The Function of Backup Pathways of Non-Homologous End Joining in Relation to Growth State and Lesion Quality in Cells of Higher Eukaryotes

DNA double strand breaks (DSBs) are toxic lesions generated by various endogenous processes (like free radicals, V(D)J recombination etc.) and by exogenous agents such as ionizing radiation (IR) and radiomimetic drugs. DSBs are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ). Defects in any of the pathways of DSB repair lead to an increase in cell death, as well as to genomic instability that eventually leads to cancer. Homologous recombination occurs only during late S and G2 phase of the cell cycle when sister chromatids are available in close proximity. In contrast, NHEJ is the major pathway for DSB repair as it functions throughout the cell cycle and does not require a homologous chromosome or chromatid. NHEJ repairs broken DNA ends with no homology requirement, although it occasionally uses micro-homologies. During this process, broken DNA ends are instantly captured by the Ku heterodimer (Ku70/80) that recruits DNA-PKcs to the site of the DSB. DNA-PKcs changes its conformation at the site of the DSB and dimerises to generate a scaffold for subsequent processing of the DNA ends. At the end of this process compatible ends are ligated by the Ligase IV/XRCC4 complex. This reaction is stimulated by XLF/Cernunnos, which interacts with XRCC4. We refer to this pathway as D-NHEJ to indicate its dependence on DNA-PK. In many cases, DNA ends are not compatible and need processing before ligation. Ionizing radiation, for example, induces a large number of ends that contain damaged bases and/or DNA backbone sugars that need processing before ligation. DNA polymerases (µ, λ), polynucleotide kinase and the Artemis nuclease are responsible for this end processing and the occasionally required subsequent DNA polymerization. Cells with mutations in components of D-NHEJ still remain capable of repairing the majority of IR induced DSBs albeit with slower kinetics and in an error prone manner. Unexpectedly, this pathway is not sensitive to mutations in genes required for homologous recombination repair (HRR). We therefore, refer to this form of DSB repair as a distinct form of end joining that is normally suppressed by D-NHEJ. However, when D-NHEJ is genetically or chemically compromised, this form of end joining acts like a backup and repairs the majority of DSBs in the genome, albeit slower and often incorrectly. We proposed the term B-NHEJ for this form of nonhomologous end joining to differentiate it from D-NHEJ and to indicate its putative backup function. The operation of B-NHEJ could be demonstrated at the chromosomal level using human peripheral blood lymphocytes and inhibitors of D-NHEJ. Biochemical studies implicate DNA Ligase III in B-NHEJ and suggest that the repair module PARP-1/XRCC1/DNA Ligase III, already known to be involved in single strand break (SSB) repair, also contributes to DSB repair. I have investigated the cell cycle dependence of B-NHEJ and found that B-NHEJ is enhanced in the G2 phase of the cell cycle. Further studies that form one focus of this thesis examine the growth state dependence of B-NHEJ. I found that B-NHEJ is compromised as cells enter the plateau phase of growth. I examined the generality of this observation using diverse D-NHEJ mutants, as well as in wild type cells after chemical inhibition of D-NHEJ. Interestingly, DNA-PKcs mutant and knockout cell lines do not show this inhibition in DNA DSB repair capacity in plateau phase of growth. This result indicates towards pathways regulatory activity of DNA-PKcs. Furthermore, I examined the consequences of this growth-state-dependent B-NHEJ inhibition by investigating cell radiosensitivity to killing. Changes in chromatin structure were investigated as possible causes of the observed effect, as well as changes in growth factor signaling. Finally, the thermal stability of lesions causing DSBs and their reparability by B-NHEJ and D-NHEJ is investigated. We found that thermally sensitive lesions transformed into breaks even at 37°C inside the cells too. The results uncover important properties of B-NHEJ that require further in depth investigation and point to characteristics of the chemical alterations in the DNA that underlie DSBs that need further characterization.


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