title: T antigen origin-binding domain of simian virus 40: determinants of specific DNA binding.
abstract: To better understand origin recognition and initiation of DNA replication, we have examined by NMR complexes formed between the origin-binding domain of SV40 T antigen (T-ag-obd), the initiator protein of the SV40 virus, and cognate and noncognate DNA oligomers. The results reveal two structural effects associated with "origin-specific" binding that are absent in nonspecific DNA binding. The first is the formation of a hydrogen bond (H-bond) involving His 203, a residue that genetic studies have previously identified as crucial to both specific and nonspecific DNA binding in full-length T antigen. In free T-ag-obd, the side chain of His 203 has a pK(a) value of approximately 5, titrating to the N(epsilon)(1)H tautomer at neutral pH (Sudmeier, J. L., et al. (1996) J. Magn. Reson., Ser. B 113, 236-247). In complexes with origin DNA, His 203 N(delta)(1) becomes protonated and remains nontitrating as the imidazolium cation at all pH values from 4 to 8. The H-bonded N(delta1)H resonates at 15.9 ppm, an unusually large N-H proton chemical shift, of a magnitude previously observed only in the catalytic triad of serine proteases at low pH. The formation of this H-bond requires the middle G/C base pair of the recognition pentanucleotide, GAGGC. The second structural effect is a selective distortion of the A/T base pair characterized by a large (0.6 ppm) upfield chemical-shift change of its Watson-Crick proton, while nearby H-bonded protons remain relatively unaffected. The results indicate that T antigen, like many other DNA-binding proteins, may employ "catalytic" or "transition-state-like" interactions in binding its cognate DNA (Jen-Jacobson, L. (1997) Biopolymers 44, 153-180), which may be the solution to the well-known paradox between the relatively modest DNA-binding specificity exhibited by initiator proteins and the high specificity of initiation.
authors.affiliation: Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Bradshaw
authors.forename: David G
authors.initials: DG
authors.lastname: Sanford
authors.forename: Xuelian
authors.initials: X
authors.lastname: Luo
authors.forename: James L
authors.initials: JL
authors.lastname: Sudmeier
authors.forename: Zachary A
authors.initials: ZA
authors.lastname: Gurard-Levin
authors.forename: Peter A
authors.initials: PA
authors.lastname: Bullock
authors.forename: William W
authors.initials: WW
authors.lastname: Bachovchin
mesh_terms: D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D018741:Replication Origin / Q000502:physiology*; D013539:Simian virus 40 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
chemical_list: D000952:Antigens, Polyomavirus Transforming; D011522:Protons; D006639:Histidine; D004247:DNA