Role of the Srs2–Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae

Shirin S. Jenkins, Steven Gore, Xiaoge Guo, Jie Liu, Christopher Ede, Xavier Veaute, Sue Jinks-Robertson, Stephen C. Kowalczykowski, Wolf Dietrich Heyer

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Saccharomyces cerevisiae Srs2, in addition to its well-documented antirecombination activity, has been proposed to play a role in promoting synthesis-dependent strand annealing (SDSA). Here we report the identification and characterization of an SRS2 mutant with a single amino acid substitution (srs2-F891A) that specifically affects the Srs2 pro-SDSA function. This residue is located within the Srs2–Rad51 interaction domain and embedded within a protein sequence resembling a BRC repeat motif. The srs2-F891A mutation leads to a complete loss of interaction with Rad51 as measured through yeast two-hybrid analysis and a partial loss of interaction as determined through protein pull-down assays with purified Srs2, Srs2-F891A, and Rad51 proteins. Even though previous work has shown that internal deletions of the Srs2–Rad51 interaction domain block Srs2 antirecombination activity in vitro, the Srs2-F891A mutant protein, despite its weakened interaction with Rad51, exhibits no measurable defect in antirecombination activity in vitro or in vivo. Surprisingly, srs2-F891A shows a robust shift from noncrossover to crossover repair products in a plasmid-based gap repair assay, but not in an ectopic physical recombination assay. Our findings suggest that the Srs2 C-terminal Rad51 interaction domain is more complex than previously thought, containing multiple interaction sites with unique effects on Srs2 activity.

Original languageEnglish (US)
Pages (from-to)1133-1145
Number of pages13
JournalGenetics
Volume212
Issue number4
DOIs
StatePublished - Jan 1 2019

Fingerprint

Saccharomyces cerevisiae
Proteins
Mutant Proteins
Amino Acid Substitution
Genetic Recombination
Plasmids
Yeasts
Mutation
In Vitro Techniques

Keywords

  • Crossover control
  • DNA repair
  • Genome stability
  • Helicase
  • Protein interaction
  • Recombination

ASJC Scopus subject areas

  • Genetics

Cite this

Jenkins, S. S., Gore, S., Guo, X., Liu, J., Ede, C., Veaute, X., ... Heyer, W. D. (2019). Role of the Srs2–Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae. Genetics, 212(4), 1133-1145. https://doi.org/10.1534/genetics.119.302337

Role of the Srs2–Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae. / Jenkins, Shirin S.; Gore, Steven; Guo, Xiaoge; Liu, Jie; Ede, Christopher; Veaute, Xavier; Jinks-Robertson, Sue; Kowalczykowski, Stephen C.; Heyer, Wolf Dietrich.

In: Genetics, Vol. 212, No. 4, 01.01.2019, p. 1133-1145.

Research output: Contribution to journalArticle

Jenkins, SS, Gore, S, Guo, X, Liu, J, Ede, C, Veaute, X, Jinks-Robertson, S, Kowalczykowski, SC & Heyer, WD 2019, 'Role of the Srs2–Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae', Genetics, vol. 212, no. 4, pp. 1133-1145. https://doi.org/10.1534/genetics.119.302337
Jenkins, Shirin S. ; Gore, Steven ; Guo, Xiaoge ; Liu, Jie ; Ede, Christopher ; Veaute, Xavier ; Jinks-Robertson, Sue ; Kowalczykowski, Stephen C. ; Heyer, Wolf Dietrich. / Role of the Srs2–Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae. In: Genetics. 2019 ; Vol. 212, No. 4. pp. 1133-1145.
@article{c190128c38204526a6f5cbbd11b884d2,
title = "Role of the Srs2–Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae",
abstract = "Saccharomyces cerevisiae Srs2, in addition to its well-documented antirecombination activity, has been proposed to play a role in promoting synthesis-dependent strand annealing (SDSA). Here we report the identification and characterization of an SRS2 mutant with a single amino acid substitution (srs2-F891A) that specifically affects the Srs2 pro-SDSA function. This residue is located within the Srs2–Rad51 interaction domain and embedded within a protein sequence resembling a BRC repeat motif. The srs2-F891A mutation leads to a complete loss of interaction with Rad51 as measured through yeast two-hybrid analysis and a partial loss of interaction as determined through protein pull-down assays with purified Srs2, Srs2-F891A, and Rad51 proteins. Even though previous work has shown that internal deletions of the Srs2–Rad51 interaction domain block Srs2 antirecombination activity in vitro, the Srs2-F891A mutant protein, despite its weakened interaction with Rad51, exhibits no measurable defect in antirecombination activity in vitro or in vivo. Surprisingly, srs2-F891A shows a robust shift from noncrossover to crossover repair products in a plasmid-based gap repair assay, but not in an ectopic physical recombination assay. Our findings suggest that the Srs2 C-terminal Rad51 interaction domain is more complex than previously thought, containing multiple interaction sites with unique effects on Srs2 activity.",
keywords = "Crossover control, DNA repair, Genome stability, Helicase, Protein interaction, Recombination",
author = "Jenkins, {Shirin S.} and Steven Gore and Xiaoge Guo and Jie Liu and Christopher Ede and Xavier Veaute and Sue Jinks-Robertson and Kowalczykowski, {Stephen C.} and Heyer, {Wolf Dietrich}",
year = "2019",
month = "1",
day = "1",
doi = "10.1534/genetics.119.302337",
language = "English (US)",
volume = "212",
pages = "1133--1145",
journal = "Genetics",
issn = "0016-6731",
publisher = "Genetics Society of America",
number = "4",

