Dataset Information

Crystal structure of the novel lesion-specific endonuclease PfuEndoQ from Pyrococcus furiosus.

ABSTRACT: Because base deaminations, which are promoted by high temperature, ionizing radiation, aerobic respiration and nitrosative stress, produce mutations during replication, deaminated bases must be repaired quickly to maintain genome integrity. Recently, we identified a novel lesion-specific endonuclease, PfuEndoQ, from Pyrococcus furiosus, and PfuEndoQ may be involved in the DNA repair pathway in Thermococcales of Archaea. PfuEndoQ recognizes a deaminated base and cleaves the phosphodiester bond 5' of the lesion site. To elucidate the structural basis of the substrate recognition and DNA cleavage mechanisms of PfuEndoQ, we determined the structure of PfuEndoQ using X-ray crystallography. The PfuEndoQ structure and the accompanying biochemical data suggest that PfuEndoQ recognizes a deaminated base using a highly conserved pocket adjacent to a Zn2+-binding site and hydrolyses a phosphodiester bond using two Zn2+ ions. The PfuEndoQ-DNA complex is stabilized by a Zn-binding domain and a C-terminal helical domain, and the complex may recruit downstream proteins in the DNA repair pathway.

SUBMITTER: Miyazono KI

PROVIDER: S-EPMC5961232 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Crystal structure of the novel lesion-specific endonuclease PfuEndoQ from Pyrococcus furiosus.

Miyazono Ken-Ichi KI Ishino Sonoko S Makita Naruto N Ito Tomoko T Ishino Yoshizumi Y Tanokura Masaru M

Nucleic acids research 20180501 9

Because base deaminations, which are promoted by high temperature, ionizing radiation, aerobic respiration and nitrosative stress, produce mutations during replication, deaminated bases must be repaired quickly to maintain genome integrity. Recently, we identified a novel lesion-specific endonuclease, PfuEndoQ, from Pyrococcus furiosus, and PfuEndoQ may be involved in the DNA repair pathway in Thermococcales of Archaea. PfuEndoQ recognizes a deaminated base and cleaves the phosphodiester bond 5' ...[more]

PMID: 29660024

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Project description:Endonuclease Q (EndoQ), a DNA repair endonuclease, was originally identified in the hyperthermophilic euryarchaeon Pyrococcus furiosus in 2015. EndoQ initiates DNA repair by generating a nick on DNA strands containing deaminated bases and an abasic site. Although EndoQ is thought to be important for maintaining genome integrity in certain bacteria and archaea, the underlying mechanism catalyzed by EndoQ remains unclear. Here, we provide insights into the molecular basis of substrate recognition by EndoQ from P. furiosus (PfuEndoQ) using biochemical approaches. Our results of the substrate specificity range and the kinetic properties of PfuEndoQ demonstrate that PfuEndoQ prefers the imide structure in nucleobases along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. The combined results for EndoQ substrate binding and cleavage activity analyses indicated that PfuEndoQ flips the target base from the DNA duplex, and the cleavage activity is highly dependent on spontaneous base flipping of the target base. Furthermore, we find that PfuEndoQ has a relatively relaxed substrate specificity; therefore, the role of EndoQ in restriction modification systems was explored. The activity of the EndoQ homolog from Bacillus subtilis was found not to be inhibited by the uracil glycosylase inhibitor from B. subtilis bacteriophage PBS1, whose genome is completely replaced by uracil instead of thymine. Our findings suggest that EndoQ not only has additional functions in DNA repair but also could act as an antiviral enzyme in organisms with EndoQ.IMPORTANCE Endonuclease Q (EndoQ) is a lesion-specific DNA repair enzyme present in certain bacteria and archaea. To date, it remains unclear how EndoQ recognizes damaged bases. Understanding the mechanism of substrate recognition by EndoQ is important to grasp genome maintenance systems in organisms with EndoQ. Here, we find that EndoQ from the euryarchaeon Pyrococcus furiosus recognizes the imide structure in nucleobases by base flipping, and the cleavage activity is enhanced by the base pair instability of the target base, along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. Furthermore, a potential role of EndoQ in Bacillus subtilis as an antiviral enzyme by digesting viral genome is demonstrated.

Dataset Information

Crystal structure of the novel lesion-specific endonuclease PfuEndoQ from Pyrococcus furiosus.

Publications

Crystal structure of the novel lesion-specific endonuclease PfuEndoQ from Pyrococcus furiosus.

Similar Datasets

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