Replisome bypass of a protein-based R-loop block by Pif1

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facil...

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Autores principales: Schauer, Grant D., Spenkelink, Lisanne M., Lewis, Jacob S., Yurieva, Olga, Mueller, Stefan H., van Oijen, Antoine M., O’Donnell, Michael E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720201/
https://www.ncbi.nlm.nih.gov/pubmed/33199603
http://dx.doi.org/10.1073/pnas.2020189117
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author Schauer, Grant D.
Spenkelink, Lisanne M.
Lewis, Jacob S.
Yurieva, Olga
Mueller, Stefan H.
van Oijen, Antoine M.
O’Donnell, Michael E.
author_facet Schauer, Grant D.
Spenkelink, Lisanne M.
Lewis, Jacob S.
Yurieva, Olga
Mueller, Stefan H.
van Oijen, Antoine M.
O’Donnell, Michael E.
author_sort Schauer, Grant D.
collection PubMed
description Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.
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spelling pubmed-77202012020-12-18 Replisome bypass of a protein-based R-loop block by Pif1 Schauer, Grant D. Spenkelink, Lisanne M. Lewis, Jacob S. Yurieva, Olga Mueller, Stefan H. van Oijen, Antoine M. O’Donnell, Michael E. Proc Natl Acad Sci U S A Biological Sciences Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks. National Academy of Sciences 2020-12-01 2020-11-16 /pmc/articles/PMC7720201/ /pubmed/33199603 http://dx.doi.org/10.1073/pnas.2020189117 Text en Copyright © 2020 the Author(s). Published by PNAS. http://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY) (http://creativecommons.org/licenses/by/4.0/) .
spellingShingle Biological Sciences
Schauer, Grant D.
Spenkelink, Lisanne M.
Lewis, Jacob S.
Yurieva, Olga
Mueller, Stefan H.
van Oijen, Antoine M.
O’Donnell, Michael E.
Replisome bypass of a protein-based R-loop block by Pif1
title Replisome bypass of a protein-based R-loop block by Pif1
title_full Replisome bypass of a protein-based R-loop block by Pif1
title_fullStr Replisome bypass of a protein-based R-loop block by Pif1
title_full_unstemmed Replisome bypass of a protein-based R-loop block by Pif1
title_short Replisome bypass of a protein-based R-loop block by Pif1
title_sort replisome bypass of a protein-based r-loop block by pif1
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720201/
https://www.ncbi.nlm.nih.gov/pubmed/33199603
http://dx.doi.org/10.1073/pnas.2020189117
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