Media 1: Manipulation of micro-particles by flexible polymer-based optically-induced dielectrophoretic devices

Originally published in Optics Express on 02 January 2012 (oe-20-1-583

H2Bub-mediated fork stalling is independent of dNTP pool size.

<p>The size of the dNTP pools in exponentially-growing cultures of WT (CFK1419), <i>htb-K123R</i> (CFK1421), <i>dun1Δ</i> (YCL023), and <i>dun1Δ htb-K123R</i> (YCL025) cells in YPD media. Two independent isogenic strains of each genotype were analyzed. (B) Graph depicting the distribution of BrdU track lengths in WT (CFK1419), <i>htb-K123R</i> (CFK1421), <i>dun1Δ</i> (YCL023), and <i>dun1Δ htb-K123R</i> (YCL025) mutants, as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004667#pgen-1004667-g001" target="_blank">Fig. 1F</a>. (C) Replication profiles of the replication origins <i>ARS207</i>, <i>ARS208/209</i>, <i>ARS718</i>, <i>ARS719/720</i>, and <i>ARS1309/1310</i> in WT (CFK1419), <i>htb-K123R</i> (CFK1421), <i>dun1Δ</i> (YCL023), and <i>dun1Δ htb-K123R</i> (YCL025) mutants. The BrdU histogram was analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004667#pgen-1004667-g001" target="_blank">Fig. 1C–E</a>. (D) Temperature sensitivity and HU resistance of the indicated genotypes (WT (CFK1204), <i>sml1Δ</i> (CFK1481), <i>dun1Δ</i> (YMW069), <i>htb-K123R</i> (CFK1231), and <i>htb-K123R</i> in combination with <i>sml1Δ</i> (CFK1482) or <i>dun1Δ</i> (YMW072)). Log-phase cells were serially diluted and spotted onto YPD plates with or without HU, and incubated at 30°C or 37°C for 2–3 days.</p

H2Bub and Sgs1 cooperatively control replication fork stalling and stability under HU.

<p>(A) Replication profiles of the early origins <i>ARS305</i> and <i>ARS607</i> in <i>sgs1Δ</i> (YCL007) and <i>sgs1Δ htb-K123R</i> (YCL008) mutants. The BrdU histogram was analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004667#pgen-1004667-g001" target="_blank">Fig. 1C–E</a>. (B) Graph depicting the distribution of BrdU track lengths in WT (CFK1419), <i>sgs1Δ</i> (YCL007), and <i>sgs1Δ htb-K123R</i> (YCL008) mutants, as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004667#pgen-1004667-g001" target="_blank">Fig. 1F</a>. (C) Cell cycle progression of these mutants in the presence of 0.2M HU was analyzed by flow cytometry. (D) The size of the dNTP pools in exponentially-growing cultures of WT (CFK1419), <i>htb-K123R</i> (CFK1421), <i>sgs1Δ</i> (YCL007), and <i>sgs1Δ htb-K123R</i> (YCL008) cells in YPD media. Two independent isogenic strains of each genotype were analyzed. (E) Survival of WT (CFK1204), <i>htb-K123R</i> (CFK1231), <i>sgs1Δ</i> (CFK1447), and <i>sgs1Δ htb-K123R</i> (CFK1453) cells in response to acute doses of HU, as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004667#pgen-1004667-g001" target="_blank">Fig. 1A</a>.</p

A model for how H2B mono-ubiquitylation facilitates fork stability under replication stress.

<p>Upon HU-induced stress, H2Bub promotes nucleosome assembly, which assists replication fork stalling, Sgs1 recruitment, and Rad53 phosphorylation. The reassembly of chromatin on nascent DNA restricts fork progression and promotes replication fork stability and its recovery after the removal of HU. In the absence of H2Bub (<i>bre1Δ/htb-K123R</i>), replication fork movement is faster than even that observed under nucleotide depletion by HU, which results in shorter tracts of RPA-coated-single-stranded DNA. This in turn reduces retention of Sgs1 at the forks, and delays phosphorylation of Rad53.</p

H2Bub promotes chromatin assembly in response to replication stress.

