Abstract
Cytosine deamination induced by stresses or enzymatic catalysis converts deoxycytidine into deoxyuridine, thereby introducing a G to A mutation after DNA replication. Base-excision repair to correct uracil to cytosine is initiated by uracil-DNA glycosylase (UDG), which recognizes and eliminates uracil from DNA. Mimivirus, one of the largest known viruses, also encodes a distinctive UDG gene containing a long N-terminal domain (N-domain; residues 1–130) and a motif-I (residues 327–343), in addition to the canonical catalytic domain of family I UDGs (also called UNGs). To understand the structural and functional features of the additional segments, we have determined the crystal structure of UNG from Acanthamoeba polyphaga mimivirus (mvUNG). In the crystal structure of mvUNG, residues 95–130 in the N-domain bind to a hydrophobic groove in the catalytic domain, and motif-I forms a short β-sheet with a positively charged surface near the active site. Circular dichroism spectra showed that residues 1–94 are in a random coil conformation. Deletion of the three additional fragments reduced the activity and thermal stability, compared to full-length mvUNG. The results suggested that the mvUNG N-domain and motif-I are required for its structural and functional integrity.
Highlights
Uracil is a base component of RNA, it is occasionally introduced into DNA by hydrolytic deamination of cytosine [1]
The plasmid was transformed into Escherichia coli strain BL21-star(DE3) (Thermo Fisher Scientific, Waltham, MA, USA), and the cells were grown in Luria-Bertani medium
The recombinant protein was expressed in E. coli and purified by Ni-affinity chromatography and size exclusion chromatography (SEC)
Summary
Uracil is a base component of RNA, it is occasionally introduced into DNA by hydrolytic deamination of cytosine [1]. Uracil generated by cytosine deamination causes a ‘G to A mutation’ in the complementary strand during DNA replication, which must be corrected to maintain genomic integrity [1]. Uracil in DNA can be repaired by the base excision repair (BER) pathway [2, 3]. In the BER pathway, uracil-DNA glycosylase (UDG) recognizes uracil which exists in DNA and creates an abasic site by cleaving the N-glycosidic bond between uracil and deoxyribose. Apurinic/apyrimidinic (AP) endonuclease removes the sugar-phosphate backbone of the abasic site. DNA polymerase adds a correct nucleotide to the site [2, 3]
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