Cargando…
CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism
The adenosine triphosphate (ATP) analog ATPγS often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATPγS and thus unable to perform DNA unwinding, as documented for certain other helicases. Conseque...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
National Academy of Sciences
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8794833/ https://www.ncbi.nlm.nih.gov/pubmed/35042821 http://dx.doi.org/10.1073/pnas.2119580119 |
_version_ | 1784640910933360640 |
---|---|
author | Yao, Nina Y. Zhang, Dan Yurieva, Olga O’Donnell, Michael E. |
author_facet | Yao, Nina Y. Zhang, Dan Yurieva, Olga O’Donnell, Michael E. |
author_sort | Yao, Nina Y. |
collection | PubMed |
description | The adenosine triphosphate (ATP) analog ATPγS often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATPγS and thus unable to perform DNA unwinding, as documented for certain other helicases. Consequently, ATPγS is often used to “preload” CMG onto forked DNA substrates without unwinding before adding ATP to initiate helicase activity. We find here that CMG does hydrolyze ATPγS and couples it to DNA unwinding. Indeed, the rate of unwinding of a 20- and 30-mer duplex fork of different sequences by CMG is only reduced 1- to 1.5-fold using ATPγS compared with ATP. These findings imply that a conformational change is the rate-limiting step during CMG unwinding, not hydrolysis. Instead of using ATPγS for loading CMG onto DNA, we demonstrate here that nonhydrolyzable adenylyl-imidodiphosphate (AMP-PNP) can be used to preload CMG onto a forked DNA substrate without unwinding. |
format | Online Article Text |
id | pubmed-8794833 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | National Academy of Sciences |
record_format | MEDLINE/PubMed |
spelling | pubmed-87948332022-02-03 CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism Yao, Nina Y. Zhang, Dan Yurieva, Olga O’Donnell, Michael E. Proc Natl Acad Sci U S A Biological Sciences The adenosine triphosphate (ATP) analog ATPγS often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATPγS and thus unable to perform DNA unwinding, as documented for certain other helicases. Consequently, ATPγS is often used to “preload” CMG onto forked DNA substrates without unwinding before adding ATP to initiate helicase activity. We find here that CMG does hydrolyze ATPγS and couples it to DNA unwinding. Indeed, the rate of unwinding of a 20- and 30-mer duplex fork of different sequences by CMG is only reduced 1- to 1.5-fold using ATPγS compared with ATP. These findings imply that a conformational change is the rate-limiting step during CMG unwinding, not hydrolysis. Instead of using ATPγS for loading CMG onto DNA, we demonstrate here that nonhydrolyzable adenylyl-imidodiphosphate (AMP-PNP) can be used to preload CMG onto a forked DNA substrate without unwinding. National Academy of Sciences 2022-01-18 2022-01-25 /pmc/articles/PMC8794833/ /pubmed/35042821 http://dx.doi.org/10.1073/pnas.2119580119 Text en Copyright © 2022 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by/4.0/This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Biological Sciences Yao, Nina Y. Zhang, Dan Yurieva, Olga O’Donnell, Michael E. CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism |
title | CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism |
title_full | CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism |
title_fullStr | CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism |
title_full_unstemmed | CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism |
title_short | CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism |
title_sort | cmg helicase can use atpγs to unwind dna: implications for the rate-limiting step in the reaction mechanism |
topic | Biological Sciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8794833/ https://www.ncbi.nlm.nih.gov/pubmed/35042821 http://dx.doi.org/10.1073/pnas.2119580119 |
work_keys_str_mv | AT yaoninay cmghelicasecanuseatpgstounwinddnaimplicationsfortheratelimitingstepinthereactionmechanism AT zhangdan cmghelicasecanuseatpgstounwinddnaimplicationsfortheratelimitingstepinthereactionmechanism AT yurievaolga cmghelicasecanuseatpgstounwinddnaimplicationsfortheratelimitingstepinthereactionmechanism AT odonnellmichaele cmghelicasecanuseatpgstounwinddnaimplicationsfortheratelimitingstepinthereactionmechanism |