Sgs1 Prevents COs in <i>zmm</i> Mutants

<div><p>(A) Schematic representation of the ectopic <i>URA3-ARG4</i> interval. Symbols are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020155#pgen-0020155-g001" target="_blank">Figure 1</a>. EcoRI (E) and XhoI (X) restrictions sites are indicated. To detect COs and DSBs, DNA is digested with XhoI and probed with <i>ARG4</i> sequences (ArgD; [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020155#pgen-0020155-b016" target="_blank">16</a>]). To detect NCOs and a CO product (CO1′), DNA is digested with EcoRI and XhoI and probed with <i>HIS4</i> sequences (HisU; [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020155#pgen-0020155-b016" target="_blank">16</a>]).</p><p>(B) Southern blot of DNA isolated from a meiotic culture of MJL3035 (<i>TRP1:SGS1</i>) at the indicated time after initiation of sporulation, digested, and probed to detect COs and DSBs. mw, HinDIII digest of phage λ DNA. DSBs occur in <i>URA3-ARG4</i> inserted at both <i>his4</i> and <i>leu2,</i> but are ~7-fold stronger in the <i>his4</i> insert than at <i>leu2</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020155#pgen-0020155-b016" target="_blank">16</a>]. Palindrome cleavage, by unidentified activities, occurs at the same time as CO formation and results in a band about the size of DSB2 (T. Allers, L. Jessop, and M. Lichten, unpublished data).</p><p>(C) Southern blot of the same samples, digested and probed to detect NCO and CO1′ recombinants. <i>HIS4</i>, <i>HIS4</i> locus lacking an insert; DSB*, DSB1 product where resection has passed the EcoRI site in <i>HIS4</i>; mw, BstEII digest of phage λ DNA.</p><p>(D) Molecular analysis of mutants. Color codes: <i>TRP1:SGS1,</i> black; <i>sgs1ΔC795,</i> grey; <i>TRP1:SGS1 zmm</i> single mutants, red; <i>sgs1ΔC795 zmm</i> double mutants, blue<i>.</i> M1+M2, percent of cells containing at least two nuclei, a sign of passage through meiosis 1. DSB, DSB1 band signal/total lane signal from Southern blots as in (B). CO, CO1′ signal/total lane signal from Southern blots as in (C). NCO, NCO band signal/total insert signal from Southern blots as in (C).</p>