UROP Symposium 2024

Interplay Between the DNA Nucleases MRE11 and Artemis During Normal and Aberrant Lymphocyte-Specific Programmed DNA Rearrangements

Delaney Christy1,2, McKenna DeFoer3, Andrea Hartlerode3, JoAnn Sekiguchi, Ph.D3

1 Undergraduate Research Opportunity Program, 2 Molecular, Cellular, and Developmental Biology Program, 3 Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA 

Defects in DNA double-strand break (DSB) repair can cause human genetic disease with phenotypes including immunodeficiency, cancer, and genome instability. The MRN complex, consisting of MRE11, RAD50, and NBS1, binds DSB ends, can initiate the DNA damage response, and has DNA repair functions. MRN is present at programmed DSBs produced during V(D)J recombination, a process that forms antigen-binding receptor genes through DNA rearrangements in developing lymphocytes. However, functions of MRN during V(D)J recombination are unknown. These DSBs contain hairpin coding ends and blunt signal ends that are repaired by the classical non-homologous end joining (C-NHEJ) pathway. The Artemis nuclease is critical for opening hairpin coding ends. Hypomorphic, recessive, disease-associated alleles ArtemisP70 and Mre11 ATLD1 result in increased interchromosomal transrearrangements, a rare and abnormal V(D)J rearrangement that can cause genome instability and immunodeficiency. To determine if MRE11 and Artemis function coordinately to suppress aberrant rearrangements, levels of interchromosomal transrearrangements between TCRβ and TCRγ loci - genetic coding segments located on different chromosomes - were assessed by nested PCR using genomic DNA from lymphocytes of single- and double-mutant ArtemisP70 and Mre11 ATLD1 mice. PCR products were visualized using gel electrophoresis, and rearrangements were validated using Southern blotting. If double-mutant mice have TCRγ-TCRβ interchromosomal transrearrangement levels consistent with those observed in single-mutant Mre11 ATLD1 or ArtemisP70 mice, it could indicate that MRE11 and Artemis operate in the same pathway during V(D)J recombination. If double-mutant mice have rearrangement levels greater than those observed in single-mutant mice, it could indicate that MRE11 and Artemis operate in different pathways during V(D)J recombination. Results of this study may lead to a better understanding of the functions of these two key DSB repair nucleases and mechanisms underlying disease phenotypes in patients.

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