Malfatti Matilde Clarissa, Balachander Sathya, Antoniali Giulia, Koh Kyung Duk, Saint-Pierre Christine, Gasparutto Didier, Chon Hyongi, Crouch Robert J, Storici Francesca, Tell Gianluca
Laboratory of Molecular Biology and DNA repair, Department of Medicine, University of Udine, Udine, Italy.
School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, USA.
Nucleic Acids Res. 2017 Nov 2;45(19):11193-11212. doi: 10.1093/nar/gkx723.
Ribonucleoside 5'-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3'-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA.
核糖核苷5'-单磷酸（rNMPs）是真核细胞DNA中最常见的非标准核苷酸，每个细胞周期有超过1亿个rNMPs短暂掺入哺乳动物基因组中。人核糖核酸酶（RNase）H2是能够切割DNA中rNMPs的主要酶。RNase H2是否能够处理掺入DNA中的无碱基或氧化的rNMPs尚不清楚。碱基切除修复（BER）途径主要负责将DNA中的氧化和无碱基位点修复。在这里，我们表明人RNase H2无法处理嵌入DNA中的无碱基rNMP（rAP位点）或核糖8-氧代鸟嘌呤（r8oxoG）位点。相反，我们发现重组纯化的人脱嘌呤/脱嘧啶内切核酸酶-1（APE1）和来自人细胞提取物的APE1能够有效处理DNA中的rAP位点，并且对r8oxoG底物具有较弱的核糖核酸内切酶和3'-外切核酸酶活性。通过生化分析，我们的结果提供了一种能够识别和处理嵌入DNA中的无碱基和氧化核糖核苷酸的人类酶的证据。