<p>(A) H2Bub is required for nucleosome assembly near replication forks under replication stress. WT (CFK1204) or <i>htb-K123R</i> (CFK1231) cells were arrested in G1 phase using α-factor, and were then released into 200 mM HU at 30°C for 60 minutes. At the indicated time, cells were collected and histone occupancy at two early origins (<i>ARS305</i> and <i>ARS607</i>) and one late origin (<i>ARS501</i>) was determined by ChIP using antibodies against H3. IP signals at ARS sequences were normalized to IP signals at TELVI-R. The results are the mean +/− SEM of three replicates. (B) Genetic interactions between H2Bub and histone chaperones (Cac1, Asf1, and Hir1) or a histone acetyl-transferase (Rtt109). Ten-fold serial dilutions of the indicated strains (WT (CFK1204), <i>htb-K123R</i> (CFK1231), <i>cac1Δ</i> (CFK1206), <i>cac1Δ htb-K123R</i> (CFK1237), <i>asf1Δ</i> (CFK1208), <i>asf1Δ htb-K123R</i> (CFK1233), <i>rtt109Δ</i> (CFK1212), <i>rtt109Δ htb-K123R</i> (CFK1241), <i>hir1Δ</i> (CFK1202), and <i>hir1Δ htb-K123R</i> (CFK1235)) were spotted onto YPD plates containing HU (0 or 50 mM), and cell growth was monitored for 2–3 days. (C) The survival of asf1Δ (CFK1208) and <i>asf1Δ htb-K123R</i> (CFK1233) cells in response to acute treatment with HU, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004667#pgen-1004667-g001" target="_blank">Fig. 1A</a>. (D) H2Bub modulates the interaction between Asf1 and Rad53 under HU stress. Asynchronous cultures of WT (YMW105) or <i>htb-K123R</i> (YMW104) cells were untreated (−) or treated (+) with 0.2M HU for 90 minutes. Protein extracts were prepared and incubated with pre-bound anti-HA-protein G beads to pull down Asf1-3×HA, and the immune-precipitates were resolved by SDS-PAGE, before being probed with either anti-HA or anti-Rad53 antibodies. (E) A working model depicting the role of H2Bub in nucleosome assembly under HU stress. H2Bub coordinates nucleosome assembly in response to replication stress by directly contributing to nucleosome formation and by indirectly regulating the availability of Asf1 during HU stress.</p

H2Bub and Sgs1 play interdependent roles in Rad53 phosphorylation.

<p>(A) Sgs1 occupancy at replication origins is unstable in <i>htb-K123R</i> cells exposed to HU. WT (CFK1764) or <i>htb-K123R</i> (CFK1765) cells were synchronized in G1 and then released into fresh YPD containing 0.2M HU for 60 minutes at 30°C. Chromatin immunoprecipitation (ChIP) was performed using antibodies against Sgs1-3×Myc. DNA was quantified by qPCR using primers adjacent to ARS305 and a region 3.5 kb distal. Sgs1 occupancy at each time point was normalized to that of G1. (B) Activation of Rad53 is impaired in the absence of both H2Bub and Sgs1. WT (CFK1204), <i>htb-K123R</i> (CFK1231), <i>sgs1Δ</i> (CFK1447), and <i>sgs1Δ htb-K123R</i> (CFK1453) cells were arrested in G1 and released into fresh media containing 0.2M HU for 90 minutes at 30°C. Whole cell lysates were prepared at the indicated time points, and analyzed by Western blot using antibodies against Rad53 (EL7), phospho-Rad53 (F9), H2B, and mono-ubiquitylated H2B (anti-FLAG). G6PDH was used as a loading control.</p