ATP-independent DNA Unwinding by the Adenovirus Single-stranded DNA Binding Protein Requires a Flexible DNA Binding Loop Job Dekker 1 , Panagiotis N. Kanellopoulos 2 Joost A. W. M. van Oosterhout 1 , Gunter Stier 3 , Paul A. Tucker 2,3 and Peter C. van der Vliet 1 * 1 Laboratory for Physiological Chemistry, Utrecht University Universiteitsweg 100, 3584 CG Utrecht, The Netherlands 2 European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY Notkestrasse 85 D22603 Hamburg, Germany 3 Structural Biology Programme, European Molecular Biology Laboratory Meyerhofstrasse 1 D69012 Heidelberg, Germany The adenovirus DNA binding protein (DBP) binds cooperatively to single-stranded (ss) DNA and stimulates both initiation and elongation of DNA replication. DBP forms protein ®laments via a C-terminal arm that hooks into a neighbouring molecule. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. Another conserved part of DBP forms an unstructured ¯exible loop that is probably directly involved in contacting DNA. By making appropriate deletion mutants that do not distort the overall DBP structure, the in¯uence of the C-terminal arm and the ¯exible loop on the kinetics of ssDNA binding and on DNA replication was studied. Employing surface plasmon resonance we show that both parts of the protein are required for high af®nity binding. Deletion of the C-terminal arm leads to an extremely labile DBP±ssDNA complex indicating the importance of multimerization. The ¯exible loop is also required for opti- mal stability of the DBP±ssDNA complex, providing additional evidence that this region forms part of the ssDNA-binding surface of DBP. Both deletion mutants are still able to stimulate initiation of DNA replica- tion but are defective in supporting elongation, which may be caused by the fact that both mutants have a reduced DNA unwinding activity. Sur- prisingly, mixtures containing both mutants do stimulate elongation. Mixing the puri®ed mutant proteins leads to the formation of mixed ®la- ments that have a higher af®nity for ssDNA than homogeneous mutant ®laments. These results provide evidence that the C-terminal arm and the ¯exible loop have distinct functions in unwinding during replication. We propose the following model for ATP-independent DNA unwinding by DBP. Multimerization via the C-terminal arm is required for the for- mation of a protein ®lament that saturates the displaced strand. A high af®nity of a DBP monomer for ssDNA and subsequent local destabiliza- tion of the replication fork requires the ¯exible loop. # 1998 Academic Press Limited Keywords: adenovirus; helix destabilizing proteins; DNA replication; DNA unwinding; surface plasmon resonance *Corresponding author Introduction Adenovirus contains a 36 kb double-stranded linear DNA genome with a terminal protein cova- lently linked to the 5 0 ends. The inverted terminal repeats contain the origins of DNA replication. Adenovirus DNA replication can be reconstituted in vitro and has been studied in great detail (for reviews, see Hay et al., 1995; Hay, 1996; van der Vliet, 1995). The adenovirus DNA polymerase (pol) is tightly associated with the precursor of the terminal protein (pTP). During initiation of DNA replication pTP functions as a primer to which the ®rst residue, a dCMP residue, is covalently Abbreviations used: ssDNA, single-stranded DNA; pol, adenovirus DNA polymerase; pTP, precursor of the terminal protein; NFI, nuclear factor I; SPR, surface plasmon resonance; DBP, DNA, binding protein; PMSF, phenylmethylsulphonyl ¯uoride. J. Mol. Biol. (1998) 277, 825±838 0022±2836/98/140825±14 $25.00/0/mb981652 # 1998 Academic Press Limited