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Title page for ETD etd-09202018-140208

Type of Document Dissertation
Author McCartney, Heather Joan
URN etd-09202018-140208
Title SAN1 - a novel nuclease involved in DNA Interstrand Crosslinnk Repair
Degree PhD
Department Cell and Developmental Biology
Advisory Committee
Advisor Name Title
William Tansey Committee Chair
Jason MacGurn Committee Member
Katherine Friedman Committee Member
Neil Osheroff Committee Member
  • nuclease
Date of Defense 2018-07-13
Availability unrestricted
Interstrand DNA cross-links (ICLs) block both replication and transcription, and are commonly repaired by the Fanconi anemia (FA) pathway. However, FA-independent repair mechanisms of ICLs remain poorly understood. Yeast two-hybrid analysis has identified a novel, uncharacterized Senataxin-interacting protein with 5’ exonuclease activity, and homology to the FEN1 nuclease domain. I found that SAN1 (Senataxin-Associated Nuclease 1) is unable to cut dsDNA, but is active against 5’ overhangs and against ssDNAs. SAN1 cleaves 3 or 7 nucleotide fragments from the 5’ end of ssDNA and utilizes magnesium as a cofactor. SAN1 also possesses a unique and conserved C terminus that has been found to interact with the nuclease domain of SAN1 and is required for activity. SAN1 also acts independently of the FA pathway in response to ICLs. Deletion of SAN1 in HeLa cells and mouse embryonic fibroblasts causes sensitivity to ICLs, which is prevented by re-expression of wild type but not nuclease-dead SAN1. SAN1 deletion causes DNA damage and radial chromosome formation following treatment with Mitomycin C, phenocopying defects in the FA pathway. However, SAN1 deletion is not epistatic with FANCD2, a core FA pathway component. Unexpectedly, SAN1 binds to Senataxin (SETX), an RNA/DNA helicase that resolves R-loops. SAN1-SETX binding is increased by ICLs, and is required to prevent cross-link sensitivity. I propose that SAN1 functions with SETX in a pathway necessary for resistance to ICLs.
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