Role of the Lig3 BRCT Domains in DNA Repair

University Of Chicago, Chicago IL

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Abstract

DESCRIPTION (provided by applicant): DNA ligase IV (Lig4) is an indispensable component of nonhomologous end-joining (NHEJ), a major DNA double-strand break repair pathway, and V(D)J recombination, a process used to generate antigen-specific lymphocyte receptors. These pathways are essential for maintaining genomic integrity and developing a functional immune system. The overall goal of this project is to study the Lig4 BRCT (BRCA1 C-Terminal) domains and their role in coordinating Lig4 activities with other components of the NHEJ complex. Studies will involve two aims: (1) To identify structural elements critical to Lig4 BRCT domain function, BRCT deletion and point mutants will be evaluated with in vivo DNA end-joining and V(D)J recombination assays. (2) To identify Lig4 BRCT-specific interactions and characterize the interaction interface, candidate proteins involved in NHEJ will be assessed for their ability to interact with the Lig4 BRCT domains. Coimmunoprecipitation (Co-IP) and immunofluorescence (IF) will be utilized to evaluate potential candidates that interact with the Lig4 BRCT domains. Interactions will be characterized by performing Co-IP and IF with BRCT mutants, determining if interactions are DNA-dependent and the kinetics of interaction.