T antigen origin-binding domain of simian virus 40: Determinants of specific DNA binding

Elizabeth M. Bradshaw, David G. Sanford, Xuelian Luo, James L. Sudmeier, Zachary A. Gurard-Levin, Peter A. Bullock, William W. Bachovchin

Research output: Contribution to journalArticle

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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 pKa value of ∼5, titrating to the Nε1H 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 Nδ1 becomes protonated and remains nontitrating as the imidazolium cation at all pH values from 4 to 8. The H-bonded Nδ1H 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.

Original languageEnglish (US)
Pages (from-to)6928-6936
Number of pages9
JournalBiochemistry
Volume43
Issue number22
DOIs
StatePublished - Jun 8 2004

Fingerprint

Simian virus 40
Viral Tumor Antigens
Viruses
DNA
Protons
Base Pairing
Chemical shift
Hydrogen
Hydrogen bonds
Polyomavirus Transforming Antigens
Biopolymers
DNA-Binding Proteins
Serine Proteases
DNA Replication
Cations
Oligomers
Proteins
Nuclear magnetic resonance

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bradshaw, E. M., Sanford, D. G., Luo, X., Sudmeier, J. L., Gurard-Levin, Z. A., Bullock, P. A., & Bachovchin, W. W. (2004). T antigen origin-binding domain of simian virus 40: Determinants of specific DNA binding. Biochemistry, 43(22), 6928-6936. https://doi.org/10.1021/bi030228+

T antigen origin-binding domain of simian virus 40 : Determinants of specific DNA binding. / Bradshaw, Elizabeth M.; Sanford, David G.; Luo, Xuelian; Sudmeier, James L.; Gurard-Levin, Zachary A.; Bullock, Peter A.; Bachovchin, William W.

In: Biochemistry, Vol. 43, No. 22, 08.06.2004, p. 6928-6936.

Research output: Contribution to journalArticle

Bradshaw, EM, Sanford, DG, Luo, X, Sudmeier, JL, Gurard-Levin, ZA, Bullock, PA & Bachovchin, WW 2004, 'T antigen origin-binding domain of simian virus 40: Determinants of specific DNA binding', Biochemistry, vol. 43, no. 22, pp. 6928-6936. https://doi.org/10.1021/bi030228+
Bradshaw EM, Sanford DG, Luo X, Sudmeier JL, Gurard-Levin ZA, Bullock PA et al. T antigen origin-binding domain of simian virus 40: Determinants of specific DNA binding. Biochemistry. 2004 Jun 8;43(22):6928-6936. https://doi.org/10.1021/bi030228+
Bradshaw, Elizabeth M. ; Sanford, David G. ; Luo, Xuelian ; Sudmeier, James L. ; Gurard-Levin, Zachary A. ; Bullock, Peter A. ; Bachovchin, William W. / T antigen origin-binding domain of simian virus 40 : Determinants of specific DNA binding. In: Biochemistry. 2004 ; Vol. 43, No. 22. pp. 6928-6936.
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