Helix-destabilizing properties of the baculovirus single-stranded DNA-binding protein (LEF-3).
The helix-destabilizing properties of a single-stranded DNA-binding protein, LEF-3, of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Partial duplexes of DNA containing single-stranded (ss) tails of different sizes and orientations were used as substrates for assay of the unwinding ability of LEF-3. Upon noncooperative binding to ssDNA, LEF-3 was capable of unwinding the duplexes with 5' ss tails. However, it did not cause melting of the duplexes containing 3' ss tails, even at oversaturation of ssDNA adjacent to the duplexes. Upon cooperative binding to long ss tails, LEF-3 also produced the polar melting effect; it unwound the duplexes with long 5' ss tails, but not those with long 3' ss tails. These data suggest that LEF-3 has a preferential direction for entry into duplex DNA, namely 5' to 3' with respect to the bound DNA strand. In agreement with its polarity, LEF-3 efficiently melted the primer-template complexes which serve as substrates for DNA polymerases. However, the formation of a complex with viral DNA polymerase before addition of LEF-3 protected the primer-templates from the destabilization effect of LEF-3. Although the destabilization effect of LEF-3 was highly sensitive to monovalent and divalent salts, the protein was capable of melting DNA duplexes in a polar manner at physiological conditions, i.e., 30 degrees C in 0.15 M NaCl. Therefore, the polar destabilization effect of LEF-3 seems to be physiologically important and may be connected, in particular, with the polar action of viral helicase holoenzyme during baculovirus replication.
Mikhailov
V S
VS
Kol'tsov Institute of Developmental Biology, Moscow, 117808, Russia. vsmikha@ibrran.msk.su
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D012831:Bombyx / Q000821:virology; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism; D015636:Magnesium Chloride / Q000494:pharmacology; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013698:Templates, Genetic; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C094647:late expression factor 3, baculovirus; D015636:Magnesium Chloride; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases