Abstract
The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands. Holliday junction cleavage requires the coordination of two active sites, necessitating the assembly of two Mus81-Mms4 heterodimers. Contrary to this expectation, we show that Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer both in solution and when bound to DNA substrates in vitro. Consistently, immunoprecipitation experiments demonstrate that Mus81-Mms4 does not multimerize in vivo. Moreover, chromatin-bound Mus81-Mms4 does not detectably form higher-order multimers. We show that Cdc5 kinase activates Mus81-Mms4 nuclease activity on 3′ flaps and Holliday junctions in vitro but that activation does not induce a preference for Holliday junctions and does not induce multimerization of the Mus81-Mms4 heterodimer. These data support a model in which Mus81-Mms4 cleaves nicked recombination intermediates such as displacement loops (D-loops), nicked Holliday junctions, or 3′ flaps but not intact Holliday junctions with four uninterrupted strands. We infer that Mus81-dependent crossing over occurs in a noncanonical manner that does not involve the coordinated cleavage of classic Holliday junctions.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3065-3080 |
| Number of pages | 16 |
| Journal | Molecular and Cellular Biology |
| Volume | 32 |
| Issue number | 15 |
| DOIs | |
| State | Published - Aug 2012 |
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ASJC Scopus subject areas
- Molecular Biology
- Cell Biology
Cite this
Mus81-Mms4 functions as a single heterodimer to cleave nicked intermediates in recombinational DNA repair. / Schwartz, Erin K.; Wright, William D.; Ehmsen, Kirk T.; Evans, James E.; Stahlberg, Henning; Heyer, Wolf Dietrich.
In: Molecular and Cellular Biology, Vol. 32, No. 15, 08.2012, p. 3065-3080.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Mus81-Mms4 functions as a single heterodimer to cleave nicked intermediates in recombinational DNA repair
AU - Schwartz, Erin K.
AU - Wright, William D.
AU - Ehmsen, Kirk T.
AU - Evans, James E.
AU - Stahlberg, Henning
AU - Heyer, Wolf Dietrich
PY - 2012/8
Y1 - 2012/8
N2 - The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands. Holliday junction cleavage requires the coordination of two active sites, necessitating the assembly of two Mus81-Mms4 heterodimers. Contrary to this expectation, we show that Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer both in solution and when bound to DNA substrates in vitro. Consistently, immunoprecipitation experiments demonstrate that Mus81-Mms4 does not multimerize in vivo. Moreover, chromatin-bound Mus81-Mms4 does not detectably form higher-order multimers. We show that Cdc5 kinase activates Mus81-Mms4 nuclease activity on 3′ flaps and Holliday junctions in vitro but that activation does not induce a preference for Holliday junctions and does not induce multimerization of the Mus81-Mms4 heterodimer. These data support a model in which Mus81-Mms4 cleaves nicked recombination intermediates such as displacement loops (D-loops), nicked Holliday junctions, or 3′ flaps but not intact Holliday junctions with four uninterrupted strands. We infer that Mus81-dependent crossing over occurs in a noncanonical manner that does not involve the coordinated cleavage of classic Holliday junctions.
AB - The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands. Holliday junction cleavage requires the coordination of two active sites, necessitating the assembly of two Mus81-Mms4 heterodimers. Contrary to this expectation, we show that Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer both in solution and when bound to DNA substrates in vitro. Consistently, immunoprecipitation experiments demonstrate that Mus81-Mms4 does not multimerize in vivo. Moreover, chromatin-bound Mus81-Mms4 does not detectably form higher-order multimers. We show that Cdc5 kinase activates Mus81-Mms4 nuclease activity on 3′ flaps and Holliday junctions in vitro but that activation does not induce a preference for Holliday junctions and does not induce multimerization of the Mus81-Mms4 heterodimer. These data support a model in which Mus81-Mms4 cleaves nicked recombination intermediates such as displacement loops (D-loops), nicked Holliday junctions, or 3′ flaps but not intact Holliday junctions with four uninterrupted strands. We infer that Mus81-dependent crossing over occurs in a noncanonical manner that does not involve the coordinated cleavage of classic Holliday junctions.
UR - http://www.scopus.com/inward/record.url?scp=84864592240&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84864592240&partnerID=8YFLogxK
U2 - 10.1128/MCB.00547-12
DO - 10.1128/MCB.00547-12
M3 - Article
C2 - 22645308
AN - SCOPUS:84864592240
VL - 32
SP - 3065
EP - 3080
JO - Molecular and Cellular Biology
JF - Molecular and Cellular Biology
SN - 0270-7306
IS - 15
ER -