Cargando…

Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity

Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarl...

Descripción completa

Detalles Bibliográficos
Autores principales: Wan, Li, Toland, Sabrina, Robinson-McCarthy, Lindsey R., Lee, Nara, Schaich, Matthew A., Hengel, Sarah R., Li, Xiaochen, Bernstein, Kara A., Van Houten, Bennett, Chang, Yuan, Moore, Patrick S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10372695/
https://www.ncbi.nlm.nih.gov/pubmed/37459531
http://dx.doi.org/10.1073/pnas.2308010120
_version_ 1785078419463077888
author Wan, Li
Toland, Sabrina
Robinson-McCarthy, Lindsey R.
Lee, Nara
Schaich, Matthew A.
Hengel, Sarah R.
Li, Xiaochen
Bernstein, Kara A.
Van Houten, Bennett
Chang, Yuan
Moore, Patrick S.
author_facet Wan, Li
Toland, Sabrina
Robinson-McCarthy, Lindsey R.
Lee, Nara
Schaich, Matthew A.
Hengel, Sarah R.
Li, Xiaochen
Bernstein, Kara A.
Van Houten, Bennett
Chang, Yuan
Moore, Patrick S.
author_sort Wan, Li
collection PubMed
description Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.
format Online
Article
Text
id pubmed-10372695
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher National Academy of Sciences
record_format MEDLINE/PubMed
spelling pubmed-103726952023-07-28 Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity Wan, Li Toland, Sabrina Robinson-McCarthy, Lindsey R. Lee, Nara Schaich, Matthew A. Hengel, Sarah R. Li, Xiaochen Bernstein, Kara A. Van Houten, Bennett Chang, Yuan Moore, Patrick S. Proc Natl Acad Sci U S A Biological Sciences Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication. National Academy of Sciences 2023-07-17 2023-07-25 /pmc/articles/PMC10372695/ /pubmed/37459531 http://dx.doi.org/10.1073/pnas.2308010120 Text en Copyright © 2023 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Wan, Li
Toland, Sabrina
Robinson-McCarthy, Lindsey R.
Lee, Nara
Schaich, Matthew A.
Hengel, Sarah R.
Li, Xiaochen
Bernstein, Kara A.
Van Houten, Bennett
Chang, Yuan
Moore, Patrick S.
Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity
title Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity
title_full Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity
title_fullStr Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity
title_full_unstemmed Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity
title_short Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity
title_sort unlicensed origin dna melting by mcv and sv40 polyomavirus lt proteins is independent of atp-dependent helicase activity
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10372695/
https://www.ncbi.nlm.nih.gov/pubmed/37459531
http://dx.doi.org/10.1073/pnas.2308010120
work_keys_str_mv AT wanli unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity
AT tolandsabrina unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity
AT robinsonmccarthylindseyr unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity
AT leenara unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity
AT schaichmatthewa unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity
AT hengelsarahr unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity
AT lixiaochen unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity
AT bernsteinkaraa unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity
AT vanhoutenbennett unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity
AT changyuan unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity
AT moorepatricks unlicensedorigindnameltingbymcvandsv40polyomavirusltproteinsisindependentofatpdependenthelicaseactivity