Epigenetic modifications, such as histone modifications, DNA methylation status, and non-coding RNAs (ncRNA), all contribute to antibody maturation during somatic hypermutation (SHM) and class-switch recombination (CSR). Histone modifications alter the chromatin landscape and, together with DNA primary and tertiary structures, they help recruit Activation-Induced Cytidine Deaminase (AID) to the immunoglobulin (Ig) locus. AID is a potent DNA mutator, which catalyzes cytosine-to-uracil deamination on single-stranded DNA to create U:G mismatches. It has been shown that alternate chromatin modifications, in concert with ncRNAs and potentially DNA methylation, regulate AID recruitment and stabilize DNA repair factors. We, hereby, assess the combination of these distinct modifications and discuss how they contribute to initiating differential DNA repair pathways at the Ig locus, which ultimately leads to enhanced antibody–antigen binding affinity (SHM) or antibody isotype switching (CSR). We will also highlight how misregulation of epigenomic regulation during DNA repair can compromise antibody development and lead to a number of immunological syndromes and cancer.
DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and regulatory components can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we have developed a quick, safe, reproducible, cost-effective, and real-time nuclease assay toolkit that could be used for small-and large-scale experimental assays. Additionally, these data can be analysed to determine each reaction's unique enzyme kinetics. We have designed a library of DNA and RNA substrates that can be used to study resecting nucleases and nickases, conferring the ability to ascertain substrate preference and enzyme directionality. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended this assay to consider analysing the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, and RNA:DNA nucleases, which are also involved in DNA repair and immune regulation, and have been associated with various disease conditions, including cancer and immune disorders.
DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA nucleases. These nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.
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