ATP-independent DNA unwinding by the adenovirus single-stranded DNA binding protein requires a flexible DNA binding loop.

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 filaments 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 flexible loop that is probably directly involved in contacting DNA. By making appropriate deletion mutants that do not distort the overall DBP structure, the influence of the C-terminal arm and the flexible 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 affinity binding. Deletion of the C-terminal arm leads to an extremely labile DBP-ssDNA complex indicating the importance of multimerization. The flexible loop is also required for optimal 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 replication but are defective in supporting elongation, which may be caused by the fact that both mutants have a reduced DNA unwinding activity. Surprisingly, mixtures containing both mutants do stimulate elongation. Mixing the purified mutant proteins leads to the formation of mixed filaments that have a higher affinity for ssDNA than homogeneous mutant filaments. These results provide evidence that the C-terminal arm and the flexible 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 formation of a protein filament that saturates the displaced strand. A high affinity of a DBP monomer for ssDNA and subsequent local destabilization of the replication fork requires the flexible loop.