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Role of Translesion Synthesis DNA Polymerases in SJG-136 Interstrand Crosslink Repair

Ahmed, Arooba(1), Liebau, Andy(2), Zagelbaum-Rose, Jennifer(2), Gautier, Jean(2)  1. Columbia College, Columbia University in the City of New York, New York, 10027 United States 2. Institute for Cancer Genetics, College of Physicians and Surgeons, Columbia University, New York, NY, 10032 USA

DNA Interstrand Crosslinks (ICLs) covalently link opposite strands of DNA and prevent cells from completing crucial processes such as replication and transcription. This quality makes them functional chemotherapeutics. However, exposure to endogenous crosslinking agents has allowed cells to develop mechanisms to repair ICLs. SJG-136, a synthetic pyrrolobenzodiazepine dimer (PBD) which forms ICLs with high efficiency, has been used to study the role of Translesion Synthesis (TLS) DNA polymerases in ICL repair. Preliminary data has shown that concomitant loss of Polymerase Eta and Kappa contribute to an inability of cells to facilitate repair, by a mechanism that is not fully characterized. Comparison of SJG-136 induced RPA and γ-H2AX ICL repair foci in U2OS cells demonstrated that SJG-136 induced ICL repair follows the expected kinetics of repair, as evidenced by other types of DNA damage. Following this confirmation, RPA and γ-H2AX foci were quantified in U2OS Polymerase Eta/Polymerase Kappa double knockout cells. I hypothesize that these cells will show a persistence in ICL repair foci, which would provide the mechanistic insight that defects in ICL repair are likely due to an impairment in the gap filling step by the TLS polymerases. To further determine the role of the TLS polymerases in ICL repair, I am generating a plasmid containing a single SJG-136 crosslink to transfect into cells containing loss-of-function combinations of Polymerase Eta, Kappa, and Iota. I anticipate that ICL repair quantification will reveal hindrance of repair in certain combinations, allowing me to visualize which polymerases are indispensable to replication-independent repair. Altogether, these studies will provide critical insight into the role of these polymerases in ICL repair, which may help determine which patients might be more susceptible to ICL chemotherapeutic treatments.




Additional Abstract Information

Presenter: Arooba Ahmed

Institution: Columbia University

Type: Poster

Subject: Biology

Status: Approved


Time and Location

Session: Poster 3
Date/Time: Mon 4:30pm-5:30pm
Session Number: 3084