Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA

Abstract

The adenovirus-encoded single-stranded DNA-binding protein (DBP) functions in viral DNA replication and several aspects of RNA metabolism. Previous studies (G. A. M. Neale and G. R. Kitchingman, J. Biol. Chem. 264:3153-3159, 1989) have defined three highly conserved regions in the carboxy-terminal domain of the protein (amino acids 178 to 186, 322 to 330, and 464 to 475) that may be involved in the binding of the protein to single-stranded DNA. We examined the role of conserved region 3 (464 to 475) by constructing nine classes of point mutants with from one to four amino acid changes. The point mutants were tested for their ability to assist adeno-associated virus DNA replication. All nine differed from wild-type DBP; seven were essentially nonfunctional, whereas two had 55 and 145%, respectively, of the wild-type DBP helper activity. Three of the mutants were found to be temperature sensitive, with significantly greater helper activity at 33°C than at 37°C. All nine mutants produced essentially wild-type levels of protein. One monoclonal antibody against the DBP, termed 2/4, did not immunoprecipitate the mutant DBPs as well as wild-type DBP, indicating either that the antibody recognized sequences around CR3 or that the conformation of the protein around the epitope recognized by 2/4 had changed. Two of the three temperature-sensitive DBP mutants bound to single-stranded DNA-cellulose with the same affinity as wild-type DBP at 4°C; the remaining mutants all showed reduced affinity. These results demonstrated that many of the residues within conserved region 3 of the DBP are important for interaction of the protein with nucleic acid.

Helix-destabilizing properties of the adenovirus DNA-binding protein

“…DBP is an anisometric protein of 529 amino acids that can be split into two domains by mild chymotrypsin treatment. In contrast to the N-terminal domain (amino acids 1 to 173), the C-terminal domain (amino acids 174 to 529) is well conserved among serotypes and harbors most of the biological functions ascribed to DBP, including its nucleic acid-binding properties (3,22,23,29).…”

Role of the Adenovirus DNA-Binding Protein in In Vitro Adeno-Associated Virus DNA Replication

“…Two sets of more definitive experiments have been done more recently. Kitchingman and colleagues (33,38) constructed a series of point mutants in Ad-DBP, including several targeted to the putative single-stranded DNA binding domain. When transfected into AAV-infected Cos cells, several of these mutants, especially those targeted to the putative DNA binding site, supported almost no synthesis of AAV DNA.…”

Functional changes in temperature-sensitive mutants of the adenovirus single-stranded DNA-binding protein are accompanied by structural alterations

“…been found by comparative sequence analysis (12) among the DBPs of various human adenovirus serotypes. Some of the mutations introduced in CR2 (amino acids 322 to 330) and CR3 (amino acids 464 to 475) substantially decrease the protein's ability to bind to ssDNA and to support adenoassociated virus DNA replication in vivo (22,23) without affecting its in vivo stability, phosphorylation, or subcellular localization. Thus, these two regions of the DBP may be directly involved in DNA binding.…”