Phosphorylation of DNA-PKcs at the S2056 cluster ensures efficient and productive lymphocyte development in XLF-deficient mice

The non-homologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of Kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters – PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But mice carrying alanine substitution at all 5 serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. XLF is a non-essential NHEJ factor. Xlf-/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, MRI, etc.) or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf-/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class switch recombination junctions from DNA-PKcsPQR/PQRXlf-/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation. Overall design: To test whether the presence of XLF masks the role of DNA-PKcs PQR phosphorylation on chromosomal NHEJ, we generated DNA-PKcsPQR/PQRXlf-/- mice. In contrast to single deficient mice, DNA-PKcsPQR/PQRXlf-/- mice display severe lymphocytopenia consistent with V(D)J recombination defects. HTGTS of chromosomal V(D)J recombination junctions reveals markedly increased end-resection, large deletions, and severely reduced SJ fidelity to less than 10%. CSR to IgG1 is also further decreased in DNA-PKcsPQR/PQRXlf-/- cells than in the Xlf-/- control.

非同源末端连接（non-homologous end-joining, NHEJ）通路是哺乳动物体内主要的DNA双链断裂修复通路，对淋巴细胞发育至关重要。Ku70与Ku80异二聚体（Ku70 and Ku80 heterodimer, KU）启动NHEJ过程，进而招募并激活DNA依赖蛋白激酶催化亚基（DNA-dependent protein kinase catalytic subunit, DNA-PKcs）。尽管DNA-PKcs缺失仅会轻度削弱末端连接反应，但表达激酶失活型DNA-PKcs会完全阻断NHEJ通路。活化的DNA-PK可在两个簇区——S2056（小鼠中为S2053）附近的PQR簇以及T2609附近的ABCDE簇——磷酸化DNA-PKcs。S2056簇位点的丙氨酸替换在质粒检测实验中仅会轻度损害末端连接效率。但携带S2056簇全部5个丝氨酸残基丙氨酸替换的小鼠（DNA-PKcs^PQR/PQR）未表现出淋巴细胞发育缺陷，使得S2056簇磷酸化的生理学意义仍不明确。
XLF是一类非必需的NHEJ因子。Xlf^-/-小鼠体内存在大量外周淋巴细胞，但该群体可因DNA-PKcs缺失、相关的共济失调毛细血管扩张突变激酶（ataxia telangiectasia mutated, ATM）缺失、其他染色质相关DNA损伤应答因子（如53BP1、MDC1、H2AX、MRI等）缺失或RAG2 C端区域突变而完全消失，这提示存在功能冗余现象。尽管ATM抑制不会进一步削弱末端连接效率，但本研究发现，在XLF缺陷的背景下，DNA-PKcs S2056簇磷酸化对正常淋巴细胞发育至关重要。
来自DNA-PKcs^PQR/PQR Xlf^-/- B细胞的染色体V(D)J重组效率可观，但常伴随大片段缺失，进而损害淋巴细胞发育。DNA-PKcs^PQR/PQR Xlf^-/-小鼠的类别转换重组（class switch recombination, CSR）连接位点效率更低，且残留连接位点的保真度下降、缺失片段增多。上述研究结果证实了DNA-PKcs S2056簇磷酸化在生理性染色体NHEJ中的作用，表明S2056簇磷酸化参与介导XLF与DNA-PKcs在末端连接过程中的协同效应。
总体实验设计：为验证XLF的存在是否掩盖了DNA-PKcs PQR磷酸化在染色体NHEJ中的作用，我们构建了DNA-PKcs^PQR/PQR Xlf^-/-小鼠。与单缺陷型小鼠相比，DNA-PKcs^PQR/PQR Xlf^-/-小鼠表现出严重的淋巴细胞减少症，这与V(D)J重组缺陷相符。对染色体V(D)J重组连接位点进行高通量全基因组易位测序（high-throughput genome-wide translocation sequencing, HTGTS）分析发现，末端切除、大片段缺失显著增加，信号连接（signal joint, SJ）保真度严重降至10%以下。与Xlf^-/-对照组相比，DNA-PKcs^PQR/PQR Xlf^-/-细胞向IgG1的类别转换重组效率进一步降低。