20th-banner-rev.png

Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

Mechanisms of R-loop-induced chromosome fragility at structure-forming repeats

CAG repeats are structure-forming repetitive DNA sequences, and expansion beyond a threshold of ~35 CAG repeats is the cause of many human diseases including Huntington’s disease and myotonic dystrophy. Expanded CAG repeats are prone to breakage, and repair of the breaks can cause repeat contractions and expansions. We utilized an expanded CAG-70 repeat inserted into a yeast artificial chromosome (YAC) system to evaluate mechanisms of repeat fragility and instability (contractions and expansions). Upon deletion of yeast RNase H genes, RNH1 and RNH201, we found an elevated level of RNA:DNA hybrids at CAG-70 repeats in vivo by using chromatin immunoprecipitation (ChIP). Through CAG repeat fragility analysis, we discovered that CAG-70 repeats show a dramatic and significant increase in fragility in the absence of RNase H genes compared to wild-type, indicating higher levels of breakage events at the expanded repeats during R-loop formation1. In contrast, fragility of another structure-forming repeat composed of an AT dinucleotide was not affected by RNase deletion or overexpression. Significant increases in CAG-70 repeat instability were also observed in the RNase H deletion background that were dependent on the base excision repair pathway1. Since disease-causing CAG repeat expansions occur in transcribed regions shown to harbor R-loops, our findings indicate that R-loop-mediated fragility is a mechanism that could cause DNA damage and repeat-length changes in human cells.

In a search for the cause of R-loop-mediated fragility, we found that a yeast cytosine deaminase, Fcy1, was enriched at the expanded CAG repeats in rnh1Δrnh201Δ cells. Deletion of Fcy1 significantly decreases CAG-70 repeat fragility in the rnh1Δrnh201Δ background, and reduces the elevated CAG repeat contractions back to the wild-type level(1). These findings provide evidence for a novel mechanism for CAG repeat fragility mediated by cytosine deamination of DNA engaged in R-loops. In addition, a second mechanism of R-loop-mediated fragility at CAG repeats was identified to be dependent on the Mlh1-Mlh3 nuclease. This result could explain the previously demonstrated role of Mlh1-Mlh3 in causing CAG repeat expansions in a mouse model(2). Thus, R-loop-mediated cleavage could be an important determinant of instability at unstable and expandable repeats in the human genome.

References

    1. Cytosine deamination and base excision repair cause R-loop-induced CAG repeat fragility and instability in Saccharomyces cerevisiae. Su XA, Freudenreich CH. Proc Natl Acad Sci U S A. (2017) Oct 3;114(40):E8392-E8401.

    2. Pinto RM, et al. (2013) Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches. PLoS Genet 9(10):e1003930.

Xiaofeng Allen Su
Simran Kaushal
Ruby Ye
Catherine H. Freudenreich

Department of Biology
Tufts University
Medford, MA 02155

Email: catherine.freudenreich@tufts.edu