Validation of genetic interactions of <i>RAD1</i>, <i>RAD10</i>, <i>RAD14</i>, and <i>RAD59</i> with <i>cdc9-1</i> by tetrad dissection.

<p>(<b>A</b>) Heat map indicating strong positive (green) or negative (red) interactions between <i>cdc9-1</i> and <i>rad14Δ</i>, <i>rad1Δ</i> or <i>rad59Δ</i>, respectively. Black indicates no genetic interaction. Gray indicates that the interaction could not be scored. Among a collection of approximately 1800 SGA queries, only <i>scl1Δ</i> mutants displayed a genetic interaction signature similar to that of <i>cdc9-1</i> with respect to these three mutants. Other query mutants that exhibited similar genetic interactions with two out of the three genes are shown as well as <i>rad27Δ</i>, which exhibited synthetic sickness with <i>rad59Δ</i>. PH indicates strains received from Phil Hieter. The heat map was generated based on previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066379#pone.0066379-Costanzo1" target="_blank">[56]</a> and new SGA screens in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066379#pone.0066379.s009" target="_blank">Table S2</a>. (<b>B–F</b>) Selected diploid strains were dissected and incubated at either 25°C or 30°C. All haploid genotypes are as indicated on the right. Four independent tetrads (1-4) are laid out horizontally. The diploid strain genotypes are as followed: (<b>B</b>) <i>CDC9/cdc9-1 rad1Δ/RAD1</i>, (<b>C</b>) <i>CDC9/cdc9-1 rad14Δ/RAD14</i>, (<b>D</b>) <i>CDC9/cdc9-1 rad10Δ/RAD10</i>, (<b>E</b>) <i>CDC9/cdc9-1 rad59Δ/RAD59,</i> (<b>F</b>) <i>CDC9/cdc9-1 slx4Δ/SLX4</i>. (<b>G</b>) A Venn diagram summarizes some of the pertinent genetic interactions with <i>cdc9-1</i> mutants identified in this study. Genes are grouped into their respective repair pathways, homologous recombination (HR), HR-mediated single-strand annealing (SSA), and nucleotide excision repair (NER). Negative and positive genetic interactions with <i>cdc9-1</i> mutants identified in our SGA screen are illustrated as red and green solid lines, respectively. Red dotted lines indicate negative interactions with <i>cdc9-1</i> mutants that were only observed from manual tetrad analysis. No genetic interaction was observed between <i>SLX4</i> and <i>cdc9-1</i>.</p

Deletion of <i>RAD59</i> in <i>cdc9-td</i> mutants displayed an increase in Mrc1 phosphorylation.

<p>(<b>A</b>) Asynchronous cultures of the indicated strains were grown at 25°C and subsequently shifted to 30°C for 90 or 180 min. Unmodified and phosphorylated Rad53 was detected using an anti-Rad53 antibody. (<b>B</b>) Asynchronous cultures were grown at 28°C and subsequently shifted to 37°C for the indicated time. Unmodified and phosphorylated 3HA-Mrc1 and Cdc9-td-HA levels were monitored using an anti-HA antibody. α-tubulin was used as a loading control.</p

Defects in DNA ligase I trigger PCNA mono- and poly-ubiquitination independently of lysine 164.

<p>(<b>A</b>) Successive 10-fold dilutions of the indicated strains were grown on Sc-His plates for 3 days at the indicated temperatures. (<b>B, C</b>) All strains shown in <b>A</b> were grown asynchronously to mid-log phase at 25°C and shifted to the restrictive temperature of 35°C for 3 hr. PCNA and its ubiquitinated forms, and Cdc9 were detected with anti-PCNA (S871) and anti-Cdc9 antibodies, respectively. α-tubulin served as a loading control. Asterisks indicate non-specific bands.</p

Overexpression of <i>Chlorella</i> virus DNA ligase fully complements <i>cdc9-1</i> temperature sensitivity and reverses PCNA ubiquitination.

