Institute of Chemical Biology and Fundamental Medicine (ICBFM) SB RAS, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.
SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France.
J Mol Biol. 2019 Jul 12;431(15):2655-2673. doi: 10.1016/j.jmb.2019.05.028. Epub 2019 May 23.
Nuclear poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) catalyze the synthesis of poly(ADP-ribose) (PAR) and use NAD as a substrate for the polymer synthesis. Both PARP1 and PARP2 are involved in DNA damage response pathways and function as sensors of DNA breaks, including temporary single-strand breaks formed during DNA repair. Consistently, with a role in DNA repair, PARP activation requires its binding to a damaged DNA site, which initiates PAR synthesis. Here we use atomic force microscopy to characterize at the single-molecule level the interaction of PARP1 and PARP2 with long DNA substrates containing a single damage site and representing intermediates of the short-patch base excision repair (BER) pathway. We demonstrated that PARP1 has higher affinity for early intermediates of BER than PARP2, whereas both PARPs efficiently interact with the nick and may contribute to regulation of the final ligation step. The binding of a DNA repair intermediate by PARPs involved a PARP monomer or dimer depending on the type of DNA damage. PARP dimerization influences the affinity of these proteins to DNA and affects their enzymatic activity: the dimeric form is more effective in PAR synthesis in the case of PARP2 but is less effective in the case of PARP1. PARP2 suppresses PAR synthesis catalyzed by PARP1 after single-strand breaks formation. Our study suggests that the functions of PARP1 and PARP2 overlap in BER after a site cleavage and provides evidence for a role of PARP2 in the regulation of PARP1 activity.
核多聚（ADP-核糖）聚合酶 1 和 2（PARP1 和 PARP2）催化多聚（ADP-核糖）（PAR）的合成，并使用 NAD 作为聚合物合成的底物。PARP1 和 PARP2 都参与 DNA 损伤反应途径，并作为 DNA 断裂的传感器发挥作用，包括 DNA 修复过程中形成的暂时性单链断裂。与 DNA 修复的作用一致，PARP 的激活需要其与受损 DNA 位点结合，这会引发 PAR 的合成。在这里，我们使用原子力显微镜在单分子水平上表征 PARP1 和 PARP2 与含有单个损伤位点的长 DNA 底物的相互作用，这些底物代表短补丁碱基切除修复（BER）途径的中间产物。我们证明，PARP1 对 BER 的早期中间产物的亲和力高于 PARP2，而两种 PARP 都能有效地与切口相互作用，并可能有助于调节最终的连接步骤。PARPs 与 DNA 修复中间体的结合涉及 PARP 单体或二聚体，具体取决于 DNA 损伤的类型。PARP 二聚化会影响这些蛋白与 DNA 的亲和力，并影响它们的酶活性：在 PARP2 的情况下，二聚体形式在 PAR 合成中更有效，但在 PARP1 的情况下则效果较差。PARP2 在单链断裂形成后抑制 PARP1 催化的 PAR 合成。我们的研究表明，PARP1 和 PARP2 在位点切割后在 BER 中的功能重叠，并为 PARP2 在调节 PARP1 活性中的作用提供了证据。
