Flap endonuclease 1 mechanism analysis indicates flap base binding prior to threading.
FEN1 cleaves 5' flaps at their base to create a nicked product for ligation. FEN1 has been reported to enter the flap from the 5'-end and track to the base. Current binding analyses support a very different mechanism of interaction with the flap substrate. Measurements of FEN1 binding to a flap substrate show that the nuclease binds with similar high affinity to the base of a long flap even when the 5'-end is blocked with biotin/streptavidin. However, FEN1 bound to a blocked flap is more sensitive to sequestration by a competing substrate. These results are consistent with a substrate interaction mechanism in which FEN1 first binds the flap base and then threads the flap through an opening in the protein from the 5'-end to the base for cleavage. Significantly, when the unblocked flap length is reduced from five to two nucleotides, FEN1 can be sequestered from the substrate to a similar extent as a blocked, long flap substrate. Apparently, interactions related to threading occur only when the flap is greater than two to four nucleotides long, implying that short flaps are cleaved without a threading requirement.
Gloor
Jason W
JW
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Balakrishnan
Lata
L
Bambara
Robert A
RA
D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045585:Flap Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
D029968:Escherichia coli Proteins; D004247:DNA; D045585:Flap Endonucleases