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 does not require a DNA helicase to unwind the double-stranded template. Elongation is dependent on the adenovirus DNA-binding protein (DBP) which has helix-destabilizing properties. DBP binds cooperatively to single-stranded DNA (ssDNA) in a non-sequence-specific manner. The crystal 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 results in a monomeric protein. The mutant binds with a greatly reduced affinity to ssDNA. The deletion mutant still stimulates initiation of DNA replication like the intact DBP. This shows that a high affinity of DBP for ssDNA is not required for initiation. On a single-stranded template, 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 synthesis on a double-stranded template is only observed in the presence of DBP. The mutant, however, does not support elongation on a double-stranded template. The unwinding activity of the mutant is highly reduced compared with intact DBP. These data suggest that protein chain formation by DBP and high affinity binding to the displaced strand drive the ATP-independent unwinding of the template during adenovirus DNA replication.
Dekker
J
J
Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
Kanellopoulos
P N
PN
Loonstra
A K
AK
van Oosterhout
J A
JA
Leonard
K
K
Tucker
P A
PA
van der Vliet
P C
PC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017384:Sequence Deletion; D018411:Spodoptera; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate