RNase H is an exo- and endoribonuclease with asymmetric directionality, depending on the binding mode to the structural variants of RNA:DNA hybrids

Abstract

RNase H is involved in fundamental cellular processes and is responsible for removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. Defects in RNase H lead to embryo lethality in mice and Aicardi-Goutieres syndrome in humans, suggesting the importance of RNase H. To date, RNase H is known to be a non-sequence-specific endonuclease, but it is not known whether it performs other functions on the structural variants of RNA:DNA hybrids. Here, we used Escherichia coli RNase H as a model, and examined its catalytic mechanism and its substrate recognition modes, using single-molecule FRET. We discovered that RNase H acts as a processive exoribonuclease on the 3′ DNA overhang side but as a distributive non-sequence-specific endonuclease on the 5′ DNA overhang side of RNA:DNA hybrids or on blunt-ended hybrids. The high affinity of previously unidentified double-stranded (ds) and single-stranded (ss) DNA junctions flanking RNA:DNA hybrids may help RNase H find the hybrid substrates in long genomic DNA. Our study provides new insights into the multifunctionality of RNase H, elucidating unprecedented roles of junctions and ssDNA overhang on RNA:DNA hybrids.

Highly Selective FRET-Aided Single-Molecule Counting of MicroRNAs Labeled by Splinted Ligation

“…Since single-stranded DNA is shorter than duplex DNA without external tension, the new peak and the intermediate broadening could be attributed to the complete and partial loss of cleaved miRNA fragments, respectively. This observation also agreed with a previous smFRET study of RNase H . Therefore, the narrow distribution at low FE indicates binding of the miRNA to the probe without degradation.…”

“…Since single-stranded DNA is shorter than duplex DNA without external tension, the new peak and the intermediate broadening could be attributed to the complete and partial loss of cleaved miRNA fragments, respectively. This observation also agreed with a previous smFRET study of RNase H. 30 Therefore, the narrow distribution at low FE indicates binding of the miRNA to the probe without degradation. While optimizing the ligation conditions, we noted that the SplintR ligase was very efficient, as reported previously; 31 although we chose 2 h of incubation for consistency, the ligation was almost complete within 1 h (Figure S4).…”

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

“…In addition, in vitro reconstitutions of mouse cell 67 and simian virus 40 68 DNA replication proposed that RNase H activity is required for efficient MOF. Remarkably, RNase H acts as a processive exoribonuclease on unmatured OF-like substrates 58 , which would predominantly degrade the initiator RNA into monoribonucleotide products, similar to NT 15 and probably to in vivo MOF products. Other pathways may involve recruiting proteins that work with Polα to pre-remove the initiator RNA immediately after primer synthesis 8 .…”

Integrating CRISPR/Cas systems with programmable DNA nanostructures for delivery and beyond

“…In a very recent breakthrough article, Lee et al. (2022) discovered the dual functionality of the E. coli RNase H, whose function crucially depends on symmetry of the DNA overhang.…”