Adenovirus Type 5 DNA Binding Protein Stimulates Binding of DNA Polymerase to the Replication Origin

Abstract

The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the K m for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.

Inhibition of androgen receptor transactivation function by adenovirus type 12 E1A undermines prostate cancer cell survival

“…Interestingly, the expression of the adenoviral DBP was markedly increased upon R1881 stimulation in LNCaP cells infected with Ad‐E1A12. DBP is required for Ad replication . As shown previously, E1A12 expression was essential for DBP expression as DBP was not expressed in LNCaP cells infected with Ad‐eGFP (Figure B).…”

“…DBP is required for Ad replication. 62 As shown previously, 38 E1A12 expression was essential for DBP expression as DBP was not expressed in LNCaP cells infected with Ad-eGFP ( Figure 8B). These results suggest that E1A12 deploys AR to stimulate viral gene expression for efficient viral replication, potentially resulting in enhanced oncolytic effects of Ad-E1A12.…”

Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

“…The pTP peptide is the precursor form of the Ad terminal protein which matures following protease cleavage [51,52]. pTP stabilizes the origins of Ad DNA replication, promoting DNA synthesis by the formation of a pTP/pol dimer and the recruitment of the transcription factors octamer-binding protein 1 (Oct-1) and nuclear factor I (NFI) which bind pTP and pol, respectively [53,54]. In AdUV, pTP is mutated at 8783, changing an encoded arginine with a positive charge at physiological pH to a hydrophobic leucine.…”

A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation

“…But how can USP7 affect late gene expression? Taking into account that USP7 not only affects E1B-55K but also, for example, E2A levels it is probable that E2A functions in promoting DNA replication might be hampered which in turn lead to the observed negative effect on late gene expression [67], [68].…”