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Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA
Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10279920/ https://www.ncbi.nlm.nih.gov/pubmed/37178921 http://dx.doi.org/10.1016/j.jbc.2023.104817 |
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author | Mustafi, Mainak Kwon, Youngho Sung, Patrick Greene, Eric C. |
author_facet | Mustafi, Mainak Kwon, Youngho Sung, Patrick Greene, Eric C. |
author_sort | Mustafi, Mainak |
collection | PubMed |
description | Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and break-induced replication. However, details of its translocation properties and the importance of amino acids residues implicated in DNA binding remain unclear. Here, we use total internal reflection fluorescence microscopy with single-molecule DNA curtain assays to directly observe the movement of fluorescently tagged Saccharomyces cerevisiae Pif1 on single-stranded DNA (ssDNA) substrates. We find that Pif1 binds tightly to ssDNA and translocates very rapidly (∼350 nucleotides per second) in the 5’→3′ direction over relatively long distances (∼29,500 nucleotides). Surprisingly, we show the ssDNA–binding protein replication protein A inhibits Pif1 activity in both bulk biochemical and single-molecule measurements. However, we demonstrate Pif1 can strip replication protein A from ssDNA, allowing subsequent molecules of Pif1 to translocate unimpeded. We also assess the functional attributes of several Pif1 mutations predicted to impair contact with the ssDNA substrate. Taken together, our findings highlight the functional importance of these amino acid residues in coordinating the movement of Pif1 along ssDNA. |
format | Online Article Text |
id | pubmed-10279920 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-102799202023-06-21 Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA Mustafi, Mainak Kwon, Youngho Sung, Patrick Greene, Eric C. J Biol Chem Research Article Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and break-induced replication. However, details of its translocation properties and the importance of amino acids residues implicated in DNA binding remain unclear. Here, we use total internal reflection fluorescence microscopy with single-molecule DNA curtain assays to directly observe the movement of fluorescently tagged Saccharomyces cerevisiae Pif1 on single-stranded DNA (ssDNA) substrates. We find that Pif1 binds tightly to ssDNA and translocates very rapidly (∼350 nucleotides per second) in the 5’→3′ direction over relatively long distances (∼29,500 nucleotides). Surprisingly, we show the ssDNA–binding protein replication protein A inhibits Pif1 activity in both bulk biochemical and single-molecule measurements. However, we demonstrate Pif1 can strip replication protein A from ssDNA, allowing subsequent molecules of Pif1 to translocate unimpeded. We also assess the functional attributes of several Pif1 mutations predicted to impair contact with the ssDNA substrate. Taken together, our findings highlight the functional importance of these amino acid residues in coordinating the movement of Pif1 along ssDNA. American Society for Biochemistry and Molecular Biology 2023-05-11 /pmc/articles/PMC10279920/ /pubmed/37178921 http://dx.doi.org/10.1016/j.jbc.2023.104817 Text en © 2023 The Authors 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 Mustafi, Mainak Kwon, Youngho Sung, Patrick Greene, Eric C. Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA |
title | Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA |
title_full | Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA |
title_fullStr | Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA |
title_full_unstemmed | Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA |
title_short | Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA |
title_sort | single-molecule visualization of pif1 helicase translocation on single-stranded dna |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10279920/ https://www.ncbi.nlm.nih.gov/pubmed/37178921 http://dx.doi.org/10.1016/j.jbc.2023.104817 |
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