Iles Natasha, Rulten Stuart, El-Khamisy Sherif F, Caldecott Keith W
Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, Sussex, United Kingdom.
Mol Cell Biol. 2007 May;27(10):3793-803. doi: 10.1128/MCB.02269-06. Epub 2007 Mar 12.
Aprataxin and polynucleotide kinase (PNK) are DNA end processing factors that are recruited into the DNA single- and double-strand break repair machinery through phosphorylation-specific interactions with XRCC1 and XRCC4, respectively. These interactions are mediated through a divergent class of forkhead-associated (FHA) domain that binds to peptide sequences in XRCC1 and XRCC4 that are phosphorylated by casein kinase 2 (CK2). Here, we identify the product of the uncharacterized open reading frame C2orf13 as a novel member of this FHA domain family of proteins and we denote this protein APLF (aprataxin- and PNK-like factor). We show that APLF interacts with XRCC1 in vivo and in vitro in a manner that is stimulated by CK2. Yeast two-hybrid analyses suggest that APLF also interacts with the double-strand break repair proteins XRCC4 and XRCC5 (Ku86). We also show that endogenous and yellow fluorescent protein-tagged APLF accumulates at sites of H(2)O(2) or UVA laser-induced chromosomal DNA damage and that this is achieved through at least two mechanisms: one that requires the FHA domain-mediated interaction with XRCC1 and a second that is independent of XRCC1 but requires a novel type of zinc finger motif located at the C terminus of APLF. Finally, we demonstrate that APLF is phosphorylated in a DNA damage- and ATM-dependent manner and that the depletion of APLF from noncycling human SH-SY5Y neuroblastoma cells reduces rates of chromosomal DNA strand break repair following ionizing radiation. These data identify APLF as a novel component of the cellular response to DNA strand breaks in human cells.
脱嘌呤嘧啶核酸内切酶和多核苷酸激酶（PNK）是DNA末端加工因子，它们分别通过与XRCC1和XRCC4的磷酸化特异性相互作用，被招募到DNA单链和双链断裂修复机制中。这些相互作用是通过一类不同的叉头相关（FHA）结构域介导的，该结构域与XRCC1和XRCC4中被酪蛋白激酶2（CK2）磷酸化的肽序列结合。在这里，我们鉴定了未表征的开放阅读框C2orf13的产物，它是该FHA结构域蛋白家族的一个新成员，我们将该蛋白命名为APLF（脱嘌呤嘧啶核酸内切酶和PNK样因子）。我们表明，APLF在体内和体外与XRCC1相互作用，且这种相互作用受到CK2的刺激。酵母双杂交分析表明，APLF还与双链断裂修复蛋白XRCC4和XRCC5（Ku86）相互作用。我们还表明，内源性和黄色荧光蛋白标记的APLF在H₂O₂或UVA激光诱导的染色体DNA损伤位点积累，这至少通过两种机制实现：一种机制需要FHA结构域介导的与XRCC1的相互作用，另一种机制独立于XRCC1，但需要位于APLF C末端的一种新型锌指基序。最后，我们证明APLF以DNA损伤和ATM依赖的方式被磷酸化，并且从非循环的人SH-SY5Y神经母细胞瘤细胞中耗尽APLF会降低电离辐射后染色体DNA链断裂的修复率。这些数据表明APLF是人类细胞对DNA链断裂的细胞反应中的一个新成分。
