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.
Highlights
Genomic instability, resulting from the loss or rearrangement of the genetic material, strongly correlates with the development of a large variety of human diseases
The precise regulation of DNA replication is necessary to avoid genome instability, the deleterious alteration of genetic information that is a hallmark of diseases like cancer
Using budding yeast as our model system, we show that such damage-avoidance likely requires direct involvement of the Mcm2-7 replicative helicase, the molecular motor that unwinds DNA during replication
Summary
Genomic instability, resulting from the loss or rearrangement of the genetic material, strongly correlates with the development of a large variety of human diseases (reviewed in [1,2,3,4,5]). A major source of such instability is DNA double-strand breaks (DSBs). These are thought to predominantly occur during replication through stochastic fork collapse [6,7,8], a process believed to result from the dissolution or inappropriate repair of stalled replication forks that have been crippled by the loss of core replication factors. Such breaks have a variety of defining features. Specific mutations in replication fork components (e.g., loss of Mrc1, [8] and references therein) generate structurally unstable forks that coordinately increase the levels of both stochastic fork collapse and DSB formation
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