A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M

<div><p>The Mcm2-7 complex is the catalytic core of the eukaryotic replicative helicase. Here, we identify a new role for this complex in maintaining genome integrity. Using both genetic and cytological approaches, we find that a specific <i>mcm</i> allele (<i>mcm2DENQ</i>) causes elevated genome instability that correlates with the appearance of numerous DNA-damage associated foci of γH2AX and Rad52. We further find that the triggering events for this genome instability are elevated levels of RNA:DNA hybrids and an altered DNA topological state, as over-expression of either RNaseH (an enzyme specific for degradation of RNA in RNA:DNA hybrids) or Topoisomerase 1 (an enzyme that relieves DNA supercoiling) can suppress the <i>mcm2DENQ</i> DNA-damage phenotype. Moreover, the observed DNA damage has several additional unusual properties, in that DNA damage foci appear only after S-phase, in G2/M, and are dependent upon progression into metaphase. In addition, we show that the resultant DNA damage is not due to spontaneous S-phase fork collapse. In total, these unusual <i>mcm2DENQ</i> phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/<i>MEC1</i>, suggesting a possible regulatory interplay between Mcm2-7 and ATR during unchallenged growth. As RNA:DNA hybrids primarily result from transcription perturbations, we suggest that surveillance-mediated modulation of the Mcm2-7 activity plays an important role in preventing catastrophic conflicts between replication forks and transcription complexes. Possible relationships among these effects and the recently discovered role of Mcm2-7 in the DNA replication checkpoint induced by HU treatment are discussed.</p></div

The <i>mcm2DENQ</i> mutant exhibits multiple <i>in vivo</i> defects.

<p>A) FACS analysis of wild-type (UPY464), <i>mcm2DENQ</i> (UPY499), and <i>mrc1Δ</i> (UPY713). Briefly, strains were arrested in G1 by addition of α-factor and released into fresh YPD (T = 0). Aliquots were taken at the indicated times and processed for FACS as described in Materials and Methods. B) Cell death assay. Asynchronous cultures of indicated strains from A) ± 3 mM hydrogen peroxide (positive control) were assayed for cell death (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006277#sec020" target="_blank">Materials and Methods</a>). Fluorescence (i.e., cell death) and phase contrast images are shown. C) Percent of dead cells observed in the indicated strains during asynchronous growth. Strains assayed as indicated in A) plus, <i>mrc1Δ rad9Δ sml1Δ</i> (UPY715).</p

Proposed model for Mcm2-7/ATR DNA damage surveillance.

<p>In wild-type cells, individual elongating replication forks pause in an ATR-dependent manner when encountering, transcription bubble via modulation of the Mcm ‘gate’. Such transient pausing provides a temporal window to allow removal of topological perturbations (putatively via Top1 and RNaseH). However, in the <i>mcm2DENQ</i> mutant, failure to regulate gate opening aggravates the accumulation of supercoiling that both stabilizes RNA:DNA hybrids and leads to subsequent DNA damage. Pos SC- positive supercoiling, neg SC- negative supercoiling.</p

The <i>mcm2DENQ</i> allele causes DNA damage.

<p>A) γH2AX assay. Asynchronous cultures of either wild-type (UPY464) or <i>mcm2DENQ</i> (UPY499) were treated ± 0.01% MMS (to induce DSBs) or 200 mM HU (to induce replication stress) in rich media for two hours, then processed for both DAPI (blue) and γH2AX immunofluorescence (green). B) Time-course analysis of γH2AX foci during the cell cycle. Culture of strains from A), plus <i>mrc1Δ</i> (UPY713) and <i>mcm2DENQ sml1Δ</i> (UPY948) were synchronized in G1 with α-factor, released into fresh YPD, and samples were processed for γH2AX immunofluorescence. C) Rad52-YFP assay. Asynchronous cultures of either wild-type (UPY938) or <i>mcm2DENQ</i> (UPY1014) strains were processed for phase contrast and Rad52-YFP fluorescence (green) as shown. To help visualize cells in the negative control panel, fluorescence in the wild-type panel has been enhanced and should not be confused with a genuine DNA-damage signal. D) Strains from C) plus <i>mrc1Δ</i> (UPY1077) were synchronized as in B), and samples were assayed for Rad52-YFP foci as noted.</p

Over-expression of RNaseH and TopI suppress formation of DNA damage foci and cell death in the <i>mcm2DENQ</i> mutant.

