Overexpression, purification and helix-destabilizing properties of Epstein-Barr virus ssDNA-binding protein.
The Epstein-Barr virus (EBV) ssDNA-binding protein (SSB) encoded by the BALF2 gene is one of the essential replication proteins in the lytic phase of EBV DNA replication. In order to obtain the amount of EBV SSB required for characterization, a recombinant baculovirus containing the complete sequence of the BALF2 open reading frame under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 130 kDa, recognized by anti-BALF2 protein-specific polyclonal antibody. The overexpressed EBV SSB was purified homogeneously from the cytosolic fraction of the recombinant virus-infected cells. The purified protein displaced short DNA strands from their complementary sequences in the single-stranded form of M13. The helix-destabilizing activity was neutralized by the anti-BALF2 protein-specific antibody. Maximum unwinding occurred at EBV SSB concentrations exceeding saturation level of the DNA substrate. The DNA unwinding reaction mediated by the EBV SSB was highly cooperative and extremely rapid. The reaction displayed no directionality and required neither ATP nor MgCl2, two essential cofactors for DNA helicase activity. The helix-destabilizing property of the EBV SSB may function to melt out secondary structures on the ssDNA template, thereby facilitating the movement of the EBV DNA polymerase.
Tsurumi
T
T
Laboratory of Viral Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan. ttsurumi@aichi-cc.pref.aichi.jp
Kishore
J
J
Yokoyama
N
N
Fujita
M
M
Daikoku
T
T
Yamada
H
H
Yamashita
Y
Y
Nishiyama
Y
Y
D000818:Animals; D000914:Antibodies, Viral / Q000378:metabolism; D017104:Bacteriophage M13 / Q000235:genetics; D002144:Callithrix; D002460:Cell Line; D002461:Cell Line, Transformed; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium; D009841:Oligonucleotides; D009994:Osmolar Concentration; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000914:Antibodies, Viral; C096364:BALF2 protein, Human herpesvirus 4; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D008274:Magnesium