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

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.

R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

“…We propose that transcription of the (GAA) 10 repeat generates an R-loop (Fig 7). During replication, the approaching replicative helicase traverses the transcription complex by displacing the RNA polymerase (Pomerantz & O'Donnell, 2010) or by reorganising the helicase itself Vijayraghavan et al, 2016). Biophysical calculations show that DNA:RNA hybrids are sufficiently thermodynamically stable to survive the accumulation of positive supercoiling generated ahead of the replicative helicase (Belotserkovskii et al, 2013).…”

Transcription-mediated replication hindrance: a major driver of genome instability

“…R-loop-accumulating yeast mutants activate the S-phase checkpoint (Gómez-González et al 2009) and head-on T-R conflicts induce ATR checkpoint activation in human cells (Hamperl et al 2017). Furthermore, R-loop-accumulating mutants require a functional S-phase checkpoint to survive (Gómez-González et al 2009), and an MCM-specific mutant impairing its checkpoint activation function was found to lead to R-loop-driven T-R conflicts (Vijayraghavan et al 2016). These results suggest that the replication checkpoint protects against the harmful effect of T-R collisions.…”

Human THO –Sin3A interaction reveals new mechanisms to prevent R‐loops that cause genome instability

“…DNA–RNA hybrids have been shown to be a replication obstacle (Wellinger et al , ; Gan et al , ; Madireddy et al , ). In the last years, it has been reported that DNA replication‐associated activities, such as those of topoisomerases, MCM2‐7 replicative helicase, and Fanconi anemia proteins, are necessary to prevent transcription–replication conflicts and R‐loop accumulation (Tuduri et al , ; Garcia‐Rubio et al , ; Schwab et al , ; Madireddy et al , ; Vijayraghavan et al , ). Since R‐loops may constitute a major source of replication stress and genome instability (Garcia‐Muse & Aguilera, ), a hallmark of tumor cells (Halazonetis et al , ), deciphering how cells prevent R‐loop accumulation is crucial to understand the origin of genome instability.…”