Telomeric protein-DNA point contacts identified by photo-cross-linking using 5-bromodeoxyuridine.
The Oxytricha telomere protein specifically recognizes single-stranded telomeric DNA, forming an extremely salt resistant and kinetically stable nucleoprotein complex. The absence of information on how this heterodimeric protein binds to DNA prompted this photo-cross-linking study. Multiple protein-DNA photo-cross-links are formed upon UV irradiation of Oxytricha telomeres reconstituted with a synthetic oligonucleotide terminating in 5'-T16T15T14T13G12G11G10G9T8T7T6T5G4G3G2G1-3'. Site-specific substitution of certain nucleotides with 5-bromodeoxyuridine (BrdU) greatly increased the photo-cross-linking yield, each substitution favoring a specific protein-DNA cross-link. For example, substitution of BrdU for T7 resulted in 25% cross-linking of the bound DNA, a 10-fold increase over the unsubstituted DNA. Both subunits of the telomere protein cross-link to, and are therefore near, the DNA. Three point contacts within this nucleoprotein complex, involving the alpha subunit, were established using BrdU substitution: Tyr239, Tyr142, and His292 cross-link to G3, T15, and T7, respectively. One photo-cross-link, Tyr239-G3, occurs amid a short acidic stretch of the alpha subunit, counter to expectations for amino acids that approach the polyanionic DNA. The two remaining cross-links are to amino acids in hydrophobic regions of the primary polypeptide sequence, consistent with the hypothesis that hydrophobic interactions account for the salt resistance (> 2 M NaCl) of this protein-DNA complex. These two photo-cross-links suggest that the telomere protein may bind telomeric single-stranded DNA by intercalation of aromatic residues into a nucleotide lattice.
Hicke
B J
BJ
Department of Molecular, Cellular, and Developmental Biology, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215.
Willis
M C
MC
Koch
T H
TH
Cech
T R
TR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001973:Bromodeoxyuridine; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007834:Lasers; D008027:Light; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004247:DNA; D001973:Bromodeoxyuridine