R-loop-induced irreparable DNA damage evades checkpoint detection in the C. elegans germline

Abstract

Accumulation of DNA–RNA hybrids in the form of R-loops can result in replication–transcription conflict that leads to the formation of DNA double strand breaks (DSBs). Using null mutants for the two Caenorhabditis elegans genes encoding for RNaseH1 and RNaseH2, we identify novel effects of R-loop accumulation in the germline. R-loop accumulation leads, as expected, to replication stress, followed by the formation of DSBs. A subset of these DSBs are irreparable. However, unlike irreparable DSBs generated in other systems, which trigger permanent cell cycle arrest, germline irreparable DSBs are propagated to oocytes. Despite DNA damage checkpoint activation in the stem cell niche, the signaling cannot be sustained and nuclei with irreparable DNA damage progress into meiosis. Moreover, unlike other forms of DNA damage that increase germline apoptosis, R-loop-generated DSBs remain undetected by the apoptotic checkpoint. This coincides with attenuation of ATM/ATR signaling in mid-to-late meiotic prophase I. These data altogether indicate that in the germline, DSBs that are generated by R-loops can lead to irreparable DSBs that evade cellular machineries designed for damage recognition. These studies implicate germline R-loops as an especially dangerous driver of germline mutagenesis.

Unscheduled epigenetic modifications cause genome instability and sterility through aberrant R-loops following starvation

“…This indicates that DNA damage is induced at these R-loops, potentially due to transcription–replication conflicts that arise in the mitotic zone and persist, or alternatively, it may represent collateral sites of damage that arise during the process of R-loop resolution or interference with normal meiotic progression ( 54 ). Moreover, we noticed that nuclei with highly abundant RAD-51 foci often appeared in the pachytene region of the germ line of AMPK mutants (Figure 4B and C ), suggesting that these nuclei may have irreparable damage and could be destined for elimination ( 55 ). The membrane bound phagocytic receptor CED-1 surrounds apoptotic cells undergoing corpse engulfment and therefore is an excellent proxy to quantify cells undergoing the final steps of apoptosis ( 36 ).…”

“…In a recent study where R-loop formation was enhanced by the genetic removal of RNH activity, R-loops result in irreparable DNA damage and activation of a damage-sensitive checkpoint. This too seems to be very context dependent where the excess presence of R-loops only triggers apoptosis when they occur in specific genes/loci ( 55 ).…”

“…Beyond the apparent reproductive phenotypes that arise in the P 0 and the F 2 generations, the possibility that these aberrant R-loops could result in long-term genetic change cannot be excluded ( 55 ). Although the increased frequency of R-loops in starved AMPK mutants is resolved in five generations, the consequences of these mutagenic structures may persist for much longer.…”

Neuronal IL-17 controlsC. elegansdevelopmental diapause through CEP-1/p53

“…3b-d). CEP-1 overexpression was achieved by crossing out the cep-1 lg12501 deletion mutation from a widely used transgenic strain expressing functional, fluorescently tagged, CEP-1, CEP-1::GFP, under its own promoter (61, 65, 66). In the cep-1 ( lg12501 ) strain, CEP-1::GFP is the only source of CEP-1/p53, rescues cep-1 ( lg12501 ) apoptosis defects and is expressed at half the levels of wild type (Supplementary Fig.…”

“…S6d for mRNA levels upon cep-1 overexpression). CEP-1 overexpression was achieved by expressing a functional, fluorescently tagged CEP-1, CEP-1::GFP, that was able to complement a cep-1 deletion, cep-1(lg12501), [66][67][68] as a genomically integrated multicopy array in a wildtype background. CEP-1/p53 overexpression not only caused dauer arrest at 25°C, but also prompted larvae to transiently enter dauer at 20°C, as seen by their SDS-resistance, phenocopying the ilc-17.1 deletion (Supplementary Fig.…”