TY - JOUR

T1 - Biochemical Characterization of R-Loop Degradation by Chloroplast-Localized RNase H1 from Arabidopsis thaliana

AU - Gavrilova, Anastasia A

AU - Kuznetsova, Aleksandra A

AU - Novopashina, Darya S

AU - Zheng, Chengxia

AU - Sun, Qianwen

AU - Kuznetsov, Nikita A

N1 - This work was supported by the Russian Science Foundation, Grant No. 23-44-00064 to N.A.K., and the National Natural Science Foundation of China, Grant No. 32261133529 to Q.S. Partial support by the Russian state-funded Project No. 121031300041-4 to N.A.K. for the routine maintenance of the equipment is also acknowledged.

PY - 2025/11/17

Y1 - 2025/11/17

N2 - 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.

AB - 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.

KW - Arabidopsis/enzymology

KW - Ribonuclease H/metabolism

KW - Chloroplasts/enzymology

KW - R-Loop Structures

KW - Substrate Specificity

KW - Arabidopsis Proteins/metabolism

KW - Kinetics

U2 - 10.3390/ijms262211125

DO - 10.3390/ijms262211125

M3 - Article

C2 - 41303608

VL - 26

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 22

M1 - 11125

ER -