Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8)

Abstract

We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.

Identification of a Region of the Herpes Simplex Virus Single-Stranded DNA-Binding Protein Involved in Cooperative Binding

“…As shown in Fig. 8, the cysteine modified at position 254 lies outside the centrally located DNA-binding domain which has been identified by other studies (16,19,35,55) and well outside a minimal DNA-binding domain (residues 563 to 636) recently identified in our laboratory (unpublished data). Thus, at least two regions of the protein are involved in different aspects of the interaction of ICP8 with singlestranded DNA, with the central region most likely being involved in intrinsic binding and the region around residue 254 being involved in cooperative binding.…”

“…These interactions, however, are considerably weaker than those observed with single-stranded DNA. The presence of ICP8 destabilizes poly(dA)-poly(dT) duplexes (55), and it has been shown that ICP8 is capable of displacing short oligonucleotides annealed to long single strands of DNA (2). Finally, ICP8 has been shown to aid in the annealing of separated DNA strands under the proper conditions (13).…”

Bombyx mori Nucleopolyhedrovirus Encodes a DNA-Binding Protein Capable of Destabilizing Duplex DNA

“…Basic and aromatic amino acids of DNA-binding proteins are thought to participate in interaction with DNA substrates. BmNPV DBP contains a motif, K 18 -R 21 -Y 33 -F 41 -Y 53 -R 64 -W 71 -K 80 , which presents a perfect match to the consensus sequence, K/RX 2-5 K/RX 4-12 F/YX 2-14 F/YX 6-13 F/YX 1-19 K/RX 3-26 F/Y/WX 6-11 R/K, found in all SSB proteins from prokaryotic and eukaryotic organisms (38) Isolation of a novel baculovirus DNA-binding protein, Bm-NPV DBP, poses intriguing question about the possible function of this protein in virus replication. Its preferential binding to ssDNA (Fig.…”

Characterization of the Single-Stranded DNA Binding Protein Encoded by the Vaccinia Virus I3 Gene

“…Although SSBs do not have a high degree of primary sequence similarity, there are common sequence motifs that have emerged from comparative analyses. A domain containing conserved aromatic and charged residues thought to mediate DNA binding is present in many SSBs and appears to be present in the I3 protein as well (11,34,51) (Fig. 11A).…”