Volume 12, Issue 6, Pages 1525-1536 (December 2003) The Saccharomyces cerevisiae Helicase Rrm3p Facilitates Replication Past Nonhistone Protein-DNA Complexes  Andreas S. Ivessa, Brian A. Lenzmeier, Jessica B. Bessler, Lara K. Goudsouzian, Sandra L. Schnakenberg, Virginia A. Zakian  Molecular Cell  Volume 12, Issue 6, Pages 1525-1536 (December 2003) DOI: 10.1016/S1097-2765(03)00456-8

Figure 1 Rrm3p Facilitates Replication Fork Movement through Centromeres, tRNA Genes, and Inactive Replication Origins DNA from asynchronous wild-type (WT) or rrm3Δ cultures was digested with restriction enzymes and analyzed by two-dimensional gel electrophoresis and Southern hybridization. (A) Schematics of the 2D gel technique: 1N, nonreplicating fragment; 2N, almost fully replicated fragment right before sister chromatids separate; P, replication pause; BU, bubble-shaped replication intermediates. (B–J) Southern blots were probed to detect the following regions (restriction enzymes and hybridization probes are noted in parentheses): (B) CEN3 (XbaI, YCL001W-B); (C) CEN4 (XbaI, YDL001W); (D) tRNAA (tA[AGC]F; BglII, HIS2); (E) tRNAY (tY[GUA]F1; EcoRV, YFR012W); (F) tRNAF (tF[GAA]F; BamHI,YFR007W); (G) ARS304 (BamHI, KAR4); (H) ARS608 (BglII, MET10); (I) HML (XhoI, YCL069W); (J) HMR (EcoRV, YCRWΔ12). Arrows indicate sites of fork pausing. BR, broken replication intermediates (Martin-Parras et al., 1992). BU (E), bubble-shaped replication intermediate arising from an active origin of replication on the DNA fragment (Brewer and Fangman, 1987). The positions of the E and I silencers at the HML and HMR loci (I and J) on the arc of forked replication intermediates are indicated. Molecular Cell 2003 12, 1525-1536DOI: (10.1016/S1097-2765(03)00456-8)

Figure 2 Replication of Most of the Genome Is Not Affected by Rrm3p 2D gels were used to examine DNA replication in twelve overlapping restriction fragments within a 105 kbp region of chromosome VI, starting 68 kbp from the left telomere of chromosome VI. (A) The locations of the 12 fragments are shown. Positions of tRNAs (T), active ARSs (A), CEN6 (C), and in vivo Rap1p binding sites (Lieb et al., 2001) (arrows) are indicated. Asterisks mark positions of replication pauses in rrm3Δ cells. (B) DNA from asynchronous wild-type (WT) or rrm3Δ cells was digested with the indicated restriction enzymes, analyzed by 2D gels, and probed for the indicated regions: (1) AatII, YFL030W; (2) XhoI, YFL030W; (3) HindIII, FRS2; (4) BglII, LPD1; (5) KpnI, CDC4; (6) PvuII/AflII, CDC4; (7) AflII, MSH4; (8) SacI, MSH4; (9) ClaI, DEG1; (10) XhoI, DEG1; (11) AflII/XmaI, YFR007W; (12) BglII, YFR012W. Arrows, sites of replication fork pausing; BR, broken replication intermediates; BU, bubble structures. The smaller arc of forked replication intermediates in panel 6 was probably due to a crosshybridizing fragment. Molecular Cell 2003 12, 1525-1536DOI: (10.1016/S1097-2765(03)00456-8)

Figure 3 Lack of Rrm3p Does Not Increase Chromosome Loss but Does Increase Interchromosomal Recombination The two copies of chromosome VII that are present in the disome strain, the genotypes of cells that undergo loss or recombination of the LYS5 bearing copy of chromosome VII, and the chromosome loss and recombination rates (± standard deviations) in wild-type and rrm3Δ cells are shown. Relevant genes are indicated by black rectangles. The difference in the chromosome loss rates between wild-type and rrm3Δ cells was not statistically significant while the difference in recombination rates is significant (p < 0.05 by the criterion of a t test). The centromere is indicated by the open circle and positions of tRNA genes on chromosome VII by vertical lines. Molecular Cell 2003 12, 1525-1536DOI: (10.1016/S1097-2765(03)00456-8)

