Biochemical characterization of the WRN-FEN-1 functional interaction.
Werner Syndrome is a premature aging disorder characterized by chromosomal instability. Recently we reported a novel interaction of the WRN gene product with human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in pathways of DNA metabolism that are important for genomic stability. To characterize the mechanism for WRN stimulation of FEN-1 cleavage, we have determined the effect of WRN on the kinetic parameters of the FEN-1 cleavage reaction. WRN enhanced the efficiency of FEN-1 cleavage rather than DNA substrate binding. WRN effectively stimulated FEN-1 cleavage on a flap DNA substrate with streptavidin bound to the terminal 3' nucleotide at the end of the upstream duplex, indicating that WRN does not require a free upstream end to stimulate FEN-1 cleavage of the 5' flap substrate. These results indicate that the mechanism whereby WRN stimulates FEN-1 cleavage is distinct from that proposed for the functional interaction between proliferating cell nuclear antigen and FEN-1. To understand the potential importance of the WRN-FEN-1(1) interaction in DNA replication, we have tested the effect of WRN on FEN-1 cleavage of several DNA substrate intermediates that may arise during Okazaki fragment processing. WRN stimulated FEN-1 cleavage of flap substrates with a terminal monoribonucleotide, a long 5' ssDNA tract, and a pseudo-Y structure. The ability of WRN to facilitate FEN-1 cleavage of DNA replication/repair intermediates may be important for the role of WRN in the maintenance of genomic stability.
D001710:Biotin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; D006801:Humans; D007700:Kinetics; D053484:RecQ Helicases; D019809:Streptavidin / Q000378:metabolism; D014898:Werner Syndrome / Q000378:metabolism; D000071657:Werner Syndrome Helicase
D001710:Biotin; D004247:DNA; D019809:Streptavidin; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase