Bernstein Nina K, Williams R Scott, Rakovszky Melissa L, Cui Diana, Green Ruth, Karimi-Busheri Feridoun, Mani Rajam S, Galicia Sarah, Koch C Anne, Cass Carol E, Durocher Daniel, Weinfeld Michael, Glover J N Mark
Department of Biochemistry, 4-74 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Mol Cell. 2005 Mar 4;17(5):657-70. doi: 10.1016/j.molcel.2005.02.012.
Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.
哺乳动物多核苷酸激酶（PNK）是碱基切除修复（BER）和非同源末端连接（NHEJ）DNA修复途径的关键组成部分。PNK作为一种5'-激酶/3'-磷酸酶，可产生5'-磷酸/3'-羟基末端，这是修复过程中连接的必要前提条件。PNK通过其N端FHA结构域与任一途径的磷酸化成分之间的相互作用被招募到修复复合物中。在此，我们描述了完整哺乳动物PNK的晶体结构以及与同源磷酸肽结合的PNK FHA结构。激酶结构域有一个宽广的底物结合口袋，它优先识别具有凹陷5'末端的双链底物。相比之下，磷酸酶结构域能有效地使单链3'-磷酸末端以及双链底物去磷酸化。FHA结构域通过一个柔性连接链与激酶/磷酸酶催化结构域相连，并且它基于结合表面与磷酸苏氨酸肽之间的静电互补性呈现出一种靶标选择模式。