Figure 4 Lack of Rrm3p Activates the Intra-S Phase Checkpoint (A) Genetic interactions between RRM3 and MEC1 or RAD9. Heterozygous diploids bearing the centromere plasmid pIA20, which contains URA3, ADE3, and RRM3, were dissected, and ade2 ade3 spore clones of the indicated genotypes and carrying pIA20 were identified, streaked on nonselective media, incubated at 23° for 5 days, and then kept at 4° for 3 days to enhance color development. An ade2 ade3 cell carrying pIA20 generates red colonies; white sectors are generated by cells that lose pIA20. White sectors in red colonies indicate that cells of the indicated genotype can lose RRM3 and retain viability; mec1Δ sml1Δ rrm3Δ was the only strain tested that did not generate white sectors and did not grow on FOA plates. (B) Rad53p is hyperphosphorylated and active in rrm3Δ cells. Cell extracts were prepared from wild-type or rrm3Δ asynchronous log phase cultures in either the VPS106 or YPH499 background. Where indicated, the YPH499 wild-type strain was treated with either 0.1% MMS or 0.2 M HU, conditions sufficient to arrest cells and activate Rad53p (Sun et al., 1996). Proteins were separated in acrylamide gels, transferred to membranes, and then analyzed either by Western blotting using a Rad53p polyclonal antibody JDI47 from J. Diffley (top panel) or by an in situ Rad53p kinase assay (bottom panel) (Pellicioli et al., 1999). Molecular Cell 2003 12, 1525-1536DOI: (10.1016/S1097-2765(03)00456-8)

Figure 5 Replication Pausing at tRNA Genes Is Dependent upon Binding of the Transcription Complex The tRNAY gene was inserted on chromosome VI and renamed tRNAY(moved). 2D gels were used to examine replication fork progression in wild-type and rrm3Δ cells at a wild-type tRNAY(moved), tRNAY(moved) with a B block mutation that reduces TFIIIC binding ∼370-fold (Baker et al., 1986), and tRNAY(moved) in which both the normal and backup terminators were mutated, which increases the size of the transcript (Allison and Hall, 1985) (brackets, transcribed region). Arrows point to the position of the wild-type and mutant tRNAY(moved) genes on the arc of replication intermediates. Molecular Cell 2003 12, 1525-1536DOI: (10.1016/S1097-2765(03)00456-8)

Figure 6 ORC and Rap1p Binding at the Synthetic HMR-E Silencer Is Needed to Make Its Replication Rrm3p Dependent Replication of the HMR locus in wild-type and rrm3Δ strains in which an 868 bp fragment containing HMR-E was replaced with a 138 bp fragment that contains either the wild-type or a mutant version of the synthetic silencer (McNally and Rine, 1991). The wild-type synthetic silencer has binding sites for ORC, Rap1p, and Abf1p in the same order and spacing as at the natural E silencer. Strains used were WT or rrm3Δ versions of JRY2879 (WT synthetic silencer), JRY2881 (synthetic silencer with mutated ACS in which 8 of the 11 bases in the ORC recognition site are altered), and JRY3937 (synthetic silencer in which 2 of the 13 bases in the Rap1p recognition site are altered). Genomic DNA was cut with HindIII and analyzed by 2D gels. Positions of E and I silencers on the arc of forked replication intermediates are indicated. Molecular Cell 2003 12, 1525-1536DOI: (10.1016/S1097-2765(03)00456-8)

Figure 7 Deleting Sir Proteins Reduces the Need for Rrm3p in Replication of the HM Loci, Telomeres, and Inactive Replication Origins (A–F) Replication through HML. XhoI-digested genomic DNA from the indicated strains was analyzed by 2D gels using a probe for HML. The positions of the E and I silencers on the arc of forked replication intermediates are indicated. (A) and (D) are the same gels as in Figure 1I. (G–L) Replication through HMR. EcoRV-digested genomic DNA from the indicated strains was analyzed by 2D gels using a probe for HMR. (G) and (J) are the same as in Figure 1J. The positions of the E and I silencers on the arc of forked replication intermediates are indicated; bracket indicates a pause not seen in rrm3Δ cells. Longer exposures of (J), (K), and (L) are shown in the corresponding panels (M), (N), and (O). (P–U) Replication through the left telomere of chromosome III. Genomic DNA from the indicated strains was digested with SphI and analyzed by 2D gels. The position of the normally inactive ARS300 in the subtelomeric X element, 1 kbp from the chromosome end, is indicated by asterisks. The telomere is indicated by arrows. BU, bubble structures. Molecular Cell 2003 12, 1525-1536DOI: (10.1016/S1097-2765(03)00456-8)