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 DN A replication. DBP forms protein filaments via a C-terminal arm that h ooks into a neighbouring molecule. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. Ano ther conserved part of DBP forms an unstructured flexible loop that is probably directly involved in contacting DNA. By making appropriate d eletion mutants that do not distort the overall DBP structure, the inf luence of the C-terminal arm and the flexible loop on the kinetics of ssDNA binding and on DNA replication was studied. Employing surface pl asmon resonance we show that both parts of the protein are required fo r high affinity binding. Deletion of the C-terminal arm leads to an ex tremely labile DBP-ssDNA complex indicating the importance of multimer ization. The flexible loop is also required for optimal stability of t he DBP-ssDNA complex, providing additional evidence that this region f orms part of the ssDNA-binding surface of DBP. Both deletion mutants a re still able to stimulate initiation of DNA replication but are defec tive in supporting elongation, which may be caused by the fact that bo th mutants have a reduced DNA unwinding activity. Surprisingly, mixtur es containing both mutants do stimulate elongation. Mixing the purifie d mutant proteins leads to the formation of mixed filaments that have a higher affinity for ssDNA than homogeneous mutant filaments. These r esults provide evidence that the C-terminal arm and the flexible loop have distinct functions in unwinding during replication. We propose th e following model for ATP-independent DNA unwinding by DBP. Multimeriz ation via the C-terminal arm is required for the formation of a protei n filament that saturates the displaced strand. A high affinity of a D BP monomer for ssDNA and subsequent local destabilization of the repli cation fork requires the flexible loop. (C) 1998 Academic Press Limite d.