Live Cell Studies of DNA Single Strand Break Repair Pathway: The Road Map

• Author / Creator

  Abdou, Ismail

• The repair of damaged DNA is critically important for maintaining a stable cellular environment to ensure proper and high fidelity segregation and transmission of genetic information from one generation to the next. DNA can be subjected to many forms of DNA damage that are handled by dedicated signaling pathways and repair complexes. The most common DNA lesions are DNA single strand breaks (SSBs). Most of our current understanding of DNA SSB repair (SSBR) has been derived from biochemical studies. In our work, we employed live cell imaging techniques in addition to biochemical approaches to better define the steps in this repair pathway, thus providing clearer insight for how SSBR is orchestrated in live cells. Our focus was mainly on (i) how rapidly different SSBR proteins accumulate at sites of DNA damage, (ii) how DNA SSBs are detected, (iii) providing mechanistic explanations for the association of different polymorphisms of the SSBR scaffold protein X-ray cross-complementing protein 1 (XRCC1) with cancer, and (iv) how the DNA SSBR end-processing enzyme, polynucleotide kinase/phosphatase (PNKP) is regulated in response to DNA damage. Importantly, we provide evidence, for the first time in live cells, that DNA ligase III (LIG3), in addition to its established downstream nick sealing activity in SSBR, functions as an alternative SSB sensor to poly-ADP-ribose polymerase 1 (PARP1). Furthermore, we observed that LIG3 and PARP1 detect different types of SSBs. Given the current success of PARP inhibitors targeting PARPs 1 and 2 in cancer therapy, our finding expands the number of potential targets for small molecule inhibitor development and drug intervention. We also show that two of the cancer associated XRCC1 polymorphisms, R194W and R280H, give rise to XRCC1 variant proteins that impede the accumulation and catalytic activity of PNKP at sites of DNA damage. This might lead to increased background mutations in cells harboring these polymorphisms providing a further driving force towards tumorigenesis. Finally, our published data in agreement with others, show that the phosphorylation of two serine residues within the linker region of PNKP tightly regulate the protein behavior at DNA damage sites. We extended this work by carrying out preliminary studies on the behavior of the FHA domain of PNKP in live cells in response to DNA damage.

• Subjects / Keywords

  • LIG3
  • XRCC1
  • DNA Single Strand Break Repair
  • XRCC1
  • LIG3
  • PNKP

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R32J68J93

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.