Analysis of Ori-S sequence of HSV-1: identification of one functional DNA binding domain

Abstract

Using gel retardation assays, we have detected an Ori-S binding activity in the nuclear extract of HSV-1 infected Vero cells. The sequence-specific DNA binding activity seems to be identical to that described by Elias et al. (Proc. Natl. Acad. Sci. USA 83: 6322-6326, 1986). This activity fails to retard a mutant origin DNA that has a 5 bp deletion in the reported protein binding site along with an A to T substitution at a position 16 base-pairs away from the site. This mutant also failed to replicate in a transient replication assay, thus correlating binding of the factor on the origin to replication efficiency. Using crude nuclear extracts as the source of the factor and with the help of footprint and gel retardation analyses, we confirmed that protection is only observed on the preferred site of binding on and near the left arm of the Ori-S palindrome. In order to analyze the sequence specificity of the binding we have generated a set of binding site mutants. Competition experiments with these mutant origins indicate that the sequence 5'-TTCGCACTT-3' is crucial for binding.