R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here we show that replication protein A (RPA), an ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability.
Bibliographical noteFunding Information:
We thank Drs. R. Bradley, R. Crouch, M.A. Lee-Kirsch, and S. Leppla for reagents, Dr.?A. Mar?chal for characterizations of reagents, and members of the Zou and Dyson labs for helpful discussions. The molecular graphic image in Figure?4C was produced using the University of California at San Francisco (UCSF) Chimera package from the Computer Graphics Laboratory, University of California, San Francisco (supported by NIH P41 RR-01081). H.D.N. is partly supported by a postdoctoral fellowship from the Medical Discovery Fund. L.Z. is the James and Patricia Poitras Endowed Chair in Cancer Research and was supported by a Jim and Ann Orr Massachusetts General Hospital Research Scholar Award. This work is supported by grants from the NIH (GM076388 and CA197779 to L.Z.) and the Evans Foundation (201407 to T.G.).
© 2017 Elsevier Inc.
- R loop
- RNase H1
- genome instability
- splicing inhibitor
- splicing mutation