Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae.
Eukaryotic Okazaki fragments are initiated by a RNA/DNA primer, which is removed before the fragments are joined. Polymerase delta displaces the primer into a flap for processing. Dna2 nuclease/helicase and flap endonuclease 1 (FEN1) are proposed to cleave the flap. The single-stranded DNA-binding protein, replication protein A (RPA), governs cleavage activity. Flap-bound RPA inhibits FEN1. This necessitates cleavage by Dna2, which is stimulated by RPA. FEN1 then cuts the remaining RPA-free flap to create a nick for ligation. Cleavage by Dna2 requires that it enter the 5'-end and track down the flap. Because Dna2 cleaves the RPA-bound flap, we investigated the mechanism by which Dna2 accesses the protein-coated flap for cleavage. Using a nuclease-defective Dna2 mutant, we showed that just binding of Dna2 dissociates the flap-bound RPA. Facile dissociation is specific to substrates with a genuine flap, and will not occur with an RPA-coated single strand. We also compared the cleavage patterns of Dna2 with and without RPA to better define RPA stimulation of Dna2. Stimulation derived from removal of DNA folding in the flap. Apparently, coordinated with its dissociation, RPA relinquishes the flap to Dna2 for tracking in a way that does not allow flap structure to reform. We also found that RPA strand melting activity promotes excessive flap elongation, but it is suppressed by Dna2-promoted RPA dissociation. Overall, results indicate that Dna2 and RPA coordinate their functions for efficient flap cleavage and preparation for FEN1.
Stewart
Jason A
JA
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Miller
Adam S
AS
Campbell
Judith L
JL
Bambara
Robert A
RA
D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D044366:Transition Temperature
D004277:DNA, Single-Stranded; C022262:Okazaki fragments; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae
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