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Published December 31, 2010 | Supplemental Material + Published
Journal Article Open

An Alternative Pathway for Okazaki Fragment Processing - Resolution of Fold-Back Flaps by Pif1 Helicase


Two pathways have been proposed for eukaryotic Okazaki fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in fragment processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki fragments initiated by fold-back flaps in vivo.

Additional Information

© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication,May 20, 2010, and in revised form, October 13, 2010. First Published on October 19, 2010. This work was supported, in whole or in part, by National Institutes of Health Grants GM024441 (to R. A. B.), GM078666 (to J. L. C.), and GM032431 (to P. M. J. B.). We thank the members of the Bambara laboratory for helpful discussions and suggestions. We thank Dr. Marc Wold for providing us with purified RPA.

Attached Files

Published - Pike2010p12721J_Biol_Chem.pdf

Supplemental Material - jbc.M110.146894-1.pdf


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