7LPI

APE1 phosphorothioate substrate complex with abasic ribonucleotide DNA

7LPI の概要

• エントリーDOI: 10.2210/pdb7lpi/pdb
• 分子名称: DNA-(apurinic or apyrimidinic site) lyase, DNA (5'-D(*GP*GP*AP*TP*CP*CP*GP*TP*CP*GP*AP*GP*CP*GP*CP*AP*TP*CP*AP*GP*C)-3'), DNA (5'-D(*GP*CP*TP*GP*AP*TP*GP*CP*GP*C)-R(P*(YA4))-D(P*CP*GP*AP*CP*GP*GP*AP*TP*CP*C)-3'), ... (4 entities in total)
• 機能のキーワード: dna repair, abasic ribonucleotide, ap-endonuclease, lyase, lyase-dna complex, lyase/dna
• 由来する生物種: Homo sapiens (Human); 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 75110.23

構造登録者

Freudenthal, B.D.,Hoitsma, N.M. (登録日: 2021-02-11, 公開日: 2021-08-04, 最終更新日: 2023-10-18)

主引用文献

Hoitsma, N.M.,Click, T.H.,Agarwal, P.K.,Freudenthal, B.D.
Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker.
Comput Struct Biotechnol J, 19:3293-3302, 2021

PubMed Abstract: Ribonucleotides (rNTPs) are predicted to be incorporated into the genome at a rate of up to 3 million times per cell division, making rNTPs the most common non-standard nucleotide in the human genome. Typically, misinserted ribonucleotides are repaired by the ribonucleotide excision repair (RER) pathway, which is initiated by RNase H2 cleavage. However, rNTPs are susceptible to spontaneous depurination generating abasic ribonucleotides (rAPs), which are unable to be processed by RNase H2. Additionally, rAPs have been found in nascent RNA and coupled to R-loops. Recent work identified that base excision repair (BER) protein AP-Endonuclease 1 (APE1) is responsible for the initial processing of rAPs embedded in DNA and in R-loops. APE1 is a well characterized AP endonuclease that cleaves 5' of abasic sites, but its ability to cleave at rAPs remains poorly understood. Here, we utilize enzyme kinetics, X-ray crystallography, and molecular dynamics simulations to provide insight into rAP processing by APE1. Enzyme kinetics were used to determine pre-steady-state rates of APE1 cleavage on DNA substrates containing rAP, revealing a decrease in activity compared to cleavage at a canonical deoxy-AP substrate. Using X-ray crystallography, we identified novel contacts between the rAP and the APE1 active site. We demonstrate that the rAP sugar pucker is accommodated in the active site in a C3'-endo conformation, influencing its position and contributing to a decrease in activity compared to the deoxy-AP site. Together, this work provides molecular level insights into rAP processing by APE1 and advances our understanding of ribonucleotide processing within genomic DNA.

PubMed: 34188778
DOI: 10.1016/j.csbj.2021.05.035

実験手法

X-RAY DIFFRACTION (2.05 Å)