Ilina E S, Kochetkova A S, Belousova E A, Kutuzov M M, Lavrik O I, Khodyreva S N
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630090 Russia.
Novosibirsk State University, Novosibirsk, 630090 Russia.
Mol Biol (Mosk). 2023 Mar-Apr;57(2):285-298.
Base excision repair (BER) is aimed at repair of damaged bases, which are the largest group of DNA lesions. The main steps of BER are recognition and removal of the aberrant base, cutting of the DNA sugar-phosphate backbone, gap processing (including dNMP insertion), and DNA ligation. The precise function of BER depends on the regulation of each step by regulatory/accessory proteins, the most important of which is poly(ADP-ribose) (PAR) polymerase 1 (PARP1). PARP1 plays an important role in DNA repair, maintenance of genome integrity, and regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. There is no systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells. Whole-cell extracts and RNA preparations obtained from the parental HEK293T cell line and its derivative HEK293T/P1-KD cell line with reduced PARP1 expression (shPARP1-expressing cells, a PARP1 knockdown) were used to assess the levels of mRNAs coding for BER proteins: PARP1, PARP2, uracil DNA glycosylase (UNG2), AP endonuclease 1 (APE1), DNA polymerase β (POLβ), DNA ligase III (LIG3), and XRCC1. Catalytic activities of the enzymes were evaluated in parallel. No significant effect of the PARP1 content was observed for the mRNA levels of UNG2, APE1, POLβ, LIG3, and XRCC1. The amount of the PARP2 mRNA proved to be reduced two times in HEK293T/P1-KD cells. Activities of these enzymes in whole-cell extracts did not differ significantly between HEK293T and HEK293T/P1-KD cells. No significant change was observed in the efficiencies of the reactions catalyzed by UNG2, APE1, POLβ, and LIG3 in conditions of PAR synthesis. A DNA PARylation pattern did not dramatically change in a HEK293T/P1-KD cell extract with a reduced PARP1 content as compared with an extract of the parental HEK293T cell line.
碱基切除修复（BER）旨在修复受损碱基，受损碱基是最大的一类DNA损伤。BER的主要步骤包括识别和去除异常碱基、切割DNA糖磷酸骨架、缺口处理（包括dNMP插入）以及DNA连接。BER的精确功能取决于调节/辅助蛋白对每个步骤的调控，其中最重要的是聚（ADP - 核糖）（PAR）聚合酶1（PARP1）。PARP1在DNA修复、基因组完整性维护以及mRNA稳定性和降解的调控中发挥重要作用。因此，PARP1可以在BER蛋白水平及其mRNA水平上影响BER。目前尚无关于PARP1含量如何影响人类细胞中关键BER蛋白活性及其mRNA水平的系统性数据。使用从亲本HEK293T细胞系及其PARP1表达降低的衍生细胞系HEK293T/P1 - KD细胞系（shPARP1表达细胞，PARP1基因敲低）获得的全细胞提取物和RNA制剂，来评估编码BER蛋白的mRNA水平：PARP1、PARP2、尿嘧啶DNA糖基化酶（UNG2）、AP核酸内切酶1（APE1）、DNA聚合酶β（POLβ）、DNA连接酶III（LIG3）和XRCC1。同时评估了这些酶的催化活性。未观察到PARP1含量对UNG2、APE1、POLβ、LIG3和XRCC1的mRNA水平有显著影响。结果证明，HEK293T/P1 - KD细胞中PARP2 mRNA的量减少了两倍。HEK293T和HEK293T/P1 - KD细胞全细胞提取物中这些酶的活性没有显著差异。在PAR合成条件下，UNG2、APE1、POLβ和LIG3催化反应的效率未观察到显著变化。与亲本HEK293T细胞系的提取物相比，PARP1含量降低的HEK293T/P1 - KD细胞提取物中的DNA PARylation模式没有显著变化。
