MULTIMERIZATION OF THE ADENOVIRUS DNA-BINDING PROTEIN IS THE DRIVING-FORCE FOR ATP-INDEPENDENT DNA UNWINDING DURING STRAND DISPLACEMENT SYNTHESIS

In contrast to other replication systems, adenovirus DNA replication d oes not require a DNA helicase to unwind the double-stranded template, Elongation is dependent on the adenovirus DNA-binding protein (DBP) w hich has helix-destabilizing properties, DBP binds cooperatively to si ngle-stranded DNA (ssDNA) in a non-sequence-specific manner. The cryst al structure of DBP shows that the protein has a C-terminal extension that hooks on to an adjacent monomer which results in the formation of long protein chains. We show that deletion of this C-terminal arm res ults in a monomeric protein. The mutant binds with a greatly reduced a ffinity to ssDNA, The deletion mutant still stimulates initiation of D NA replication like the intact DBP, This shows that a high affinity of DBP for ssDNA is not required for initiation, On a single-stranded te mplate, elongation is also observed in the absence of DBP, Addition of DBP or the deletion mutant has no effect on elongation, although both proteins stimulate initiation on this template, Strand displacement s ynthesis on a double-stranded template is only observed in the presenc e of DBP, The mutant, however, does not support elongation on a double -stranded template, The unwinding activity of the mutant is highly red uced compared with intact DBP, These data suggest that protein chain f ormation by DBP and high affinity binding to the displaced strand driv e the ATP-independent unwinding of the template during adenovirus DNA replication.