Apr 24, 2020

C-terminal HA tags compromise function and exacerbate phenotypes of Saccharomyces cerevisiae Bloom's helicase homolog Sgs1 SUMOylation-associated mutants

BioRxiv : the Preprint Server for Biology
M. Cohen, Michael Lichten

Abstract

The Sgs1 helicase and Top3-Rmi1 decatenase form a complex that affects homologous recombination outcomes during the mitotic cell cycle and during meiosis. Previous studies have reported that Sgs1-Top3-Rmi1 function is regulated by SUMOylation that is catalyzed by the Smc5-Smc6-Mms21 complex. These studies used strains in which SGS1 was C-terminally tagged with three or six copies of a human influenza hemagglutinin-derived epitope tag (3HA and 6HA). They identified SGS1 mutants that affect its SUMOylation, which we will refer to as SGS1 SUMO-site mutants. In previous work, these mutants showed phenotypes consistent with substantial loss of Sgs1-Top3-Rmi1 function during the mitotic cell cycle. We find that the reported phenotypes are largely due to the presence of the HA epitope tags. Untagged SGS1 SUMO-site mutants show either wild-type or weak hypomorphic phenotypes, depending on the assay. These phenotypes are exacerbated by both 6HA and 3HA epitope tags in two different S. cerevisiae strain backgrounds. Importantly, a C-terminal 6HA tag confers strong hypomorphic or null phenotypes on an otherwise wild-type Sgs1 protein. Taken together, these results suggest that the HA epitope tags used in previous studies seriously comprom...Continue Reading

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Mentioned in this Paper

Study
Immune Response
Craniosynostosis
VANGL1 protein, human
Hindgut
PDZD2
Genes
Malignant Neoplasm of Spinal Cord
Pancreatic Carcinoma
Sacral Agenesis

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