<p>(<b>A</b>) Successive 10-fold dilutions of the indicated strains were grown on Sc-His plates for 3 days at the indicated temperatures. Expression of <i>Chlorella</i> virus DNA ligase from the p<i>ChVLig-3HA</i> plasmid was under the control of the <i>CDC9</i> promoter. (<b>B</b>) All strains shown in <b>A</b> were grown asynchronously to mid-log phase at 25°C. Subsequently, cultures were shifted to the restrictive temperature of 35°C for 3 hr. PCNA and its ubiquitinated forms, Cdc9 and ChVLig-3HA were detected with anti-PCNA (S871), anti-Cdc9, and anti-HA antibodies, respectively. (<b>C</b>) Successive 10-fold dilutions of the indicated strains were grown on Sc-His plates containing either 2% glucose or 2% galactose for 5 days at 25°C and 35°C. Overexpression of <i>Chlorella</i> virus DNA ligase from the pRS423gal-<i>ChVLig-3HA</i> plasmid was under the control of the <i>GAL10</i> promoter. (<b>D</b>) Yeast strains shown in <b>C</b> were grown asynchronously to mid-log phase in medium containing 2% raffinose at 25°C. Cultures were split and grown in the presence of either 2% glucose or 2% galactose at the restrictive temperature of 35°C for 3 hr. PCNA and its ubiquitinated forms, Cdc9 and ChVLig-3HA were detected with anti-PCNA (S871), anti-Cdc9, and anti-HA antibodies, respectively. In <b>B</b> and <b>D</b>, α-tubulin served as a loading control. Asterisks indicate non-specific bands.</p

PCNA ubiquitination at K107 is crucial for the S phase arrest in <i>cdc9-1</i> mutants.

<p>(<b>A</b>) Asynchronous cultures of the indicated strains were grown at 25°C and shifted to 30°C for 1.5 and 3 hr. (<b>B</b>) Asynchronous cultures of the indicated strains were grown at 25°C and shifted to 35°C for 1.5 and 3 hr in the presence of 2% galactose. On the right, the same asynchronous <i>cdc9-1</i>+ p<i>Rad53-KD</i> culture was split and grown in the presence of 2% glucose. In <b>A</b> and <b>B</b>, DNA content was stained with Sytox Green and monitored by flow cytometry. The vertical line indicates a 2C DNA content.</p

Spotting assay of different <i>cdc9-1</i> single and double mutants.

<p>(<b>A</b>) Successive 10-fold dilutions of the indicated strains were grown on YPD plates for 2 days at 25°C and 30°C. (<b>B</b>) Asynchronous cultures of the indicated strains were grown to mid-log phase at 25°C and subsequently shifted to the restrictive temperature of 35°C for 3 hr. Cdc9 expression was detected with anti-Cdc9 antibody and α-tubulin was used as a loading control. The asterisk indicates non-specific bands. Extracts from <i>cdc9-1 slx4Δ</i> mutants were fractionated on a separate gel, as indicated by the vertical lines.</p

Hypothetical models to explain how Rad53 might be regulated at PCNA^K107-Ub-flagged replication forks.

<p>(<b>A</b>) In the absence of Cdc9 activity, we suggest that PCNA ubiquitination, in conjunction with Exo1, promotes the generation of ssDNA on the lagging strand template (left). ssDNA regions recruit Mec1 to the stalled replication fork. Mec1 then phosphorylates Mrc1 at the fork, which leads to Rad53 hyper-phosphorylation to delay cells in S phase (right). Rad53, in turn, has been shown to phosphorylate Exo1 to inhibit further nascent strand degradation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066379#pone.0066379-Morin1" target="_blank">[62]</a>. (<b>B</b>) Alternatively, pol-δ may displace the downstream Okazaki fragment, generating a 5′-flap (left). The 5′-flap may bind to RPA (not shown), thereby recruiting Mec1 to chromatin in order to phosphorylate Mrc1 at the fork. In both, <b>A</b> and <b>B</b>, Rad59 acts to suppress Mrc1 phosphorylation at the fork. As described in more detail in the text, Rad59 acts in concert with Rad52.</p

AdditionalDataS5

This file contains the complete list of yeast strains present on the diagnostic array, which was used for genetic interaction screens in this study

Legislative Documents

Also, variously referred to as: Senate bills; Senate documents; Senate legislative documents; legislative documents; and General Court documents

AdditionalDataS6

This file contains the trigenic interactions list of MDY2-MTC1 and digenic interaction list of MDY2 and MTC1 corresponding to Fig. 3. The ‘Tetrad Analysis’ tab contains confirmations results obtained from tetrad analysis: SS is synthetic sick, SL is synthetic lethal. The ‘Genetic interactions’ tab contains columns that are annotated with ‘CellMap’ since they contain genetic interactions from (7) downloaded from theCellMap.org (26) as well as scores derived in this study