Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA.
In eukaryotic cells, a 5' flap DNA endonuclease activity and a ds DNA 5'-exonuclease activity exist within a single enzyme called FEN-1 [flap endo-nuclease and 5(five)'-exo-nuclease]. This 42 kDa endo-/exonuclease, FEN-1, is highly homologous to human XP-G, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1. These structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap, and its derivative called a pseudo Y-structure. FEN-1 is essential for lagging strand DNA synthesis in Okazaki fragment joining. FEN-1 also appears to be important in mismatch repair. Here we find that human PCNA, the processivity factor for eukaryotic polymerases, physically associates with human FEN-1 and stimulates its endonucleolytic activity at branched DNA structures and its exonucleolytic activity at nick and gap structures. Structural requirements for FEN-1 and PCNA loading provide an interesting picture of this stimulation. PCNA loads on to substrates at double-stranded DNA ends. In contrast, FEN-1 requires a free single-stranded 5' terminus and appears to load by tracking along the single-stranded DNA branch. These physical constraints define the range of DNA replication, recombination and repair processes in which this family of structure-specific nucleases participate. A model explaining the exonucleolytic activity of FEN-1 in terms of its endonucleolytic activity is proposed based on these observations.
Wu
X
X
Department of Pathology, Washington University School of Medicine, St Louis, MO 63110, USA.
Li
J
J
Li
X
X
Hsieh
C L
CL
Burgers
P M
PM
Lieber
M R
MR
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D018076:DNA, Complementary / Q000032:analysis; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D045585:Flap Endonucleases; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human