A complementary single-stranded docking site is required for enhancement of strand exchange by human immunodeficiency virus nucleocapsid protein on substrates that model viral recombination.
Enhancement of strand exchange by nucleocapsid protein (NC) is proposed to occur during retroviral recombination. The mechanism was examined using an RNA (donor)-DNA hybrid that mimicked a retrovirus replication intermediate. This consisted of a 25 base pair hybrid region flanked on each side by single-stranded RNA or DNA. A second set of acceptor RNAs that could bind to the 25-base hybrid region and to various lengths of additional bases on the DNA was used to displace the donor by hybridizing with the DNA. Displacement required a complementary single-stranded DNA region outside the donor-DNA 25-nucleotide hybrid region. NC enhanced displacement slightly when the acceptor could bind 10 nucleotides and significantly when binding 22 or more nucleotides in the single-stranded region. Two mutated acceptors that bound over 47 total nucleotides on the DNA (22 in the single-stranded region plus 25 in the hybrid region) were constructed. One had three mismatches in the hybrid region; the other, three in the single-stranded region and one in the hybrid region. Each acceptor bound the DNA with approximately equal thermodynamic stability, yet NC stimulated exchange with the former and actually inhibited with the latter. This emphasized the importance of the single-stranded region in NC stimulation. The results support a mechanism where NC enhances the docking of the acceptor to the single-stranded region and then the acceptor "zippers" through the hybrid and displaces the donor. Results with the mutated acceptors indicate that NC may actually inhibit strand exchange between genomes in nonhomologous regions.
Heath
Megan J
MJ
Department of Cell Biology and Molecular Genetics, University of Maryland College Park, College Park, Maryland 20742, USA.
Destefano
Jeffrey J
JJ
D020029:Base Pairing / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics*; D008956:Models, Chemical; D009693:Nucleic Acid Hybridization; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013816:Thermodynamics
D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D019590:Nucleocapsid Proteins; D012367:RNA, Viral