TY - JOUR
T1 - Pif1 helicase directs eukaryotic okazaki fragments toward the two-nuclease cleavage pathway for primer removal
AU - Rossi, Marie L.
AU - Pike, Jason E.
AU - Wang, Wensheng
AU - Burgers, Peter M.J.
AU - Campbell, Judith L.
AU - Bambara, Robert A.
PY - 2008/10/10
Y1 - 2008/10/10
N2 - Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase δ (Pol δ) extends the upstream Okazaki fragment and displaces the 5′-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase δ only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.
AB - Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase δ (Pol δ) extends the upstream Okazaki fragment and displaces the 5′-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase δ only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.
UR - http://www.scopus.com/inward/record.url?scp=55549146815&partnerID=8YFLogxK
U2 - 10.1074/jbc.M804550200
DO - 10.1074/jbc.M804550200
M3 - Article
C2 - 18689797
AN - SCOPUS:55549146815
VL - 283
SP - 27483
EP - 27493
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 41
ER -