TY - JOUR

T1 - Correlated Target Search by Vaccinia Virus Uracil-DNA Glycosylase, a DNA Repair Enzyme and a Processivity Factor of Viral Replication Machinery

AU - Diatlova, Evgeniia A

AU - Mechetin, Grigory V

AU - Yudkina, Anna V

AU - Zharkov, Vasily D

AU - Torgasheva, Natalia A

AU - Endutkin, Anton V

AU - Shulenina, Olga V

AU - Konevega, Andrey L

AU - Gileva, Irina P

AU - Shchelkunov, Sergei N

AU - Zharkov, Dmitry O

N1 - Funding: This research was supported by the Russian Science Foundation (grant 21-64-00017, all biochemical experiments) and the Russian Ministry of Higher Education and Science (grant 121031300056-8 to D.O.Z.). Quench-flow experiments were supported by the Russian Ministry of Higher Education and Science (grant 075-15-2021-1360 to A.L.K.).

PY - 2023/5/23

Y1 - 2023/5/23

N2 - The protein encoded by the vaccinia virus D4R gene has base excision repair uracil-DNA N-glycosylase (vvUNG) activity and also acts as a processivity factor in the viral replication complex. The use of a protein unlike PolN/PCNA sliding clamps is a unique feature of orthopoxviral replication, providing an attractive target for drug design. However, the intrinsic processivity of vvUNG has never been estimated, leaving open the question whether it is sufficient to impart processivity to the viral polymerase. Here, we use the correlated cleavage assay to characterize the translocation of vvUNG along DNA between two uracil residues. The salt dependence of the correlated cleavage, together with the similar affinity of vvUNG for damaged and undamaged DNA, support the one-dimensional diffusion mechanism of lesion search. Unlike short gaps, covalent adducts partly block vvUNG translocation. Kinetic experiments show that once a lesion is found it is excised with a probability ~0.76. Varying the distance between two uracils, we use a random walk model to estimate the mean number of steps per association with DNA at ~4200, which is consistent with vvUNG playing a role as a processivity factor. Finally, we show that inhibitors carrying a tetrahydro-2,4,6-trioxopyrimidinylidene moiety can suppress the processivity of vvUNG.

AB - The protein encoded by the vaccinia virus D4R gene has base excision repair uracil-DNA N-glycosylase (vvUNG) activity and also acts as a processivity factor in the viral replication complex. The use of a protein unlike PolN/PCNA sliding clamps is a unique feature of orthopoxviral replication, providing an attractive target for drug design. However, the intrinsic processivity of vvUNG has never been estimated, leaving open the question whether it is sufficient to impart processivity to the viral polymerase. Here, we use the correlated cleavage assay to characterize the translocation of vvUNG along DNA between two uracil residues. The salt dependence of the correlated cleavage, together with the similar affinity of vvUNG for damaged and undamaged DNA, support the one-dimensional diffusion mechanism of lesion search. Unlike short gaps, covalent adducts partly block vvUNG translocation. Kinetic experiments show that once a lesion is found it is excised with a probability ~0.76. Varying the distance between two uracils, we use a random walk model to estimate the mean number of steps per association with DNA at ~4200, which is consistent with vvUNG playing a role as a processivity factor. Finally, we show that inhibitors carrying a tetrahydro-2,4,6-trioxopyrimidinylidene moiety can suppress the processivity of vvUNG.

KW - Uracil-DNA Glycosidase/genetics

KW - Vaccinia virus/genetics

KW - DNA/metabolism

KW - Virus Replication

KW - DNA Repair

KW - Uracil/metabolism

KW - DNA, Viral/genetics

KW - DNA Replication

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85161586267&origin=inward&txGid=69ecaad2d610428ea26e67f12e7fa836

U2 - 10.3390/ijms24119113

DO - 10.3390/ijms24119113

M3 - Article

C2 - 37298065

VL - 24

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 11

M1 - 9113

ER -