A zinc-binding motif located between amino acids 273 and 286 in the adenovirus DNA-binding protein is necessary for ssDNA binding.
The human adenovirus single-stranded (ss) DNA-binding protein (DBP) possesses a highly conserved carboxyl domain which contains a putative zinc-binding motif between amino acids (aa) 273 and 286. Using a zinc blotting technique DBP was shown to bind 65Zn at levels similar to other documented zinc metalloproteins. In competition experiments, DBP bound specifically to the zinc ion even in the presence of other divalent ions such as Ca+2, Mg+2, Cd+2, Co+2, and Mn+2. The zinc-binding ability of DBP was also confirmed by zinc affinity chromatography. Site-directed mutagenesis was utilized to construct a mutant which deleted the entire zinc region (pKZNdl 273-286) and a mutant which contained a Cys to Ser substitution at aa residue 284 (pKZNpt 284). The deletion mutant was unable to bind zinc, and the point mutant showed limited binding suggesting that aa 273-286 are responsible for the interaction of DBP with zinc. The DBP zinc mutants were also examined for their ability to bind to ssDNA. The deletion mutant was unable to bind ssDNA cellulose while the point mutant exhibited decreased affinity. Thus, the region between aa 273 and 286 which mediates zinc binding also appears fundamental for the ssDNA-binding function of DBP.
Eagle
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Waksman Institute, Rutgers, State University New Jersey, Piscataway 08855.
Klessig
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D000256:Adenoviridae / Q000737:chemistry*; D017456:Adenovirus Early Proteins; D000595:Amino Acid Sequence; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers
D017456:Adenovirus Early Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D009856:Oncogene Proteins, Viral; D015032:Zinc