Cloning and characterization of uracil-DNA glycosylase and the biological consequences of the loss of its function in the nematode Caenorhabditis elegans.
Uracil arises in DNA from spontaneous deamination of cytosine and through incorporation of dUMP by DNA polymerase during DNA replication. Excision of uracil by the action of uracil-DNA glycosylase (Ung) initiates the base excision repair pathway to counter the promutagenic base modification. In this study, we cloned a cDNA-encoding Caenorhabditis elegans homologue (CeUng-1) of Escherichia coli Ung. There was 49% identity in amino acid sequence between E.coli Ung and CeUng-1. Purified CeUng-1 removed uracil from both U:G and U:A base pairs in DNA. It also removed uracil from single-stranded oligonucleotide substrate less efficiently than double-stranded oligonucleotide. The CeUng-1 activity was inhibited by Bacillus subtilis Ung inhibitor, indicating that CeUng-1 is a member of the family-1 Ung group. The mutation in the ung-1 gene did not affect development, fertility and lifespan in C.elegans, suggesting the existence of backup enzyme. However, we could not detect residual uracil excision activity in the extract derived from the ung-1 mutant. The present experiments also showed that the ung-1 mutant of C.elegans was more resistant to NaHSO(3)-inducing cytosine deamination than wild-type strain.
Nakamura
Nobuya
N
Department of Biological Sciences, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Morinaga
Hironobu
H
Kikuchi
Masahiro
M
Yonekura
Shin-Ichiro
S
Ishii
Naoaki
N
Yamamoto
Kazuo
K
Yonei
Shuji
S
Zhang
Qiu-Mei
QM
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003596:Cytosine / Q000378:metabolism; D003641:Deamination; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D013447:Sulfites / Q000633:toxicity; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000378:metabolism*
D013447:Sulfites; D003596:Cytosine; D051981:Uracil-DNA Glycosidase; C009279:sodium bisulfite