br DNA damage and response DDR DNA in

DNA damage and response (DDR) DNA in cells of living organisms is continuously exposed to devastating factors present endogenously and exogenously which can introduce DNA damage thereby resulting in loss of genome integrity [30]. Genome integrity is affected by DNA replication errors, endogenous genotoxic stress from reactive oxygen species (ROS) produced during cellular metabolism and exogenous insults such ultraviolet light (UV), ionizing radiation (IR) or DNA damaging agents [31] (Fig. 1). Mutations due to DNA damage can cause loss and alterations of gene function that lead to diseases [32]. In order to counter these harmful effects of DNA damage, cells have developed an array of DNA repair mechanisms to remove different types of damages. The mechanisms involved in DNA repair are highly coordinated and are collectively known as DNA damage response (DDR) [30], [33], [34]. DDR senses the damage and initiates the repair mechanism. It involves various proteins whose functions are classified as DNA damage sensors, transducers, mediators and effectors [35]. DDRs are composed of a number of repair proteins and checkpoints that coordinate to form a complex signaling cascade in order to repair DNA damage either by ml to moles calculator arrest providing time for DNA repair or by triggering apoptosis (Fig. 2). DDR also includes chromatin modifications at the sites of DNA damage and transcriptional activation and post translational modifications of DNA repair and checkpoint proteins that influences DNA repair [34], [36], [37]. Chromatin modifications that play a critical role in the repair of DNA damage involves covalent modifications of histones, ATP dependent remodeling of chromatin and incorporation of histone variants [38]. Two key DDR signaling components in mammalian cells are two protein kinases from PIKK (Phosphatidylinositol 3 kinase related kinase) family, ATM (Ataxia Telangiectasia Mutated) and ATR (Ataxia Telangiectasia RAD3 Related) which are recruited and activated by double stranded breaks (DSB’s) and replication protein A (RPA) coated ssDNA respectively [39]. ATR gets activated in response to DNA lesions that generate ssDNA (single stranded DNA), like stalled replication forks whereas ATM gets activated in response to double stranded chromosomal break [40], [41]. On their respective activation, ATM and ATR phosphorylates a network of proteins that are required for the signaling cascade in response to DNA damage [42]. The substrates of ATM includes p53, CHK1 (checkpoint kinase 1), CHK2 (checkpoint kinase 2), the Fanconi anemia protein FANCD2, SMC 1 (part of the cohesion multi-protein complex involved in sister chromatids cohesion), the DNA helicase BLM1, BRCA1 (Breast cancer associated gene 1), histone H2AX (γH2AX), the replication fork associated MCM (mini chromosome maintenance) proteins and the MRN (Mre11, Rad50 and Nbs1) complex [43], [44] (Fig. 2). Similarly ATR phosphorylates CHK1, BRCA1 and BLM. These proteins acts as transducers affecting apoptosis (p53, p38 and CHK2), DNA repair (BLM, BRCA1, H2AX, MRN and SMC1), cell cycle arrest (p53, p38, FANCD2, SMS1, CHK1 and CHK2) [45], [46]. CHK2 and CHK1 on phosphorylation by ATM/ATR reduce cyclin dependent kinase activity (CDK) (Fig. 2). Inhibition of CDK’s retards or arrests cell cycle progression at G1/S, intra S and G2/M cell cycle checkpoints thus increasing the time available for DNA repair before replication and mitosis [44], [45]. CHK1 plays a key role in DDR pathway after it is activated by phosphorylation at Ser 345 and Ser 317 [47]. CHK1 phosphorylates CDC25A phosphatase causing ubiquitination and degradation of CDC25A. In non-phosphorylated state CDC25A de-phosphorylates cyclin dependent kinases CDK1 and CDK2, causing progression through S phase and leads to catastrophic consequences in a cell that harbors DNA damage. CHK1 also phosphorylates CDC25C at Ser 216 causing its cytoplasmic sequestration by 14-3-3 protein and prevents it from acting on CDK1 which results in arrest of cell cycle at G2/M checkpoint [48]. Like CHK1, CHK2 is also activated in response to DNA damage [49]. In response to DSBs, ATM phosphorylates Thr 68 on CHK2 which leads to oligomerization of monomers of CHK2 and subsequent trans-phosphorylation at Thr 383 and Thr 387 that result in its kinase activity. CHK2 then phosphorylates a number of substrates which can regulate apoptosis in response to DNA damage by phosphorylating PML, p53, E2F-1 [45], [50], [51], [52]. Signaling by ATM/ATR also enhances repair by inducing DNA repair proteins during transcription and post transcriptionally by recruitment of repair factors to the damage site. It has been reported that stimuli inducing DNA double strand breaks activates p38 MAPKs leading to its nuclear translocation. Phosphorylation of p38 MAPK within the active site triggers a conformational change which causes p38 MAPK to translocate inside the nucleus. Upon DNA damage p38 MAPK gets phosphorylated that leads to nuclear accumulation of p38 MAPK that induces activation of G2/M cell cycle checkpoint and repair of DNA [53].