QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

Vol. 20, Issue 4, 1241-1251, February 15, 2009

This Article Full Text Full Text (PDF) Supplemental Materials All Versions of this Article: E08-06-0659v1 E08-06-0659v2 20/4/1241    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Xiong, L. Articles by Johnson, E. S. Search for Related Content PubMed PubMed Citation Articles by Xiong, L. Articles by Johnson, E. S.

Deficient SUMO Attachment to Flp Recombinase Leads to Homologous Recombination-dependent Hyperamplification of the Yeast 2 µm Circle Plasmid

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107

Submitted June 30, 2008; Revised November 18, 2008; Accepted December 8, 2008
Monitoring Editor: Charles Boone

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 µm circle plasmid (2 µm). Here we show that accumulation of 2 µm in the SUMO pathway mutants siz1 siz2, slx5, and slx8 is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 µm. Characterization of this species from siz1 siz2 showed that it contains tandem copies of the 2 µm sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 µm–encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 µm sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir° strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 µm amplification.

* These authors contributed equally to this work.

Address correspondence to: Erica S. Johnson (erica.johnson{at}jefferson.edu).

Abbreviations used: 2 µm, yeast 2-µm circle plasmid; BIR, break-induced replication; DSB, double-strand break; EtBr, ethidium bromide; FRT, Flp recombination target; GFP, green fluorescent protein; HJ, Holliday junction; HMW, high molecular weight; HRR, homologous recombination repair; NPC, nuclear pore complex; QAOS, quantitative analysis of single-stranded DNA; RF, replication fork; ssDNA, single-stranded DNA; wt, wild-type.