The Mus81-Mms4 structure-selective endonuclease requires nicked DNA junctions to undergo conformational changes and bend its DNA substrates for cleavage

Sucheta Mukherjee, William Douglass Wright, Kirk Tevebaugh Ehmsen, Wolf Dietrich Heyer

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Mus81-Mms4/EME1 is a DNA structure-selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81-Mms4 requires inherent rotational flexibility in DNA junctions for optimal catalysis. Förster Resonance Energy Transfer experiments further reveal that recognition of 3′-flap and nicked Holliday junction substrates by Mus81-Mms4 involves induction of a sharp bend with a 100° angle between two duplex DNA arms. In addition, thiol crosslinking of Mus81-Mms4 bound to DNA junctions demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches suggest a model for catalysis by Mus81-Mms4 in which initial DNA binding is based on minimal structural requirements followed by a rate-limiting conformational transition of the substrate and protein. This leads to a sharply kinked DNA molecule that may fray the DNA four base pairs away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotide. These data suggest that mutually compatible conformational changes of Mus81-Mms4 and its substrates tailor its incision activity to nicked junction molecules.

Original languageEnglish (US)
Pages (from-to)6511-6522
Number of pages12
JournalNucleic Acids Research
Volume42
Issue number10
DOIs
StatePublished - 2014

Fingerprint

DNA Cleavage
Endonucleases
DNA
Catalysis
Joints
Cruciform DNA
Saccharomycetales
Homologous Recombination
Energy Transfer
Sulfhydryl Compounds
Base Pairing
Nucleotides

ASJC Scopus subject areas

  • Genetics

Cite this

The Mus81-Mms4 structure-selective endonuclease requires nicked DNA junctions to undergo conformational changes and bend its DNA substrates for cleavage. / Mukherjee, Sucheta; Wright, William Douglass; Ehmsen, Kirk Tevebaugh; Heyer, Wolf Dietrich.

In: Nucleic Acids Research, Vol. 42, No. 10, 2014, p. 6511-6522.

Research output: Contribution to journalArticle

Mukherjee, Sucheta ; Wright, William Douglass ; Ehmsen, Kirk Tevebaugh ; Heyer, Wolf Dietrich. / The Mus81-Mms4 structure-selective endonuclease requires nicked DNA junctions to undergo conformational changes and bend its DNA substrates for cleavage. In: Nucleic Acids Research. 2014 ; Vol. 42, No. 10. pp. 6511-6522.
@article{ebfb773fa17e4882b13fe57e7a9e3e95,
title = "The Mus81-Mms4 structure-selective endonuclease requires nicked DNA junctions to undergo conformational changes and bend its DNA substrates for cleavage",
abstract = "Mus81-Mms4/EME1 is a DNA structure-selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81-Mms4 requires inherent rotational flexibility in DNA junctions for optimal catalysis. F{\"o}rster Resonance Energy Transfer experiments further reveal that recognition of 3′-flap and nicked Holliday junction substrates by Mus81-Mms4 involves induction of a sharp bend with a 100° angle between two duplex DNA arms. In addition, thiol crosslinking of Mus81-Mms4 bound to DNA junctions demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches suggest a model for catalysis by Mus81-Mms4 in which initial DNA binding is based on minimal structural requirements followed by a rate-limiting conformational transition of the substrate and protein. This leads to a sharply kinked DNA molecule that may fray the DNA four base pairs away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotide. These data suggest that mutually compatible conformational changes of Mus81-Mms4 and its substrates tailor its incision activity to nicked junction molecules.",
author = "Sucheta Mukherjee and Wright, {William Douglass} and Ehmsen, {Kirk Tevebaugh} and Heyer, {Wolf Dietrich}",
year = "2014",
doi = "10.1093/nar/gku265",
language = "English (US)",
volume = "42",
pages = "6511--6522",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "10",

}

TY - JOUR

T1 - The Mus81-Mms4 structure-selective endonuclease requires nicked DNA junctions to undergo conformational changes and bend its DNA substrates for cleavage

AU - Mukherjee, Sucheta

AU - Wright, William Douglass

AU - Ehmsen, Kirk Tevebaugh

AU - Heyer, Wolf Dietrich

PY - 2014

Y1 - 2014

N2 - Mus81-Mms4/EME1 is a DNA structure-selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81-Mms4 requires inherent rotational flexibility in DNA junctions for optimal catalysis. Förster Resonance Energy Transfer experiments further reveal that recognition of 3′-flap and nicked Holliday junction substrates by Mus81-Mms4 involves induction of a sharp bend with a 100° angle between two duplex DNA arms. In addition, thiol crosslinking of Mus81-Mms4 bound to DNA junctions demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches suggest a model for catalysis by Mus81-Mms4 in which initial DNA binding is based on minimal structural requirements followed by a rate-limiting conformational transition of the substrate and protein. This leads to a sharply kinked DNA molecule that may fray the DNA four base pairs away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotide. These data suggest that mutually compatible conformational changes of Mus81-Mms4 and its substrates tailor its incision activity to nicked junction molecules.

AB - Mus81-Mms4/EME1 is a DNA structure-selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81-Mms4 requires inherent rotational flexibility in DNA junctions for optimal catalysis. Förster Resonance Energy Transfer experiments further reveal that recognition of 3′-flap and nicked Holliday junction substrates by Mus81-Mms4 involves induction of a sharp bend with a 100° angle between two duplex DNA arms. In addition, thiol crosslinking of Mus81-Mms4 bound to DNA junctions demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches suggest a model for catalysis by Mus81-Mms4 in which initial DNA binding is based on minimal structural requirements followed by a rate-limiting conformational transition of the substrate and protein. This leads to a sharply kinked DNA molecule that may fray the DNA four base pairs away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotide. These data suggest that mutually compatible conformational changes of Mus81-Mms4 and its substrates tailor its incision activity to nicked junction molecules.

UR - http://www.scopus.com/inward/record.url?scp=84903173997&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84903173997&partnerID=8YFLogxK

U2 - 10.1093/nar/gku265

DO - 10.1093/nar/gku265

M3 - Article

C2 - 24744239

AN - SCOPUS:84903173997

VL - 42

SP - 6511

EP - 6522

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 10

ER -