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 st able nucleoprotein complex. The absence of information on how this het erodimeric protein binds to DNA prompted this photo-cross-linking stud y. Multiple protein-DNA photo-cross-links are formed upon UV irradiati on of Oxytricha telomeres reconstituted with a synthetic oligonucleoti de terminating in G(11)G(10)G(9)T(8)T(7)T(6)T(5)G(4)G(3)G(2)G(1)-3'. S ite-specific substitution of certain nucleotides with 5-bromodeoxyurid ine (BrdU) greatly increased the photo-cross-linking yield, each subst itution favoring a specific protein-DNA cross-link. For example, subst itution of BrdU for T-7 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, an d His292 cross-link to G(3), T-15, and T-7, respectively. One photo-cr oss-link, Tyr239-G(3), occurs amid a short acidic stretch of the alpha subunit, counter to expectations for amino acids that approach the po lyanionic DNA. The two remaining cross-links are to amino acids in hyd rophobic regions of the primary polypeptide sequence, consistent with the hypothesis that hydrophobic interactions account for the salt resi stance (> 2 M NaCl) of this protein-DNA complex. These two photo-cross -links suggest that the telomere protein may bind telomeric single-str anded DNA by intercalation of aromatic residues into a nucleotide latt ice.