NIH grant: Combative or Collaborative? Relationships between homologous and non-homologous pathways of double-strand break repair in human cells (1R35GM161701-01)

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NIH grant 1R35GM161701-01: Combative or Collaborative? Relationships between homologous and non-homologous pathways of double-strand break repair in human cells
Institute: {'code': 'GM', 'abbreviation': 'NIGMS', 'name': 'National Institute of General Medical Sciences', 'admin_org_id': '12500024', 'admin_funding_url': ''}. PI: TANYA T PAULL. Award: $485765.
Start: 2026-08-15T00:00:00.
Summary
DNA damage in the form of double-strand breaks (DSBs) occurs in all organisms and requires
immediate and accurate repair to safeguard the integrity of the genome. In eukaryotes, this
repair occurs through two primary pathways: homologous recombination, requiring replication-
based mechanisms to restore the breakpoint, and non-homologous end joining (NHEJ), which
occurs through ligation. These pathways have been historically viewed as opposing outcomes
that are independent from each other and compete for DNA ends; however, our data suggests
that the protein machines that recognize DNA double-strand breaks interact with and regulate
each other. These interactions have important outcomes: genetic mutations in genes encoding
repair enzymes give rise to cancer predisposition as well as neurological disease in humans.
 This proposal is guided by the view that the DSB repair pathways are not competing with
each other in a simple way, but instead are working together on the same ends through a
sequential series of DNA binding and phosphorylation reactions. In (A), the role of NHEJ in
regulating the initiation of homologous recombination is investigated, focusing on recruitment
and regulation of the Mre11-Rad50-Nbs1 (MRN) complex. We will determine the key interfaces
that regulate interactions between MRN and the NHEJ machinery, with the hypothesis that
phosphorylation of Xrcc4 recruits MRN through an evolutionarily conserved bipartite motif. We
will investigate how and when this interaction is exchanged for a structurally similar interaction
between Nbs1 and phosphorylated CtIP, and will pursue the atomic structure of MRN bound to
DNA ends with NHEJ proteins to understand these interactions at a mechanistic level.
 In (B), we investigate how NHEJ factors recognize breaks during replication, and how
this process is dysregulated in the absence of efficient homologous recombination. Using Cas9-
directed breaks in human cells we will determine if the core NHEJ machinery recognizes one-
ended and two-ended double-strand breaks differently during replication. We will determine how
the loss of homologous recombination factors affects the recruitment of NHEJ proteins during
replication, and will determine the basis of replication-dependent, "toxic NHEJ" that occurs
under these conditions. Lastly, we will map and analyze endogenous DSB break sites that occur
during replication stress in cells lacking homologous recombination, to understand how these
lesions generate the high level of cell death that is observed. Through this analysis we will
understand the functional relationships between NHEJ and homologous recombination at a
molecular level, and determine how these interactions ultimately control repair outcomes.

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