Structure of reaction intermediates formed during Saccharomyces cerevisiae Rad51-catalyzed strand transfer.
The process by which the Saccharomyces cerevisiae strand transfer protein, Rad51, seeks out homologous sequences in vivo can be modeled by an in vitro reaction between a single-stranded DNA circle and a double-stranded linear DNA. In addition to the substrates and products, electrophoresis of reaction mixtures resolves two groups of low mobility bands. Here we show that the low mobility bands formed during strand transfer by Rad51 (or Escherichia coli RecA) represent joint molecules (JM) between the two substrates. One group, which we name JM1, is an obligatory reaction intermediate in which the complementary strand from the duplex substrate has been partially transferred to the single-stranded circle. Our assignment is based on pulse-chase and restriction enzyme digestion experiments and verified by electron microscopy. The slower moving group of bands, designated JM2, is formed by an unexpected reaction between JM1 and a second double-stranded linear substrate. Strand transfer of the second duplex initiates noncanonically from the end where the complementary strand is recessed. Thus JM2 is formed by two strand transfer reactions with the same single-stranded circular substrate but with opposite polarities. Finally, we show that the multiple sharp bands in JM1 and JM2 are the result of substrate sequences that pause strand transfer.
Holmes
Victor F
VF
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Scandellari
Francesca
F
Benjamin
Kirsten R
KR
Cozzarelli
Nicholas R
NR
D004247:DNA / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D051135:Rad51 Recombinase; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D051135:Rad51 Recombinase; D011938:Rec A Recombinases; D004262:DNA Restriction Enzymes