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Journal of Virology, January 2003, p. 915-922, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.915-922.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Adenovirus Type 5 DNA Binding Protein Stimulates Binding of DNA Polymerase to the Replication Origin

Bas van Breukelen, Arjan B. Brenkman, P. Elly Holthuizen, and Peter C. van der Vliet*

Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands

Received 20 August 2002/ Accepted 29 September 2002

The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the Km for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.

* Corresponding author. Mailing address: UMCU, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Phone: (31) 302538989. Fax: (31) 302539035. E-mail: P.C.vanderVliet{at}med.uu.nl.

Journal of Virology, January 2003, p. 915-922, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.915-922.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Mysiak, M. E., Wyman, C., Holthuizen, P. E., van der Vliet, P. C. (2004). NFI and Oct-1 bend the Ad5 origin in the same direction leading to optimal DNA replication. Nucleic Acids Res 32: 6218-6225 [Abstract] [Full Text]  


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