On the roles of Saccharomyces cerevisiae Dna2p and Flap endonuclease 1 in Okazaki fragment processing.
Short DNA segments designated Okazaki fragments are intermediates in eukaryotic DNA replication. Each contains an initiator RNA/DNA primer (iRNA/DNA), which is converted into a 5'-flap and then removed prior to fragment joining. In one model for this process, the flap endonuclease 1 (FEN1) removes the iRNA. In the other, the single-stranded binding protein, replication protein A (RPA), coats the flap, inhibits FEN1, but stimulates cleavage by the Dna2p helicase/nuclease. RPA dissociates from the resultant short flap, allowing FEN1 cleavage. To determine the most likely process, we analyzed cleavage of short and long 5'-flaps. FEN1 cleaves 10-nucleotide fixed or equilibrating flaps in an efficient reaction, insensitive to even high levels of RPA or Dna2p. On 30-nucleotide fixed or equilibrating flaps, RPA partially inhibits FEN1. CTG flaps can form foldback structures and were inhibitory to both nucleases, however, addition of a dT(12) to the 5'-end of a CTG flap allowed Dna2p cleavage. The presence of high Dna2p activity, under reaction conditions favoring helicase activity, substantially stimulated FEN1 cleavage of tailed-foldback flaps and also 30-nucleotide unstructured flaps. Our results suggest Dna2p is not used for processing of most flaps. However, Dna2p has a role in a pathway for processing structured flaps, in which it aids FEN1 using both its nuclease and helicase activities.
Kao
Hui-I
HI
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA. robert_bambara@urmc.rochester.edu
Veeraraghavan
Janaki
J
Polaczek
Piotr
P
Campbell
Judith L
JL
Bambara
Robert A
RA
D000251:Adenosine Triphosphatases / Q000502:physiology*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry / Q000502:physiology*; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases / Q000502:physiology*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C022262:Okazaki fragments; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae