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Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker
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) pat...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Research Network of Computational and Structural Biotechnology
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8207216/ https://www.ncbi.nlm.nih.gov/pubmed/34188778 http://dx.doi.org/10.1016/j.csbj.2021.05.035 |
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author | Hoitsma, Nicole M. Click, Timothy H. Agarwal, Pratul K. Freudenthal, Bret D. |
author_facet | Hoitsma, Nicole M. Click, Timothy H. Agarwal, Pratul K. Freudenthal, Bret D. |
author_sort | Hoitsma, Nicole M. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8207216 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Research Network of Computational and Structural Biotechnology |
record_format | MEDLINE/PubMed |
spelling | pubmed-82072162021-06-28 Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker Hoitsma, Nicole M. Click, Timothy H. Agarwal, Pratul K. Freudenthal, Bret D. Comput Struct Biotechnol J Research Article 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. Research Network of Computational and Structural Biotechnology 2021-05-25 /pmc/articles/PMC8207216/ /pubmed/34188778 http://dx.doi.org/10.1016/j.csbj.2021.05.035 Text en © 2021 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Hoitsma, Nicole M. Click, Timothy H. Agarwal, Pratul K. Freudenthal, Bret D. Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker |
title | Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker |
title_full | Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker |
title_fullStr | Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker |
title_full_unstemmed | Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker |
title_short | Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker |
title_sort | altered ape1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8207216/ https://www.ncbi.nlm.nih.gov/pubmed/34188778 http://dx.doi.org/10.1016/j.csbj.2021.05.035 |
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