Macrae Chloe J, McCulloch Richard D, Ylanko Jarkko, Durocher Daniel, Koch C Anne
Ontario Cancer Institute, University Health Network, Toronto, ON, Canada.
DNA Repair (Amst). 2008 Feb 1;7(2):292-302. doi: 10.1016/j.dnarep.2007.10.008.
Nonhomologous end-joining (NHEJ) is the major mammalian DNA double-strand break (DSB) repair pathway of DSBs induced by DNA damaging agents. NHEJ is initiated by the recognition of DSBs by the DNA end-binding heterodimer, Ku, and the final step of DNA end-joining is accomplished by the XRCC4-DNA ligase IV complex. We demonstrate that Aprataxin and PNK-like factor (APLF), an endo/exonuclease with an FHA domain and unique zinc fingers (ZFs), interacts with both Ku and XRCC4-DNA ligase IV in human cells. The interaction of APLF with XRCC4-DNA ligase IV is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. In contrast, APLF associates with Ku independently of the FHA and ZF domains, and APLF complexes with Ku at DNA ends. APLF undergoes ionizing radiation (IR) induced ATM-dependent hyperphosphorylation at serine residue 116, which is highly conserved across mammalian APLF homologues. We demonstrate further that depletion of APLF in human cells by siRNA is associated with impaired NHEJ. Collectively, these results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and XRCC4-DNA ligase IV.
非同源末端连接（NHEJ）是哺乳动物中由DNA损伤剂诱导产生的DNA双链断裂（DSB）的主要修复途径。NHEJ由DNA末端结合异二聚体Ku识别DSB启动，DNA末端连接的最后一步由XRCC4-DNA连接酶IV复合物完成。我们证明，Aprataxin和PNK样因子（APLF），一种具有FHA结构域和独特锌指（ZF）的核酸内切酶/核酸外切酶，在人类细胞中与Ku和XRCC4-DNA连接酶IV相互作用。APLF与XRCC4-DNA连接酶IV的相互作用是FHA和磷酸化依赖性的，并且在体外由CK2对XRCC4的磷酸化介导。相比之下，APLF与Ku的结合独立于FHA和ZF结构域，并且APLF在DNA末端与Ku形成复合物。APLF在丝氨酸残基116处经历电离辐射（IR）诱导的ATM依赖性超磷酸化，该丝氨酸残基在哺乳动物APLF同源物中高度保守。我们进一步证明，通过siRNA耗尽人类细胞中的APLF与NHEJ受损有关。总的来说，这些结果表明APLF是一个ATM靶点，参与NHEJ并促进DSB修复，可能是通过与Ku和XRCC4-DNA连接酶IV相互作用来实现的。
