The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage

Abstract

RNA-DNA hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1’s helicase activity plays a crucial role in responding to persistent RNA-DNA hybrids.

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

“…These results suggest not only that fork protection and/or replication-associated repair could have a role in resolving T-R conflicts (Bhatia et al 2017) but also that an important cause of replication fork blockage are R loops, whose dissolution by specialized pathways such as FA is critical. Indeed, the FA helicase FANCM and its yeast counterpart, Mph1, play a role in R-loop removal in vitro and in vivo (Schwab et al 2015;Lafuente-Barquero et al 2017). The differential role of FANCM, senataxin, and other helicases recently described to remove DNA-RNA hybrids, such as DHX9 A B Figure 4.…”

The many types of heterogeneity in replicative senescence

“…Because replication through the repetitive G‐rich telomeric sequences is difficult (Ivessa, Zhou, Schulz, Monson, & Zakian, ; Makovets, Herskowitz, & Blackburn, ; Miller, Rog, & Cooper, ; Sfeir et al, ), occasional replication errors at telomeres would fit the apparent stochastic nature of nonterminal arrests. This is supported by data that implicate multiple DNA and RNA processing enzymes and replication checkpoints in the onset of senescence (Azam et al, ; Balk et al, ; Fallet et al, ; Gao, Moss, Parke, Tatum, & Lustig, ; Grandin & Charbonneau, ; Lafuente‐Barquero et al, ), as discussed in Teixeira ().…”

RNA at DNA Double‐Strand Breaks: The Challenge of Dealing with DNA:RNA Hybrids

“…Depletion of either RNase H, SETX or EXOSC10 results in increased levels of DNA:RNA hybrids and genomic instability, as described above, [ 29,49,54,63,64 ] which suggests that both RNase H and the SETX‐exosome system are required to prevent the accumulation of DNA:RNA hybrids in the DNA. However, the reason why both nucleolytic systems are needed is not understood yet.…”

“…[ 52,57 ] Second, depletion of RNase H2 results in increased DNA damage and increased persistence of DNA:RNA hybrids in the DNA. [ 63,64 ] Third, the DNA repair factor BRCA2 recruits RNase H2 to DSBs, and the impaired localization of RNase H2 to DSBs upon BRCA2 inactivation results in increased DNA:RNA levels at DSBs. [ 57 ] In summary, these observations show that RNase H2 plays an active role in HR repair by resolving DNA:RNA hybrids at DSBs.…”