RNase H activities counteract a toxic effect of Polymerase η in cells replicating with depleted dNTP pools

Abstract

RNA:DNA hybrids are transient physiological intermediates that arise during several cellular processes such as DNA replication. In pathological situations, they may stably accumulate and pose a threat to genome integrity. Cellular RNase H activities process these structures to restore the correct DNA:DNA sequence. Yeast cells lacking RNase H are negatively affected by depletion of deoxyribonucleotide pools necessary for DNA replication. Here we show that the translesion synthesis DNA polymerase η (Pol η) plays a role in DNA replication under low deoxyribonucleotides condition triggered by hydroxyurea. In particular, the catalytic reaction performed by Pol η is detrimental for RNase H deficient cells, causing DNA damage checkpoint activation and G2/M arrest. Moreover, a Pol η mutant allele with enhanced ribonucleotide incorporation further exacerbates the sensitivity to hydroxyurea of cells lacking RNase H activities. Our data are compatible with a model in which Pol η activity facilitates the formation or stabilization of RNA:DNA hybrids at stalled replication forks. However, in a scenario where RNase H activity fails to restore DNA, these hybrids become highly toxic for cells.

Molecular and physiological consequences of faulty eukaryotic ribonucleotide excision repair

“…This would also be consistent with a genetic rescue by the RNH201-RED allele. Recent work has demonstrated that translesion polymerase eta (Pol g) can incorporate consecutive rNMP stretches at stalled replication forks in the presence of HU (Meroni et al, 2019). Consistently, the deletion of Pol g rescues the HU sensitivity of rnh1D rnh201D cells.…”

“…This indicates that lethality in this strain is likely mediated by an accumulation of toxic R-loops and implies that yeast cells can in principle tolerate the rNMPs that accumulate in the genome when both RER and Top1-dependent repair are compromised. Recent work has demonstrated that translesion polymerase eta (Pol g) can incorporate consecutive rNMP stretches at stalled replication forks in the presence of HU (Meroni et al, 2019). In cells that lack both RNase H1 and RNase H2, the postreplicative repair pathway has been found to be crucial (Lazzaro et al, 2012), but it has not been addressed to which extent this is due to either R-loops or rNMPs, leaving the involvement of this pathway in the bypass of lethality allowed by the RNH201-RED allele an interesting hypothesis to test.…”

One, No One, and One Hundred Thousand: The Many Forms of Ribonucleotides in DNA

“…Family Role In rNMPs Insertion pol ε B replication/repair undamaged leading strand [16] pol δ B replication/repair undamaged lagging strand [16] pol α B replication/repair undamaged lagging strand [16] pol ζ B translesion synthesis (TLS); mitochondrial replication rare [25] pol β X repair/TLS undamaged template, CPDs [26] 8-oxo-Gs [27] pol λ X repair/TLS 8-oxo-Gs [27] pol µ X repair NHEJ ends [28][29][30] Tdt X repair N-regions of V(D)J ends [31] pol η Y TLS; lesion-independent replication stress undamaged template [32][33][34]; 8-oxo-Gs, CPDs, cis-PtGG, 8-methyl-2 -deoxyGs [32,33]…”

“…The wild type S. cerevisiae pol η just shows a minimal rate of rNMP insertion on undamaged and damaged DNA; by contrast, the steric gate mutant pol η-F35A readily incorporates the correct rNMP opposite both templates, and in vivo experiments suggest that it may catalyze the incorporation of stretches of ribonucleotides in DNA [48,49]. Moreover, genetic evidence points towards the idea that under low dNTP conditions, either the wild type pol η or, even more, pol η-F35A inserts consecutive ribonucleotides, which become toxic in the absence of RNase H activity [34]. Differently from its yeast counterpart, the wild type human pol η inserts rNMPs opposite both undamaged and damaged DNA templates, even if maintaining base selectivity [32,33].…”

“…Moreover, under particular stress conditions, also wild type replicative and/or reparative DNA polymerases may incorporate consecutive rNMPs. This is what has been suggested for the S. cerevisiae pol η. Meroni et al found that, upon replication stress induced by hydroxyurea, pol η was recruited at stalled replication forks, where it facilitated the formation of stretches of rNMPs that became highly toxic for cells, when not properly replaced with DNA [34].…”