Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

Abstract

The Sae2/CtIP protein is required for efficient processing of DNA double-strand breaks that initiate homologous recombination in eukaryotic cells. Sae2/CtIP is also important for survival of single-stranded Top1-induced lesions and CtIP is known to associate directly with transcription-associated complexes in mammalian cells. Here we investigate the role of Sae2/CtIP at single-strand lesions in budding yeast and in human cells and find that depletion of Sae2/CtIP promotes the accumulation of stalled RNA polymerase and RNA-DNA hybrids at sites of highly expressed genes. Overexpression of the RNA-DNA helicase Senataxin suppresses DNA damage sensitivity and R-loop accumulation in Sae2/CtIP-deficient cells, and a catalytic mutant of CtIP fails to complement this sensitivity, indicating a role for CtIP nuclease activity in the repair process. Based on this evidence, we propose that R-loop processing by 5’ flap endonucleases is a necessary step in the stabilization and removal of nascent R-loop initiating structures in eukaryotic cells.

Human XPG nuclease structure, assembly, and activities with insights for neurodegeneration and cancer from pathogenic mutations

“…Multiple specialized structure-specific nucleases act in DDRs and maintenance of genome integrity, and the key to their different activities and functions is in their DNA and protein binding, catalytic specificities, and regulation. Besides its important roles in NER, XPG has key functions in BER (11)(12)(13) and HRR (10), and its loss is implicated in accumulation of R-loops (15)(16)(17)62). A molecular understanding of the diverse XPG functions will be greatly facilitated by the combined XPGcat crystal structure and solution measurements (SAXS, EM, and biochemical, mutational, and cell biology analyses) presented here that reveal DNA-specificity elements, dimer assembly, bubble DNA bending, and the location of pathogenic mutations.…”

Transcription-induced formation of extrachromosomal DNA during yeast ageing

“…Topoisomerase II activity at highly transcribed sites can cause DSBs [72], or replication forks colliding with R-loops can be processed to DSBs, though it is not clear that Sae2 would be required for processing the latter (reviewed in [73]). R-loops may also be directly involved as Sae2 and Mre11 can remove R-loops via a DSB intermediate that could itself undergo intrachromosomal recombination [74]. Whichever the mechanism, transcriptional stimulation of any recombination process has the potential to bias recombination patterns depending on the gene expression pattern of the cell.…”

Poly-ADP-ribosylation drives loss of protein homeostasis in ATM and Mre11 deficiency

“…To test for effects of R-loop removal in cells, we used inducible expression of the C-terminal half of the RNA-DNA helicase Senataxin (SETX), an enzyme that removes RNA species from genomic DNA and also promotes transcription termination (Lavin et al, 2013). We have previously found that recombinant SETX expression reduces high levels of R-loops in CtIP-deficient human cells and in Δsae2 budding yeast (Makharashvili et al, 2018). Here we found that doxycyclineinduced SETX expression drastically reduced the levels of single-strand breaks in comet assays with ATM depletion (Fig.…”

“…To test this hypothesis further, we measured RNA-DNA hybrids at several genomic loci in human U2OS cells using DNA-RNA hybrid immunoprecipitation (DRIP) followed by quantitative PCR. A representative set of loci previously shown to be prone to R-loop formation (Bhatia et al, 2014;Hatchi et al, 2015;Makharashvili et al, 2018) was used for this analysis.…”