DNA molecules. If not properly repaired, this could lead to genomic instability and tumorigenesis. Cells repair DSBs by two repair pathways — homology-directed repair (HDR) and non-homologous end-joining (NHEJ). Recently a back-up NHEJ pathway has been reported and is referred to as alternate-NHEJ (Alt-NHEJ). Although Alt-NHEJ is advantageous for damaged cells, it can mutate the DNA sequences at the junctions, which may lead to an altered genome that can have severe biological consequences. Mre11 is an essential component of the MRN complex and plays a key role in DSB repair pathways as HDR, NHEJ and Alt-NHEJ. Mre11 possesses single-strand endonuclease activity and double-strand-specific 3\u27-5\u27 exonuclease activity which are essential for DNA end-processing. Recently Mre11 is shown to be overexpressed in breast cancer and high Mre11 expression was associated with a more malignant behavior in breast cancer. Furthermore, germline mutations of the Mre11 gene was identified in a recent screening of hereditary susceptibility to breast and/or ovarian cancer. Mutations in DNA repair genes can have a significant impact on cancer prognosis and treatment. We are focusing to determine whether germline and somatic mutations of Mre11 can lead to tumorigenesis and alter responses to chemotherapies

Molecular Characterization and Elevated Cancer Risk Associated with DNA Repair Gene Variants

DNA is the repository of genetic information, and its integrity is crucial for genome stability. Several exogenous and endogenous factors damage the DNA and if not repaired lead to mutagenesis, genomic instability, and cancer. Several databases, including 1000 Genomes, Cbioportal, The Cancer Genome Atlas (TCGA), and the Catalog of Somatic Mutations in Cancer (COSMIC), continue to catalog several cancer-associated variants of DNA repair genes. However, the biological functions of these mutations and the effect in response to therapy are not known. We focus on studying: 1) how cancer-associated variants of DNA repair genes (such as MRE11 and POLQ) induce genomic instability; 2) cellular response of the cancer-associated variants to DNA damaging agents; and 3) how cancer-associated variants impact the efficacy of therapeutics. This particular research provides a commentary on the initial molecular cellular assays we employ to generate some of the cancer-associated variants of MRE11 and POLQ in vitro and their expression in mammalian cell lines

Elucidating the role of Exo1 and Mre11 mutations in DNA damage response

DNA double strand breaks (DSBs) are one of the most deleterious types of DNA damage that cells combat. DSBs are processed by the 3′-to-5′ exonuclease activity of the DSB repair nuclease, Mre11, to generate protruding 3′ single stranded DNA (ssDNA) at DSBs. Exonuclease 1 (EXO1) is an evolutionarily, well-conserved exonuclease. Its ability to resect DNA in the 5′-3′ direction has been extensively characterized and shown to be implicated in several genomic DNA metabolic processes such as replication stress response, double strand break repair (DSBR), mismatch repair, nucleotide excision repair and telomere maintenance. Both Mre11 and Exo1 play a critical role in the DNA resection in DSBR. Mre11 is responsible for the initial short-range resection, which is followed by the long resection by Exo1 before strand invasion in DSBR. However, the regulation of this switch between Mre11 and Exo1 is not well understood. The purpose of this study is to test the hypothesis that EXO1 and MRE11 gene variants contribute to genomic instability by aberrant DNA repair. This project seeks to understand the potential role of disease-associated variants of Exo1 and Mre11 in defective resection function, thus inducing genomic instability