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  • br Conflict of interest statement br Funding br Acknowledgme


    Conflict of interest statement
    Acknowledgments We thank the team at Medical and Collider-Accelerator Departments at BNL and for their support at NSRL. We thank Dr. Janice Pluth and Professor Peter O’Neill for useful discussions related to our work.
    Introduction The DNA damage response (DDR) pathway plays a critical role in maintaining genomic stability and preventing carcinogenesis [1]. DDR invoked by genotoxic stress results in nkh arrest, enhanced DNA repair, changes in transcription, and apoptosis. Activation of the checkpoint arrests the cell cycle to allow repair of the damaged DNA. If the damage is excessive and beyond repair, apoptosis is triggered. NER is a versatile DNA repair pathway that can remove a broad range of structurally unrelated lesions including UV-induced bulky DNA adducts cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) [2]. One sub-pathway of NER, global genome NER (GG-NER), removes damage from the entire genome, whereas DNA damage in the transcribed strand of active genes is preferentially eliminated by transcription-coupled NER (TC-NER) [3]. In GG-NER, damage is recognized by the UV-DDB (DDB1 and DDB2) and XPC-RAD23B complexes [4], [5]. DDB1 participates in NER through DDB2 DNA-binding and cullin 4A ubiquitin ligase activity. The DDB1-CUL4-ROC1 complex ubiquitylates XPC, which may enhance DNA binding by XPC and promotes NER [6], [7]. The DDB complex initially recognizes the CPD lesions and recruits XPC [4], [5], [8], [9], whereas XPC can independently recognize 6-4PP lesions [5]. Cullin 4A-mediated proteolysis of DDB2 protein at DNA damage sites regulates lesion recognition by XPC. In turn, XPC helps in recruiting XPA, XPG, and TFIIH components that enable opening of the DNA helix around the damage site to form a bubble [8]. XPA stabilizes the bubble and aids in positioning the XPF and XPG endonucleases for respective 5′ and 3′ incisions to excise out a 24–32bp oligonucleotide containing damaged lesion. The resulting gap is filled by repair synthesis, and finally the nick is ligated to complete NER [2], [10]. Importantly, the defects in components of the NER pathway result in Xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy which are characterized by sensitivity to UV irradiation and predisposition to skin cancers [11], [12]. The phosphoinositide-3-kinase-like kinases family of protein kinases including ATR (Ataxia telangiectasia- and Rad3-related) and ATM (Ataxia telangiectasia mutated) are the principal checkpoint kinases activated by DNA damage [13], [14]. Seckel (ATR-defective) and AT (ATM-deficient) cells show impaired signaling due to the defects in checkpoint activation. Activation of ATR and ATM triggers a phosphorylation-mediated cascade of events that lead to cell cycle arrest and stimulation of DNA repair. ATR is the primary sensor of single-stranded (ssDNA) breaks (SSB) caused by UV damage and replication stress. It has been shown that DNA damage and replication intermediates increase the unwinding of DNA, leading to the accumulation of RPA-coated ssDNA, which recruits ATR [15], [16]. ATR phosphorylates Chk1, which results in checkpoint activation during G1, S, and G2/M phases. Activated Chk1 phosphorylates Cdc25 phosphatases to inhibit their function, and the cells delay progression through the cell cycle [17]. Although DNA double-strand break (DSB) primarily activates the ATM pathway, recent studies including ours have implicated a participatory role of ATM in the NER pathway [18], [19]. ATM phosphorylates the checkpoint kinase Chk2, which also triggers degradation of Cdc25A phosphatases to delay the cell cycle [20]. ATR and ATM phosphorylate histone H2AX, which spreads along the DNA up to 200–400kb, and helps in the recruitment of proteins involved in DNA damage repair and checkpoint activation [21]. Moreover, ATR- and ATM-mediated phosphorylation of BRCA1 and H2AX [22], [23] is required for S and G2/M phase checkpoints and homologous recombination (HR)-mediated DNA repair during S and G2 phases. During DNA replication, other ssDNA gaps are generated by the stalling of replication forks at unrepaired damage sites. Repair of these gaps may involve post-replicative recombinational repair [24]. If not repaired, stalled fork gaps can evolve into DSB [24]. Besides BRCA1, BRCA2 and Rad51 are also required for HR-mediated DNA repair and replication fork maintenance [25], [26]. Both Chk1 and Chk2 regulate the functional nkh associations between BRCA1, BRCA2, and Rad51 proteins in response to DNA damage, and thus promote HR-mediated repair of stalled replication forks [27], [28].