Project Summary/Abstract Meiotic recombination is essential for the reductional cell division in mammalian germ cells and thus for the development of haploid gametes, i.e., sperm and eggs. Recombination is initiated by hundreds of DNA double- strand breaks (DSBs) introduced genome-wide that are catalyzed by the SPO11 protein. Faithful transmission of the genome to subsequent generations requires proper repair of these numerous DSBs, primarily through recombination with the homolog. DSB formation is regulated in meiotic cells by the ATM kinase, which is known to be a primary responder to DSBs in mitotic cells, such that in the absence of ATM, meiotic DSBs increase ~10-fold. We recently discovered that meiotic DSBs are at risk for provoking germline rearrangements, in particular deletions and tandem duplications involving nonhomologous end-joining, especially in the absence of ATM. These events are consequential in terms of disrupting the genes in which these hotspots occur as well as the associated PRDM9 binding sites that govern recombination at those loci. Thus, our findings reveal a previously hidden potential for germline mutagenesis that is likely to affect human health and genome evolution. In humans, recent long-range sequencing of Icelanders supports this impact. This proposal pursues aims to understand the mechanisms that give rise to these events, the range of events at meiotic DSBs, and the effect of age. We hypothesize that other rearrangements are possible at meiotic DSBs than what we have previously identified. Thus, in the first aim, we propose to determine the range of mutagenic outcomes that can arise from meiotic DSBs, including long-range deletions and duplications and chromosomal translocations. In the second aim, we examine factors that may impact the formation of deletions. We focus on the effect of DNA end processing at two steps, SPO11 removal and end processing, and recombination. Further, we address whether gaps formed at nearby DSBs are substrates for homologous recombination and the impact of paternal age in the rearrangement events at meiotic DSBs.