Nonhomologous End-Joining (NHEJ)

The nonhomologous end joining (NHEJ) pathway is initiated in response to the formation of DNA double-strand breaks (DSBs) induced by DNA-damaging agents, such as ionizing radiation. DNA DSBs are recognized by the MRN complex (MRE11A:RAD50:NBN), leading to ATM activation and ATM-dependent recruitment of a number of DNA damage checkpoint and repair proteins to DNA DSB sites (Lee and Paull 2005). The ATM phosphorylated MRN complex, MDC1 and H2AFX-containing nucleosomes (gamma-H2AX) serve as scaffolds for the formation of nuclear foci known as ionizing radiation induced foci (IRIF) (Gatei et al. 2000, Paull et al. 2000, Stewart et al. 2003, Stucki et al. 2005). Ultimately, both BRCA1:BARD1 heterodimers and TP53BP1 (53BP1) are recruited to IRIF (Wang et al. 2007, Pei et al. 2011, Mallette et al. 2012), which is necessary for ATM-mediated CHEK2 activation (Wang et al. 2002, Wilson et al. 2008). In G1 cells, TP53BP1 promotes NHEJ by recruiting RIF1 and PAX1IP, which displaces BRCA1:BARD1 and associated proteins from the DNA DSB site and prevents resection of DNA DSBs needed for homologous recombination repair (HRR) (Escribano-Diaz et al. 2013, Zimmermann et al. 2013, Callen et al. 2013). TP53BP1 also plays an important role in ATM-mediated phosphorylation of DCLRE1C (ARTEMIS) (Riballo et al. 2004, Wang et al. 2014). Ku70:Ku80 heterodimer (also known as the Ku complex or XRCC5:XRCC6) binds DNA DSB ends, competing away the MRN complex and preventing MRN-mediated resection of DNA DSB ends (Walker et al. 2001, Sun et al. 2012). The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs, PRKDC) is then recruited to DNA-bound Ku to form the DNA-PK holoenzyme. Two DNA-PK complexes, one at each side of the break, bring DNA DSB ends together, joining them in a synaptic complex (Gottlieb 1993, Yoo and Dynan 2000). DNA-PK complex recruits DCLRE1C (ARTEMIS) to DNA DSB ends (Ma et al. 2002). PRKDC-mediated phosphorylation of DCLRE1C, as well as PRKDC autophosphorylation, enables DCLRE1C to trim 3'- and 5'-overhangs at DNA DSBs, preparing them for ligation (Ma et al. 2002, Ma et al. 2005, Niewolik et al. 2006). The binding of inositol phosphate may additionally stimulate the catalytic activity of PRKDC (Hanakahi et al. 2000). Other factors, such as polynucleotide kinase (PNK), TDP1 or TDP2 may remove unligatable damaged nucleotides from 5'- and 3'-ends of the DSB, converting them to ligatable substrates (Inamdar et al. 2002, Gomez-Herreros et al. 2013). DNA ligase 4 (LIG4) in complex with XRCC4 (XRCC4:LIG4) is recruited to ligatable DNA DSB ends together with the XLF (NHEJ1) homodimer and DNA polymerases mu (POLM) and/or lambda (POLL) (McElhinny et al. 2000, Hsu et al. 2002, Malu et al. 2002, Ahnesorg et al. 2006, Mahajan et al. 2002, Lee et al. 2004, Fan and Wu 2004). After POLL and/or POLM fill 1- or 2-nucleotide long single strand gaps at aligned DNA DSB ends, XRCC4:LIG4 performs the ligation of broken DNA strands, thus completing NHEJ. The presence of NHEJ1 homodimer facilitates the ligation step, especially at mismatched DSB ends (Tsai et al. 2007). Depending on other types of DNA damage present at DNA DSBs, NHEJ can result in error-free products, produce dsDNA with microdeletions and/or mismatched bases, or result in translocations (reviewed by Povrik et al. 2012).

