R-loops: targets for nuclease cleavage and repeat instability

Abstract

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

Initiation of homologous recombination at DNA nicks

“…The quadruplex may promote persistence of a stable RNA/DNA hybrid (R-loop) between the sRNA and the transcribed C-rich strand. R-loops are resolved by two pathways in which nicks are intermediates, BER and MMR (21,22), and it is possible that HDR may leverage the free 3′ ends borne by these intermediates to promote sequence variation at pilE .…”

The role of fork stalling and DNA structures in causing chromosome fragility

“…There is evidence for the formation of stable R‐loops at many repetitive secondary structure‐forming sequences, such as CAG, CGG, GAA, and G 4 C 2 repeats (reviewed in Refs. ). The stabilization of one strand in an RNA:DNA hybrid can promote secondary structure formation on the exposed non‐template DNA strand .…”

“…In addition, treatment with RNaseH1 reduced the number of fork stalls in the FRA16D and FRA6E regions in FANCD2 −/− cells, implicating R‐loops in causing fork stalling in this region . R‐loops form stably at several structure‐forming repetitive DNA sequences and there is evidence of an interplay between R‐loops, DNA structure formation, and fragility . R‐loops are associated with both replication fork stalling and DNA breaks.…”

“…For example, at CAG repeats, R‐loop‐dependent cytosine deamination occurs, leading to BER processing and APE1/Apn1‐dependent breaks . Nucleotide excision repair (NER) nucleases XPF and XPG can also process R‐loop DNA structures, as well as cruciform and triplex DNA structures, resulting in DSBs and genomic instability . Given the interplay between DNA structure and R‐loop formation, it is possible that these two mechanisms are working together to cause fragility, or they could be independent events.…”

“…Certain DNA sequences, especially sequences that are Grich on the non-template strand, form stable R-loops, which can stall both replication and transcription and lead to DNA damage and genome instability (reviewed in Refs. [25][26][27]. There is evidence for the formation of stable R-loops at many repetitive secondary structure-forming sequences, such as CAG, CGG, GAA, and G 4 C 2 repeats (reviewed in Refs.…”