Supplemental Figure S1. Pfh1 promotes replication through single and multiple tRNA genes in a cluster. (A) tRNACYS.01 on chromosome I. N, NcoI. The NcoI digestion results in a 5.1 kb DNA fragment. The schematic on the right illustrates replication intermediates formed during replication of tRNACYS.01. (B) 2D gel analysis of nmt-pfh1-GFP cells grown in thiamine for 0 (-thiamine) or 12 h (+12 h thiamine). The letters on the gel illustrate replication intermediates shown in (A). (C) tRNAGLY.05 and tRNAARG.04 on chromosome II. A, AgeI. AgeI digested DNA yields a 5.5 kb fragment. (D) 2D gel analysis of tRNAGLY.05 and tRNAARG.04 shown as in (B). bu: bubble arc. Asterisks indicate the bubble arc. tRNAARG.04 and tRNAGLY.05 are separated by only 300 bps. We detected three pause sites in the arc formed by forked replication intermediates (marked b-d), all of which were increased two- to threefold in the absence of Pfh1. The two closely spaced tRNA genes did not seem to be more difficult for replication fork progression and resolution than replication through single tRNA genes. In the absence of Pfh1, a new pause site was detected (marked a), and two sites of converged forks were visible (marked e and f).
Supplemental Figure S2.-H2A antibody is specific for phosporylated H2A. Wild type (WT) and hta1 hta2 (htax) cells were grown in the presence or absence of 30 µM camptothecin (CPT) for 2 h before cross-linking the cells in formaldehyde. The cells were lysed and chromatin immunoprecipitated using an anti- -H2A antibody (a kind gift from C. Redon). The immunoprecipitated DNA was analyzed using quantitative PCR with primers specific for act1+. The data are presented on a logarithmic scale as immunoprecipitated DNA divided by input DNA. Data represent the mean of three independent cultures, with error bars indicating one standard deviation. The fold difference above each bar was calculated by dividing mean values for WT, WT+CPT, htax+CPT or htax over the htax mean value. Notably, -H2A levels in CPT-treated wild type cells increased twofold in comparison to untreated wild type cells, but was unchanged in CPT-treated htax cells compared to untreated htax cells.