A sequence in the N-terminal region of human uracil-DNA glycosylase with homology to XPA interacts with the C-terminal part of the 34-kDa subunit of replication protein A.
Uracil-DNA glycosylase releases free uracil from DNA and initiates base excision repair for removal of this potentially mutagenic DNA lesion. Using the yeast two-hybrid system, human uracil-DNA glycosylase encoded by the UNG gene (UNG) was found to interact with the C-terminal part of the 34-kDa subunit of replication protein A (RPA2). No interaction with RPA4 (a homolog of RPA2), RPA1, or RPA3 was observed. A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame. In this part of UNG a 23-amino acid sequence has a significant homology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair. Trimeric RPA did not enhance the activity of UNG in vitro on single- or double-stranded DNA. A part of the N-terminal region of UNG corresponding in size to the complete presequence was efficiently removed by proteinase K, leaving the proteinase K-resistant compact catalytic domain intact and fully active. These results indicate that the N-terminal part constitutes a separate structural domain required for RPA binding and suggest a possible function for RPA in base excision repair.
Nagelhus
T A
TA
UNIGEN Center for Molecular Biology, The Medical Faculty, Norwegian University of Science and Technology, N-7005 Trondheim, Norway.
Haug
T
T
Singh
K K
KK
Keshav
K F
KF
Skorpen
F
F
Otterlei
M
M
Bharati
S
S
Lindmo
T
T
Benichou
S
S
Benarous
R
R
Krokan
H E
HE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000201:enzymology; D045647:DNA Glycosylases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051381:Rats; D051716:Replication Protein A; D051981:Uracil-DNA Glycosidase; D014983:Xeroderma Pigmentosum / Q000235:genetics; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; C497168:Rpa1 protein, mouse; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C497315:Xpa protein, mouse; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase