The Oct-1 POU homeodomain stabilizes the adenovirus preinitiation complex via a direct interaction with the priming protein and is displaced when the replication fork passes.
Initiation of adenovirus DNA replication is strongly enhanced by two cellular transcription factors, NFI and Oct-1, which bind to the auxiliary origin and tether the viral precursor terminal protein-DNA polymerase (pTP.pol) complex to the core origin. NFI acts through a direct contact with the DNA polymerase, but the mode of action of Oct 1 is unknown. Employing glutathione S-transferase-POU pull-down assays and protein affinity chromatography, we have established that the POU domain contacts pTP rather than pol. The POU homeodomain is responsible for this interaction. The protein-protein contacts lead to increased binding of pTP-pol to the core origin, which is caused by a reduced off-rate. The enhanced formation of a pTP.pol.POU complex on the origin correlates with stimulation of replication. Using an immobilized replication system, we have studied the kinetics of dissociation of the Oct-1 POU domain during replication. In contrast to NFI, which dissociates very early in initiation, Oct-1 dissociates only when the binding site is rendered single-stranded upon translocation of the replication fork. Our data indicate that NFI and Oct-1 enhance initiation synergistically by touching different targets in the preinitiation complex and dissociate independently after initiation.
van Leeuwen
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HC
Laboratory for Physiological Chemistry, Utrecht University, Stratenum, P. O. Box 80042, 3508 TA Utrecht, The Netherlands.
Rensen
M
M
van der Vliet
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PC
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D018398:Homeodomain Proteins / Q000378:metabolism*; D051863:Host Cell Factor C1; D008958:Models, Molecular; D050811:Octamer Transcription Factor-1; D050809:POU Domain Factors; D010750:Phosphoproteins / Q000378:metabolism; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D014764:Viral Proteins; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; D050809:POU Domain Factors; D010750:Phosphoproteins; D011498:Protein Precursors; D014157:Transcription Factors; D014764:Viral Proteins; C085476:pTP protein, adenovirus