非同源末端连接（NHEJ）途径在DNA损伤剂，如电离辐射诱导的DNA双链断裂（DSBs）形成后启动。DNA双链断裂被MRN复合体（MRE11A:RAD50:NBN）识别，导致ATM激活，并依赖于ATM招募大量DNA损伤检查点和修复蛋白至DNA双链断裂位点（Lee and Paull 2005）。ATM磷酸化的MRN复合体、MDC1和含有H2AFX的核小体（gamma-H2AX）作为离子辐射诱导的焦点（IRIF）形成的支架（Gatei et al. 2000, Paull et al. 2000, Stewart et al. 2003, Stucki et al. 2005）。最终，BRCA1:BARD1异源二聚体和TP53BP1（53BP1）被招募至IRIF（Wang et al. 2007, Pei et al. 2011, Mallette et al. 2012），这对于ATM介导的CHEK2激活至关重要（Wang et al. 2002, Wilson et al. 2008）。在G1细胞中，TP53BP1通过招募RIF1和PAX1IP促进NHEJ，这些因子取代BRCA1:BARD1及其相关蛋白从DNA双链断裂位点，并阻止DNA双链断裂的重组修复（HRR）所需的重组（Escribano-Diaz et al. 2013, Zimmermann et al. 2013, Callen et al. 2013）。TP53BP1还在ATM介导的DCLRE1C（ARTEMIS）磷酸化中发挥重要作用（Riballo et al. 2004, Wang et al. 2014）。Ku70:Ku80异源二聚体（亦称为Ku复合体或XRCC5:XRCC6）与DNA双链断裂端结合，竞争性地去除MRN复合体，防止MRN介导的DNA双链断裂端的切除（Walker et al. 2001, Sun et al. 2012）。随后，DNA依赖性蛋白激酶（DNA-PKcs，PRKDC）的催化亚基被招募至结合DNA的Ku，形成DNA-PK全酶。每个断裂两侧都有一个DNA-PK复合体，将DNA双链断裂端拉近，在突触复合体中连接它们（Gottlieb 1993, Yoo and Dynan 2000）。DNA-PK复合体招募DCLRE1C（ARTEMIS）至DNA双链断裂端（Ma et al. 2002）。PRKDC介导的DCLRE1C磷酸化以及PRKDC的自身磷酸化，使DCLRE1C能够修剪DNA双链断裂端的3'-和5'-突出端，为连接做准备（Ma et al. 2002, Ma et al. 2005, Niewolik et al. 2006）。肌醇磷酸的结合还可以进一步刺激PRKDC的催化活性（Hanakahi et al. 2000）。其他因素，如多核苷酸激酶（PNK）、TDP1或TDP2，可能从DSB的5'-和3'-端移除无法连接的损伤核苷酸，将它们转化为可连接的底物（Inamdar et al. 2002, Gomez-Herreros et al. 2013）。DNA连接酶4（LIG4）与XRCC4（XRCC4:LIG4）复合体，连同XLF（NHEJ1）同源二聚体以及DNA多聚酶mu（POLM）和/或lambda（POLL）被招募至可连接的DNA双链断裂端（McElhinny et al. 2000, Hsu et al. 2002, Malu et al. 2002, Ahnesorg et al. 2006, Mahajan et al. 2002, Lee et al. 2004, Fan and Wu 2004）。在POLL和/或POLM填充对齐的DNA双链断裂端的1-或2个核苷酸长度的单链间隙后，XRCC4:LIG4执行断裂DNA链的连接，从而完成NHEJ。NHEJ1同源二聚体的存在有助于连接步骤，尤其是在错配的DSB端（Tsai et al. 2007）。根据DNA双链断裂处存在的其他类型DNA损伤，NHEJ可能产生无错产物，产生具有微缺失和/或错配碱基的dsDNA，或导致易位（Povrik et al. 2012综述）。