}

TY - JOUR

T1 - Role of the Srs2–Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae

AU - Jenkins, Shirin S.

AU - Gore, Steven

AU - Guo, Xiaoge

AU - Liu, Jie

AU - Ede, Christopher

AU - Veaute, Xavier

AU - Jinks-Robertson, Sue

AU - Kowalczykowski, Stephen C.

AU - Heyer, Wolf Dietrich

PY - 2019/1/1

Y1 - 2019/1/1

N2 - Saccharomyces cerevisiae Srs2, in addition to its well-documented antirecombination activity, has been proposed to play a role in promoting synthesis-dependent strand annealing (SDSA). Here we report the identification and characterization of an SRS2 mutant with a single amino acid substitution (srs2-F891A) that specifically affects the Srs2 pro-SDSA function. This residue is located within the Srs2–Rad51 interaction domain and embedded within a protein sequence resembling a BRC repeat motif. The srs2-F891A mutation leads to a complete loss of interaction with Rad51 as measured through yeast two-hybrid analysis and a partial loss of interaction as determined through protein pull-down assays with purified Srs2, Srs2-F891A, and Rad51 proteins. Even though previous work has shown that internal deletions of the Srs2–Rad51 interaction domain block Srs2 antirecombination activity in vitro, the Srs2-F891A mutant protein, despite its weakened interaction with Rad51, exhibits no measurable defect in antirecombination activity in vitro or in vivo. Surprisingly, srs2-F891A shows a robust shift from noncrossover to crossover repair products in a plasmid-based gap repair assay, but not in an ectopic physical recombination assay. Our findings suggest that the Srs2 C-terminal Rad51 interaction domain is more complex than previously thought, containing multiple interaction sites with unique effects on Srs2 activity.

AB - Saccharomyces cerevisiae Srs2, in addition to its well-documented antirecombination activity, has been proposed to play a role in promoting synthesis-dependent strand annealing (SDSA). Here we report the identification and characterization of an SRS2 mutant with a single amino acid substitution (srs2-F891A) that specifically affects the Srs2 pro-SDSA function. This residue is located within the Srs2–Rad51 interaction domain and embedded within a protein sequence resembling a BRC repeat motif. The srs2-F891A mutation leads to a complete loss of interaction with Rad51 as measured through yeast two-hybrid analysis and a partial loss of interaction as determined through protein pull-down assays with purified Srs2, Srs2-F891A, and Rad51 proteins. Even though previous work has shown that internal deletions of the Srs2–Rad51 interaction domain block Srs2 antirecombination activity in vitro, the Srs2-F891A mutant protein, despite its weakened interaction with Rad51, exhibits no measurable defect in antirecombination activity in vitro or in vivo. Surprisingly, srs2-F891A shows a robust shift from noncrossover to crossover repair products in a plasmid-based gap repair assay, but not in an ectopic physical recombination assay. Our findings suggest that the Srs2 C-terminal Rad51 interaction domain is more complex than previously thought, containing multiple interaction sites with unique effects on Srs2 activity.

KW - Crossover control

KW - DNA repair

KW - Genome stability

KW - Helicase

KW - Protein interaction

KW - Recombination

UR - http://www.scopus.com/inward/record.url?scp=85071350423&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85071350423&partnerID=8YFLogxK

U2 - 10.1534/genetics.119.302337

DO - 10.1534/genetics.119.302337

M3 - Article

C2 - 31142613

AN - SCOPUS:85071350423

VL - 212

SP - 1133

EP - 1145

JO - Genetics

JF - Genetics

SN - 0016-6731

IS - 4

ER -