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

PLoS Genet. 2016 Aug 24;12(8):e1006277. doi: 10.1371/journal.pgen.1006277. eCollection 2016 Aug.

Abstract

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.

MeSH terms

  • Cell Cycle Checkpoints / genetics
  • DNA Damage / genetics*
  • DNA Helicases / genetics
  • DNA Replication / genetics*
  • Genomic Instability / genetics
  • Intracellular Signaling Peptides and Proteins / genetics*
  • Minichromosome Maintenance Proteins / genetics*
  • Mutation
  • Phosphorylation
  • Protein Serine-Threonine Kinases / genetics*
  • RNA / genetics
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / genetics*

Substances

  • Intracellular Signaling Peptides and Proteins
  • Saccharomyces cerevisiae Proteins
  • RNA
  • MEC1 protein, S cerevisiae
  • Protein Serine-Threonine Kinases
  • DNA Helicases
  • Minichromosome Maintenance Proteins