<p>A) Plate assay of strains carrying RNaseH over-expression vectors. Strains ± P_gal-RNH1over-expression plasmid (pUP1230) were tested on rich media under non-induced (-Gal) or induced (+Gal) conditions. Strains tested were wild-type without plasmid (UPY938) or with <i>RNH1</i>-expressing plasmid (UPY1289), <i>mcm2DENQ</i> without plasmid (UPY1014), or with <i>RNH1</i>-expressing plasmid (UPY1290). B) Cell death as a function of either Rnh1 or Top1 over-expression. Assays were conducted as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006277#pgen.1006277.g001" target="_blank">Fig 1B</a>, except the strains contained the indicated over-expression plasmid. Asynchronous cultures of the indicated strains were grown with either glucose (-Gal) or galactose (+Gal), and apoptotic cells were counted. Strains with the <i>RNH1</i> plasmid were wild-type (UPY1336), <i>mcm2DENQ</i> (UPY1337), <i>mrc1Δ</i> (UPY1338), while strains with the <i>TOP1</i> plasmid were wild-type (UPY1339), <i>mcm2DENQ</i> (UPY1340), <i>mrc1Δ</i> (UPY1341). C) Bar graph showing levels of cells containing γH2AX and Rad52-YFP foci following over-expression of either Rnh1 or Top1. Asynchronous cultures of the indicated strains were grown with either raffinose (-Gal) or galactose (+Gal), and cells containing γH2AX or Rad52-YFP foci were counted. Strains with the <i>RNH1</i> plasmid were wild-type (UPY1289), <i>mcm2DENQ</i> (UPY1290), <i>mrc1Δ</i> (UPY1304) and were assayed for both γH2AX and Rad52 foci, and strains containing the <i>TOP1</i> plasmid include wild-type (UPY1342), <i>mcm2DENQ (UPY1343)</i>, and <i>mrc1Δ</i> (UPY1344). D) Time course experiment examining γH2AX and Rad52-YFP foci in a <i>mcm2DENQ</i> strain (UPY1290) in the presence (+Gal) and absence (-Gal, growth in raffinose) of RNaseH1 over-expression. To initially maintain the <i>RNH1</i> expression plasmid, the culture was grown in selective media containing either 2% raffinose or 2% galactose for four hours; cells were subsequently transferred to rich media containing either 2% raffinose or 2% galactose for α-factor arrest and subsequent timecourse analysis.</p

Analysis of Gross Chromosome Rearrangement.

<p>Analysis of Gross Chromosome Rearrangement.</p

The <i>mcm2DENQ</i> mutant accumulates RNA:DNA hybrids.

<p>A) The mcm<i>2DENQ</i> mutant (UPY1014) was assayed as chromosome spreads for total DNA (DAPI, blue) and indirect immunofluorescence of RNA:DNA hybrids (red) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006277#sec020" target="_blank">Materials and Methods</a>). Differences in the co-localization of these signals allow assignment of individual cells into one of three distinguishable classes (representative types shown). B) Quantitation of both the level of each individual type of RNA:DNA hybrid as well as the sum (total) of all three types from asynchronous cultures of wild-type (UPY938), <i>mcm2DENQ</i> (UPY1014), and <i>mrc1Δ</i> (UPY1077). C) Total percent of all types of RNA:DNA hybrids from the <i>mcm2DENQ</i> mutant (UPY1014) arrested either in G1 (α-factor) or S-phase (200 mM HU).</p