Reconstituted Okazaki fragment processing indicates two pathways of primer removal.

Eukaryotic Okazaki fragments are initiated by an RNA/DNA primer and extended by DNA polymerase delta (pol delta) and the replication clamp proliferating cell nuclear antigen (PCNA). Joining of the fragments by DNA ligase I to generate the continuous double-stranded DNA requires complete removal of the RNA/DNA primer. Pol delta extends the upstream Okazaki fragment and displaces the downstream RNA/DNA primer into a flap removed by nuclease cleavage. One proposed pathway for flap removal involves pol delta displacement of long flaps, coating of those flaps by replication protein A (RPA), and sequential cleavage of the flap by Dna2 nuclease followed by flap endonuclease 1 (FEN1). A second pathway involves reiterative single nucleotide or short oligonucleotide displacement by pol delta and cleavage by FEN1. We measured the length of FEN1 cleavage products on flaps strand-displaced by pol delta in an oligonucleotide system reconstituted with Saccharomyces cerevisiae proteins. Results showed that in the presence of PCNA and FEN1, pol delta displacement synthesis favors formation and cleavage of primarily short flaps, up to eight nucleotides in length; still, a portion of flaps grows to 20-30 nucleotides. The proportion of long flaps can be altered by mutations in the relevant proteins, sequence changes in the DNA, and reaction conditions. These results suggest that FEN1 is sufficient to remove a majority of Okazaki fragment primers. However, some flaps become long and require the two-nuclease pathway. It appears that both pathways, operating in parallel, are required for processing of all flaps.