Mutational analysis of the damage-recognition and catalytic mechanism of human SMUG1 DNA glycosylase.
Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), previously thought to be a backup enzyme for uracil-DNA glycosylase, has recently been shown to excise 5-hydroxyuracil (hoU), 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU) bearing an oxidized group at ring C5 as well as an uracil. In the present study, we used site-directed mutagenesis to construct a series of mutants of human SMUG1 (hSMUG1), and tested their activity for uracil, hoU, hmU, fU and other bases to elucidate the catalytic and damage-recognition mechanism of hSMUG1. The functional analysis of the mutants, together with the homology modeling of the hSMUG1 structure based on that determined recently for Xenopus laevis SMUG1, revealed the crucial residues for the rupture of the N-glycosidic bond (Asn85 and His239), discrimination of pyrimidine rings through pi-pi stacking to the base (Phe98) and specific hydrogen bonds to the Watson-Crick face of the base (Asn163) and exquisite recognition of the C5 substituent through water-bridged (uracil) or direct (hoU, hmU and fU) hydrogen bonds (Gly87-Met91). Integration of the present results and the structural data elucidates how hSMUG1 accepts uracil, hoU, hmU and fU as substrates, but not other oxidized pyrimidines such as 5-hydroxycytosine, 5-formylcytosine and thymine glycol, and intact pyrimidines such as thymine and cytosine.
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
Mayumi
M
Matsubara
Tamon
T
Tanaka
Hiroaki
H
Terato
Eiji
E
Ohmae
Shunsuke
S
Izumi
Katsuo
K
Katayanagi
Hiroshi
H
Ide
D000595:Amino Acid Sequence; D002384:Catalysis; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004260:DNA Repair; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010432:Pentoxyl / Q000031:analogs & derivatives* / Q000378:metabolism; D040681:Structural Homology, Protein; D014498:Uracil / Q000031:analogs & derivatives* / Q000378:metabolism; D051981:Uracil-DNA Glycosidase; D029867:Xenopus Proteins
D029867:Xenopus Proteins; C007777:5-formyluracil; C008055:5-hydroxymethyluracil; C045975:5-hydroxyuracil; D014498:Uracil; D010432:Pentoxyl; D045647:DNA Glycosylases; C498096:SMUG1 protein, Xenopus laevis; C498095:SMUG1 protein, human; D051981:Uracil-DNA Glycosidase