PROPERTIES OF THE ADENOVIRUS DNA POLYMERASE AND HELA DNA POLYMERASE ALPHA (REPLICATION)

Abstract

The 140,000 dalton Adenovirus encoded DNA polymerase (Ad Pol) is the only DNA polymerase involved in viral DNA replication. The Ad Pol was characterized by studying its catalytic properties and its interaction with other replication proteins. The Ad Pol was capable of DNA synthesis by itself on a variety of DNA templates. However, when poly(dT)(.)oligo(dA) (or oligo(rA)) was used as a template(.)primer, DNA synthesis by the Ad Pol was specifically stimulated 10-100 fold by the adenovirus DNA binding protein (Ad DBP), another viral protein required for replication. The poly(dA) products formed in the reaction with poly(dT)(.)oligo(dA) were about 30,000 nucleotides long suggesting that the Ad Pol can be highly processive. The displacement reaction was studied using (SLASHCIRC)X174 RFIII DNA tailed with poly(dT) at the 3' ends by terminal deoxynucleotidyl transferase. In the presence of Ad DBP and an oligo(dA) primer, the Ad Pol initiated DNA synthesis on the poly(dT) tails and proceeded through the duplex regions yielding full length (SLASHCIRC)X174 DNA with the expected restriction analysis pattern. The role of ATP in this reaction was also studied. In addition to its polymerizing activity the Ad Pol contains an exonuclease activity capable of hydrolyzing single stranded DNA in the 3' (--->) 5' direction.;Studies with HeLa DNA polymerase (alpha) revealed that it contained a primase activity. In the presence of ribonucleoside triphosphates this activity initiated DNA synthesis de novo on the synthetic polymers poly(dC) and poly(dT) as well as on single stranded DNA. DNA chains initiated by the primase contained RNA oligomers about 10 nucleotides in length covalently attached to their 5'-ends. The primase activity did not separate from DNA polymerase (alpha) activity over a 200 fold enrichment. However, primase was less stable than polymerase (alpha).