Understanding The Repair Mechanisms at Ionizing Radiation-induced Damage in The Human Genome

Ionizing radiation (IR) such as X-rays induce damage clusters in the genome that include DNA double-strand breaks (DSB) with unligatable dirty ends, along with more frequent oxidized bases and single-strand breaks (SSB). While nonhomologous end joining and homologous recombination are major DSB repair pathways which have been extensively characterized over the past decades, contribution of error-prone alternative end joining (Alt-EJ) at X-ray-induced DNA damage is poorly characterized and underestimated. Moreover, how repair of oxidative base lesions and DSB are coordinated at damage clusters is an important unanswered question. I used recircularization of linearized plasmid reporters to monitor repair of DSBs with 3P-blocked termini, which mimic X-ray-induced strand breaks, both in cell and in vitro, with repair complexes and measured relative efficiency of NHEJ vs. Alt-EJ based on sequence analysis of the joint site. Although NHEJ was the predominant pathway for DSB repair, Alt-EJ was significantly enhanced in pre-irradiated cells. This stimulation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2) that enhanced the interaction of XRCC1 with the end resection enzymes Mre11 and CtIP. The XRCC1 immunocomplex isolated from U2OS cells had Alt-EJ activity in vitro; this activity was significantly higher in the immunocomplex from pre-irradiated cells. Our studies thus suggest that activation of Alt-EJ proficient repair complexes after irradiation in surviving cells could contribute to radioresistance and could be therapeutically targeted. In a separate study, we showed that there is a hierarchy in repair of DSBs by NHEJ followed by base excision repair of oxidized bases at IR-induced damage clusters, coordinated by scaffold attachment factor-A (SAF-A), that is crucial to maintain genomic integrity.

Name thesis:degree_name

Biochemistry and Molecular Biology (Doctoral)

Level thesis:degree_level

Doctoral

Grantor

The University of Texas Medical Branch at Galveston

Author dc:creator

• Dutta, Arijit

Advisor dc:contributor.advisor

• Sowers, Lawrence C

Committee members dc:contributor.committeemember

• Mitra, Sankar
• Hegde, Muralidhar L
• Hazra, Tapas
• Pazdrak, Konrad
• Wiederhold, Lee R
• Pandita, Tej K

• Clustered DNA damage
• Ionizing radiation
• Double strand break
• Non-homologous end joining
• Alternative end joining
• Genomic instability
• Repair complex

Handle dc:identifier.uri

https://hdl.handle.net/2152.3/11146

OAI identifier oai:identifier

oai:utmb-ir.tdl.org:2152.3/11146