Replication of adenovirus and SV40 chromosomes in vitro

Abstract

As an approach to studying the mechanisms involved in the replication of eukaryotic chromosomes, we have developed and characterized cell-free replication systems for the animal viruses, adenovirus and SV40. In this report we summarize recent work on the proteins required for the initiation of DNA synthesis in these two systems. The adenovirus origin of DNA replication was shown to consist of three functionally distinct sequence domains. Cellular proteins that specifically recognize each of these domains were purified and characterized. Initiation of adenovirus DNA replication was reconstituted from two virus-encoded and three cell-encoded factors. The SV40 origin of replication consists of a 65 base pair DNA segment that contains a high affinity binding site for the viral initiation protein T antigen. Evidence is presented that the first step in initiation of SV40 DNA replication involves the specific binding of T antigen to the origin, followed by the local unwinding of the two strands of the template. The unwinding reaction is specific for DNA templates containing the SV40 origin and requires ATP hydrolysis. In addition to T antigen, efficient unwinding requires a cellular factor(s) that can be replaced by the single-stranded DNA binding protein of Escherichia coli. These results indicate that the recently discovered helicase activity of T antigen plays a central role in initiation of viral DNA synthesis.

Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity

“…MCV is related to the rhesus macaque SV40 polyomavirus that has been an extensively studied model for eukaryotic DNA replication for over 50 y. The first in vitro eukaryotic DNA replication studies were performed using the LT protein and DNA origin of SV40 ( 18 , 19 ), leading to the discovery of critical cellular factors in eukaryotic replication ( 20 , 21 ). SV40 LT helicase assembles as a head-to-head, double-hexameric homopolymer that is reported to unwind less than a single turn of DNA as it assembles through a mechanism requiring ATP binding but not hydrolysis ( 22 ).…”