Formation of joint DNA molecules by two eukaryotic strand exchange proteins does not require melting of a DNA duplex.
We have examined whether DNA strand exchange activities from nuclear extracts of HeLa cells or Drosophila melanogaster embryos have detectable helicase or melting activities. The partially purified recombinases have been shown to recognize homologous single strand and double strand DNA molecules and form joint molecules in a DNA strand exchange reaction. The joint molecule product consists of a linear duplex joined at one end by a region of DNA heteroduplex to a homologous single strand circular DNA. Using two different partially duplex helicase substrates, we are unable to detect any melting of duplex regions under conditions that promote joint molecule formation. One substrate consists of a 32P-labeled oligonucleotide 20 or 30 bases long annealed to M13mp18 circular single strand DNA. The second substrate consists of a linear single strand region flanked at each end by short duplex regions. We observe that even in the presence of excess recombinase protein or after prolonged incubation no helicase activity is apparent. Control experiments rule out the possibility that a helicase is masked by reannealing of displaced single strand fragments. Based on these findings and other data, we conclude that the human and D. melanogaster recombinases recognize and pair homologous sequences without significant melting of duplex DNA prior to strand exchange.
Hsieh
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Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland.
Camerini-Otero
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D000818:Animals; D004265:DNA Helicases / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D004625:Embryo, Nonmammalian / Q000201:enzymology; D006367:HeLa Cells / Q000201:enzymology*; D006358:Hot Temperature; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D011995:Recombination, Genetic; D013379:Substrate Specificity
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D004254:DNA Nucleotidyltransferases; D004265:DNA Helicases