Structural and functional analysis of the baculovirus single-stranded DNA-binding protein LEF-3.
The single-stranded DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus consists of 385 amino acid residues, forms oligomers, and promotes Mg2+-independent unwinding of DNA duplexes and annealing of complementary DNA strands. Partial proteolysis revealed that the DNA-binding domain of LEF-3 is located within a central region (residues 28 to 326) that is relatively resistant to proteolysis. In contrast, the N-terminus (27 residues) and C-terminal portion (59 residues) are not involved in interaction with DNA and are readily accessible to proteolytic digestion. Circular dichroism analyses showed that LEF-3 is a folded protein with an estimated alpha-helix content of more than 40%, but it is structurally unstable and undergoes unfolding in aqueous solutions at temperatures near 50 degrees C. Unfolding eliminated the LEF-3 domains that are resistant to proteolysis and randomized the digestion pattern by trypsin. The structural transition was irreversible and was accompanied by the generation of high molecular weight (MW) complexes. The thermal treatment inhibited DNA-binding and unwinding activity of LEF-3 but markedly stimulated its annealing activity. We propose that the shift in LEF-3 activities resulted from the generation of the high MW protein complexes, that specifically stimulate the annealing of complementary DNA strands by providing multiple DNA-binding sites and bringing into close proximity the interacting strands. The unfolded LEF-3 was active in a strand exchange reaction suggesting that it could be involved in the production of recombination intermediates.
Mikhailov
Victor S
VS
Department of Microbiology, Oregon State University, Nash Hall 220, Corvallis, OR 97331-3804, USA. Victor.Mikhailov@orst.edu
Okano
Kazuhiro
K
Rohrmann
George F
GF
D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000737:chemistry* / Q000502:physiology*; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D014764:Viral Proteins; C094647:late expression factor 3, baculovirus; D014357:Trypsin