Understanding the Sequence Dependence of NHEJ Mediated Double-strand Break Repair, and Identification of Novel DNA Ligase Inhibitors and their Potential Use as Cancer Therapeutics

Repair of DNA breaks is essential for maintenance of genomic integrity. DNA double-strand breaks (DSBs) are considered as the most harmful DNA lesions within the cells which if left unrepaired or are misrepaired, can lead to a wide variety of genetic alterations, chromosomal rearrangements either culminating in oncogenic transformation or apoptosis. Therefore, the cell adopts two major pathways, Homologous recombination (HR) and nonhomologous end joining (NHEJ), in order to repair these DSBs thereby maintaining genome stability. HR mediated DSB repair is error-free and is functional during the S and G2 phases of cell cycle and requires the participation of a sister chromatid to act as a template in the repair process. However, in higher eukaryotes, DSBs are primarily repaired by nonhomologous DNA end joining (NHEJ), which is an error-prone DNA repair mechanism and is active throughout the cell cycle. During NHEJ, KU70/KU80 heterodimer binds to the broken DNA ends and recruits DNA-PKcs, Artemis, and Pol µ or ι to the repair site, resulting in processing of broken DNA ends. Following the end processing, ligation occurs with the help of Ligase IV, XRCC4, XLF and PAXX complex. In general, NHEJ is considered as a DSB repair pathway that does not have a sequence preference and hence thought to be random. Using an oligomer based, cell-free assay system, we evaluated the role of DNA sequence during the end-to-end joining of broken DNA, both at the overhangs and at its flanking regions. Using radiolabeled double-stranded oligomeric DNA substrates, possessing DSBs with different overhang sequences, we observed that there is a distinct difference in joining efficiency of mammalian extracts (rat testis, rat lungs, rat brain and rat heart) when overhangs with G:C rich sequences were studied. In contrast, we observed an enhanced efficiency in DNA end joining when A:T-rich flanking sequences were examined. Specifically, when flanking sequences were homopolymers of A:T or sequences enriched with A:T (adenines and/or thymines), joining efficiency was higher compared to that of random or G:C-rich flanking sequences. In order to investigate the factors that contribute towards the observed sequence specificity of NHEJ, we have overexpressed and purified one of the essential factors involved during NHEJ, the Ligase IV/XRCC4 complex. Ligase IV has a multidomain architecture, consisting of a conserved DNA binding domain at N-terminus and a tandem BRCT domain at C-terminus. The central catalytic domain comprises of adenylation (AD) and oligo-binding (OB-fold) domains. The N-terminal DNA binding domain (DBD) of Ligase IV is crucial for its interaction with DNA. Two hypomorphic mutations (A3V and T9I) in the DBD are observed in Ligase IV syndrome patients. Previous studies suggested that recruitment of Ligase IV/XRCC4 complex is dependent on KU protein complex. Interestingly, we observed that binding of Ligase IV/XRCC4 complex can occur independent of KU70/80, however, in a sequence dependent manner. In the present study, we uncover…