DNA damage occurs from natural or environmental causes, such as exposure to tobacco smoke or ultraviolet rays from the sun. The cells harboring unrepaired DNA are prevented from multiplying and pushed to cell death through apoptosis pathway. However, if not successful, it may lead to cells multiplying with a mutation and initiation of cancer. A DNA double-stranded break occurs when both the strands of double helix are cleaved and it can lead to the loss of sections of chromosomes or even cell death (Fig 1.). Eukaryotic cells follow a rule of thumb for repair of damaged DNA throughout cell cycle. During the S-phase and G2 phase of cell cycle, when sister chromatid (homologous sequence) is present, Homologous Recombination Repair (HRR) ensues as a faithful repair mode to maintain the genomic integrity of the damaged cell. It prevents loss of heterozygosity during sister chromatid exchange (Fig 2.). The central enzymes in this pathway are RAD54L(paralog), a DNA motor ATPase and RAD51, a recombinase.

Regulation of this HR repair is complex and mediated by multiple enzymes. One of the key questions is how are these enzymes recruited at the damage foci for the repair?  And how these events are regulated in a temporal manner?

  My research focusses in addressing a part of this question through Tousled-like Kinases (TLK) which have been long implicated in DNA repair of mammalian cells. Our novel finding that TLK1 interacts with a central player of HRR, RAD54L has fired up questions on the functional consequence of this interaction? 

We find that following exposure of cells with gamma-irradiation, TLK1 interacts with RAD54L (mammalian cells have two homologs, RAD54L and RAD54B) and this leads to phosphorylation of RAD54L at novel sites. Studies have shown that upon DNA damage induction, RAD54L interacts with its partner Rad51, a recombinase, to mediate strand exchange with donor template. Disassembly of Rad51 post strand exchange marks the completion of HRR. My current study focusses on probing these sites of phosphorylation of RAD54L to find functional consequence on HRR. We use various techniques in lab like biochemical techniques (immunoprecipitation and western blotting), molecular cloning, cell biology-based assays (DR-GFP or HR assay and Proximity Ligation Assay) with the help of epifluorescence microscopy to address these fundamental mechanistic questions. We expect TLK1-RAD54L axis will enlighten a unique regulation mechanism in the field of DNA damage repair and this will be helpful to understand the cancer biology in a better way (Fig 3.).

Working model in progress: (developed from Heyer et al., 2006. Rad54: the Swiss Army knife of homologous recombination?)