Biochemical Characterization of R-Loop Degradation by Chloroplast-Localized RNase H1 from Arabidopsis thaliana

R-loops are three-stranded nucleic acid structures implicated in genome regulation and stability. In Arabidopsis thaliana, the chloroplast-localized RNase H1 enzyme (AtRNH1C) is important for chloroplast development and genome integrity; however, its molecular activity has not been experimentally verified. In the present study, we characterized the enzymatic activity of recombinant AtRNH1C toward model R-loops of various structures. Using a set of synthetic R-loop substrates, we demonstrate that AtRNH1C cleaves the RNA within DNA/RNA hybrids with a strong preference for purine-rich sequences, most notably at G↓X dinucleotides. Kinetic assays showed that the enzyme's efficiency is highly dependent on the length of the hybrid duplex but is not affected by a G-quadruplex structure in the single-stranded DNA flap of the R-loop. The most rapid degradation was observed for an R-loop with an 11 nt DNA/RNA hybrid region. This study provides a comparative analysis of chloroplast-localized RNase H1 activity and elucidates its substrate preferences, suggesting that an R-loop with a heteroduplex length closest to the native size found in transcription elongation complexes is the most efficient substrate. These findings suggest that the enzymatic activity of AtRNH1C is sufficient to perform its function in maintaining chloroplast genome stability by the degradation of R-loops in DNA.