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RPA transforms RNase H1 to a bidirectional exoribonuclease for processive RNA-DNA hybrid cleavage.

Yanan LiChao LiuXinshuo JiaLulu BiZhiyun RenYilin ZhaoXia ZhangLijuan GuoYanling BaoCong LiuWei LiBo Sun
Published in: Nature communications (2024)
RNase H1 has been acknowledged as an endoribonuclease specializing in the internal degradation of the RNA moiety within RNA-DNA hybrids, and its ribonuclease activity is indispensable in multifaceted aspects of nucleic acid metabolism. However, the molecular mechanism underlying RNase H1-mediated hybrid cleavage remains inadequately elucidated. Herein, using single-molecule approaches, we probe the dynamics of the hybrid cleavage by Saccharomyces cerevisiae RNase H1. Remarkably, a single RNase H1 enzyme displays 3'-to-5' exoribonuclease activity. The directional RNA degradation proceeds processively and yet discretely, wherein unwinding approximately 6-bp hybrids as a prerequisite for two consecutive 3-nt RNA excisions limits the overall rate within each catalytic cycle. Moreover, Replication Protein A (RPA) reinforces RNase H1's 3'-to-5' nucleolytic rate and processivity and stimulates its 5'-to-3' exoribonuclease activity. This stimulation is primarily realized through the pre-separation of the hybrids and consequently transfers RNase H1 to a bidirectional exoribonuclease, further potentiating its cleavage efficiency. These findings unveil unprecedented characteristics of an RNase and provide a dynamic view of RPA-enhanced processive hybrid cleavage by RNase H1.
Keyphrases
  • nucleic acid
  • single molecule
  • dna binding
  • saccharomyces cerevisiae
  • living cells
  • cell free
  • circulating tumor
  • small molecule
  • mass spectrometry
  • atomic force microscopy
  • fluorescent probe