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.

Fig 1. Different sources of DNA damage that elicits specific DNA repair pathways. Defects in any of these pathways leads to physiological disorders. (Adapted from Kiwerska and Szyfter, Journal of Applied Genetics 60, 329–334, 2019)

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? 

Fig 2. Rule of DNA repair in cells throughout cell cycle. NHEJ pathway occurs in G1 phase and also throughout cell cycle. HRR, predominant in S and G2 phase, restores genomic integrity. (Adapted from Shibata.A and Jeggo P.A, Clinical Oncology 26, 243-249, 2014)

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?)

Fig 3. During DSB damage induction, RAD51 enters nucleus via transporters.

Step1: DNA DSB is recognized by exonucleases and end-resected to generate 3’ overhangs.
Step2: Multiple RAD51 molecules are recruited on the 3’ overhangs by factors like BRCA2-PALB2 complex or RAD51C paralog to form RAD51 nucleofilament.
Step3: RAD51 nucleofilament formation is synergistically stabilized by UAF1-RAD51AP1
Step4: RAD54 interacts with RAD51 in presence of various cellular factors (NUCKS1 in diagram).
Step5: We hypothesize phosphorylated RAD54 ( by TLK1) regulates the function of RAD54 in stabilizing RAD51nucleofilament and strand -exchange activity of RAD51 to D loop formation.


Ishita Ghosh Research