Adenovirus DNA polymerase is a phosphoprotein

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Protein phosphorylation is a key regulator of eukaryotic DNA replication.
• Investigating adenovirus DNA polymerase (AdPol) phosphorylation is challenging due to low synthesis levels in infected cells.

Purpose of the Study:

• To investigate the in vivo phosphorylation of AdPol.
• To identify the specific site(s) of AdPol phosphorylation and its functional significance.

Main Methods:

• Overproduction of AdPol using recombinant vaccinia virus and baculovirus systems.
• Metabolic labeling of adenovirus 2-infected HeLa cells.
• Phosphoamino acid analysis, tryptic peptide mapping, HPLC, and protein sequencing.
• In vitro DNA replication initiation assays.

Main Results:

• In vivo phosphorylation of AdPol was demonstrated using overexpressed and native forms.
• Phosphoserine was identified as the primary phosphoamino acid.
• Tryptic peptide mapping revealed common phosphopeptides between native and recombinant AdPol.
• Serine 67 was identified as a major phosphorylation site, located near the nuclear localization signal.
• Dephosphorylation significantly reduced AdPol activity in DNA replication initiation.

Conclusions:

• Phosphorylation, specifically at serine 67, is critical for AdPol's biological activity in DNA replication initiation.
• The identified phosphorylation site suggests regulation by specific kinase families.
• This study provides insights into the post-translational modification and regulation of viral DNA replication machinery.