**3.4 Future perspectives**

488 DNA Repair

Another possibility is that XRCC4 moves to a DSB site autonomously due to its intrinsic DNA end-binding activity (Modesti et al., 1999). Furthermore, XRCC4 was shown to interact with polynucleotide kinase (PNK) (Koch et al., 2004) or aprataxin (APTX) (Clements et al., 2004), depending on the phosphorylation by casein kinase II. Unexpectedly, unphosphorylated XRCC4 interacts with PNKP, although with a lower affinity, but CKII mediated XRCC4 phosphorylation inhibited the PNKP activity (Mani et al., 2010). In addition, XRCC4 has been shown to undergo monoubiquitination (Foster et al., 2006) and SUMOylation (Yurchenko et al., 2006), the former of which was shown to be DNA damageinducible. The role of such posttranslational modifications on the chromatin-recruitment of

Additionally, studies by several groups have suggested that NHEJ is more sophisticated than thought initially and involves many proteins other than DNA-PKcs, Ku, XRCC4-DNA ligase IV, XLF/Cernunnos. In order to investigate into the entirety of the complex compounding several molecules from NHEJ and particularly from other repair or physiological pathways: XRCC4 associated complex bound to chromatin, supposedly at the

One of the other speculations is that the unwinding may be carried out by Ku since it possesses helicase activity in an ATP dependent manner (Blier et al., 1993) and is supposedly the earliest protein in repair hierarchy. Certain studies have shown a functional interaction between the Ku heterodimer and WRN (Karmakar et al., 2002) emphasizing its significance in DNA repair and metabolism pathways. The exonuclease but not the helicase activity of WRN is stimulated by physical interaction with XRCC4-ligase IV (Kusumoto et

ATM and Artemis, together with Nbs1, Mre11 and 53BP1, function in a subpathway of NHEJ that repairs approximately 10% of DSBs, probably those require DNA end processing (Riballo et. al, 2004). Another study suggested three parallel, but mutually crosstalking, pathways of NHEJ, *i.e.*, core pathway mediated by DNA-PKcs and Ku, ATM-Artemis pathway and 53BP1 pathway, all of which finally converge on XRCC4-DNA ligase IV (Iwabuchi et al., 2006). Recent studies indicated the requirement of chromatin remodeling factors, like ALC1 and ACF1, for the recruitment of NHEJ molecules to DSB (Ahel et al.,

It will be of interest to investigate whether all of the above mentioned proteins play some role in the recruitment of XRCC4-DNA Ligase IV to DSB sites or, conversely, are recruited to DSB sites through interaction with XRCC4. This entire conglomerate of proteins has yet to reveal complex mechanisms and cross-talk between other repair and

These questions may be addressed by examining the chromatin-recruitment of deletion or point mutants of XRCC4 and by applying siRNA or inhibitors of the above listed molecules in experimental systems. They could then be optimized for use as adjuvants in

Proteomic analysis is one of the vital instruments to examine any kinase network involving *in vivo* substrates. Such modern technologies have helped to understand that the DNA damage repair response is much sophisticated and complicated than anticipated earlier. It connects NHEJ with chromatin remodelling as well as transcription processes which are also pivotal to cellular functions; thereby aspiring to investigate the cross-talks involved in the

last step of resealing the DNA nicks and gaps, can be isolated and analysed.

XRCC4 is of another interest.

al., 2008).

2009; Lan et al., 2010).

cellular pathways.

radiotherapy.

repair mechanics.

There have been several studies including ours, demonstrating various mechanisms for the dynamics and assembly of the repair machinery on the damaged DNA site in response to various forms of endogenous and exogenous stress. A certain study also suggests that the DNA damage response does not require the DNA damage but the stable association of the repair factors for a prolonged period of time with chromatin which is likely a critical step in triggering, amplifying and maintaining the DNA damage response signal (Soutoglou and Misteli, 2008). It will thus be interesting to investigate the capricious questions as to what are the exact signalling mechanisms to trigger the DSB repair response or the role of several macromolecules involved in different cellular processes. Thus, the assembly of nonhomologous end joining protein complex at DSB was not as simple as thought in classical models and further studies are warranted to fully elucidate the processes. Another important aspect, not clarified, is to understand the hierarchy and mechanism of the disassembly of the repair machinery, involved in NHEJ or from cross-talk pathways, from the site of refurbished DNA. Finally, understanding the mechanisms of DNA repair at molecular levels might bring us a new approach to be applied in cancer radiotherapy or chemotherapy.
