TY - JOUR

T1 - Kinetic Features of Degradation of R-Loops by RNase H1 from Escherichia coli

AU - Kuznetsova, Aleksandra A.

AU - Kosarev, Iurii A.

AU - Timofeyeva, Nadezhda A.

AU - Novopashina, Darya S.

AU - Kuznetsov, Nikita A.

N1 - This work was supported by the Russian Science Foundation, grant No. 23-44-00064. Partial support by Russian state-funded project No. 121031300041-4 for the routine maintenance of the equipment is also acknowledged.

PY - 2024/11

Y1 - 2024/11

N2 - R-loops can act as replication fork barriers, creating transcription–replication collisions and inducing replication stress by arresting DNA synthesis, thereby possibly causing aberrant processing and the formation of DNA strand breaks. RNase H1 (RH1) is one of the enzymes that participates in R-loop degradation by cleaving the RNA strand within a hybrid RNA–DNA duplex. In this study, the kinetic features of the interaction of RH1 from Escherichia coli with R-loops of various structures were investigated. It was found that the values of the dissociation constants Kd were minimal for complexes of RH1 with model R-loops containing a 10–11-nt RNA–DNA hybrid part, indicating effective binding. Analysis of the kinetics of RNA degradation in the R-loops by RH1 revealed that the rate-limiting step of the process was catalytic-complex formation. In the presence of RNA polymerase, the R-loops containing a ≤16-nt RNA–DNA hybrid part were efficiently protected from cleavage by RH1. In contrast, R-loops containing longer RNA–DNA hybrid parts, as a model of an abnormal transcription process, were not protected by RNA polymerase and were effectively digested by RH1.

AB - R-loops can act as replication fork barriers, creating transcription–replication collisions and inducing replication stress by arresting DNA synthesis, thereby possibly causing aberrant processing and the formation of DNA strand breaks. RNase H1 (RH1) is one of the enzymes that participates in R-loop degradation by cleaving the RNA strand within a hybrid RNA–DNA duplex. In this study, the kinetic features of the interaction of RH1 from Escherichia coli with R-loops of various structures were investigated. It was found that the values of the dissociation constants Kd were minimal for complexes of RH1 with model R-loops containing a 10–11-nt RNA–DNA hybrid part, indicating effective binding. Analysis of the kinetics of RNA degradation in the R-loops by RH1 revealed that the rate-limiting step of the process was catalytic-complex formation. In the presence of RNA polymerase, the R-loops containing a ≤16-nt RNA–DNA hybrid part were efficiently protected from cleavage by RH1. In contrast, R-loops containing longer RNA–DNA hybrid parts, as a model of an abnormal transcription process, were not protected by RNA polymerase and were effectively digested by RH1.

KW - R-loop

KW - RNA polymerase

KW - RNase H1

KW - enzyme activity

KW - enzyme kinetics

KW - transcription

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85210442672&origin=inward&txGid=e33557775aab0eacbd25078eeca05427

UR - https://www.mendeley.com/catalogue/558bd29b-1a89-338b-b575-0a4fe57b0119/

U2 - 10.3390/ijms252212263

DO - 10.3390/ijms252212263

M3 - Article

C2 - 39596330

VL - 25

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 22

M1 - 12263

ER -