REGULATION OF GENOME STABILITY VIA MCM2-7 ATPASE ACTIVE SITES IN SACCHAROMYCES CEREVISIAE

Genome stability is vital to the survival and health of eukaryotic organisms. Consequently, many complex mechanisms coordinate with each other in an intricate fashion to ensure that genomes are preserved during duplication and its subsequent propagation. Despite the vast number of factors involved in these processes, their coordinated regulation hinges on a few key components. One such factor is the eukaryotic replicative helicase Mcm2-7, which is a multi-subunit enzyme complex that unwinds DNA during S-phase and paves the way for nascent DNA synthesis by the polymerases. As an essential and highly versatile replisome component, Mcm2-7 is well-suited as the ideal hub for the regulation of not only DNA replication but other fork-related activities such as S-phase checkpoints and sister chromatid cohesion. While all members of the Mcm2-7 complex are highly conserved and essential in all eukaryotes, their contributions towards DNA unwinding are unequal and distinct, and the in vivo functions of most of the Mcm ATPase active sites has remained largely unknown. We conducted an in vivo analysis of a viable mcm2 ATPase active site allele in Saccharomyces cerevisiae and found that under conditions of genotoxic stress it is deficient in the DNA replication checkpoint (DRC) activation, upstream of the Rad53/CHK2 effector kinase. Furthermore, this allele also exhibited a peculiar cell-cycle specific DNA damage phenotype and defective sister chromatid cohesion (SCC) under conditions that are normally conducive to growth. Importantly, these phenotypes manifest from an apparent defect in ATP hydrolysis rather than a qualitative reduction in Mcm2 protein abundance, stability or complex integrity. Therefore, our study demonstrates for the first time that Mcm2-7 can coordinate DNA replication with genome stability through discrete ATPase active sites. Curiously, these functions appear to be separable from general replication defects as shown through a different subset of mcm mutants, indicating that different active sites of Mcm2-7 pleiotropically coordinate various aspects of genome integrity during S-phase

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

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 mcm allele (mcm2DENQ) 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 mcm2DENQ 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 mcm2DENQ phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/MEC1, 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

OsMADS1, a rice MADS-box factor, controls differentiation of specific cell types in the lemma and palea and is an early-acting regulator of inner floral organs

Grass flowers are highly derived compared to their eudicot counterparts. To delineate OsMADS1 functions in rice floret organ development we have examined its evolution and the consequences of its knockdown or overexpression. Molecular phylogeny suggests the co-evolution of OsMADS1 with grass family diversification. OsMADS1 knockdown perturbs the differentiation of specific cell types in the lemma and palea, creating glume-like features, with severe derangements in lemma differentiation. Conversely, ectopic OsMADS1 expression suffices to direct lemma-like differentiation in the glume. Strikingly, in many OsMADS1 knockdown florets glume-like organs occupy all the inner whorls. Such effects in the second and third whorl are unexplained, as wild-type florets do not express OsMADS1 in these primordia and because transcripts for rice B and C organ-identity genes are unaffected by OsMADS1 knockdown. Through a screen for OsMADS1 targets we identify a flower-specific Nt-gh3 type gene, OsMGH3, as a downstream gene. The delayed transcription activation of OsMGH3 by dexamethasone-inducible OsMADS1 suggests indirect activation. The OsMGH3 floret expression profile suggests a novel role for OsMADS1 as an early-acting regulator of second and third whorl organ fate. We thus demonstrate the differential contribution of OsMADS1 for lemma versus palea development and provide evidence for its regulatory function in patterning inner whorl organs

OsMADS1, a rice MADS-box factor, controls differentiation of specific cell types in the lemma and palea and is an early-acting regulator of inner floral organs

Grass flowers are highly derived compared to their eudicot counterparts. To delineate OsMADS1 functions in rice floret organ development we have examined its evolution and the consequences of its knockdown or over expression. Molecular phylogeny suggests the co-evolution of OsMADS1 with grass family diversification. OsMADS1 knockdown perturbs the differentiation of specific cell types in the lemma and palea, creating glume-like features, with severe derangements in lemma differentiation. Conversely, ectopic OsMADS1 expression suffices to direct lemma-like differentiation in the glume. Strikingly,in many OsMADS1 knockdown florets glume-like organs occupy all the inner whorls. Such effects in the second and third whorl are unexplained, as wild-type florets do not express OsMADS1 in these primordia and because transcripts for rice B and C organ-identity genes are unaffected by OsMADS1 knockdown. Through a screen for OsMADS1 targets we identify a flower-specific Nt-gh3 type gene, OsMGH3, as a downstream gene. The delayed transcription activation of OsMGH3 by dexamethasone-inducible OsMADS1 suggests indirect activation. TheOsMGH3 floret expression profile suggests a novel role for OsMADS1 as an early-acting regulator of second and third whorl organ fate. We thus demonstrate the differential contribution of OsMADS1 for lemma versus palea development and provide evidence for its regulatory function in patterning inner whorl organs

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

Analysis of Gross Chromosome Rearrangement.

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

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

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

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

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