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Reconstituted Okazaki fragment processing indicates two pathways of primer removal
Marie L Rossi1, Robert A Bambara
1Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
The Journal of Biological Chemistry
|July 14, 2006
Summary
DNA ligase I requires complete RNA/DNA primer removal for Okazaki fragment joining. Studies show flap endonuclease 1 (FEN1) removes most primers, but longer flaps necessitate a two-nuclease pathway for efficient DNA replication.
Area of Science:
- Molecular Biology
- DNA Replication
- Biochemistry
Background:
- Okazaki fragments are short DNA sequences synthesized during DNA replication.
- RNA/DNA primers initiate Okazaki fragment synthesis, requiring removal before ligation.
Purpose of the Study:
- To investigate the mechanisms of RNA/DNA primer removal during Okazaki fragment processing.
- To determine the roles of DNA polymerase delta (pol delta), proliferating cell nuclear antigen (PCNA), and flap endonuclease 1 (FEN1) in flap processing.
Main Methods:
- Reconstitution of an oligonucleotide system with Saccharomyces cerevisiae proteins.
- Measurement of FEN1 cleavage products on DNA flaps displaced by pol delta.
Main Results:
- Pol delta and FEN1 primarily generate short flaps (up to 8 nucleotides).
- A subset of flaps reach 20-30 nucleotides, requiring a two-nuclease pathway.
- Flap length is influenced by protein mutations, DNA sequence, and reaction conditions.
Conclusions:
- FEN1 is sufficient for processing the majority of Okazaki fragment primers.
- A parallel two-nuclease pathway is essential for processing longer flaps.
- Both pathways are required for complete Okazaki fragment processing in eukaryotic DNA replication.