title: Serial intermediates with a 1 nt 3'-flap and 5' variable-length flaps are formed by cooperative functioning of Pyrococcus horikoshii FEN-1 with either B or D DNA polymerases.
abstract: Flap endonuclease-1 (FEN-1) plays important roles with DNA polymerases in DNA replication, repair and recombination. FEN-1 activity is elevated by the presence of a 1 nucleotide expansion at the 3' end in the upstream primer of substrates called "structures with a 1 nt 3'-flap", which appear to be the most preferable substrates for FEN-1; however, it is unclear how such substrates are generated in vivo. Here, we show that substrate production occurred by the cooperative function of FEN-1(phFEN-1) and Pyrococcus horikoshii DNA polymerase B (phPol B) or D (phPol D). Using various substrates, the activities of several phFEN-1 F79 mutants were compared with those of the wild type. Analysis of the activity profiles of these mutants led us to discriminate "structures with a 1 nt 3'-flap" from substrates with a 3' -projection longer than 2 nt or from those without a 3'-projection. When phFEN-1 processed a gap substrate with phPol B or phPol D, "structures with a 1 nt 3'-flap" were assumed the reaction intermediates. Furthermore, the phFEN-1 cleavage products with phPol B or D were from 1mer to 7mer, corresponding to the sizes of the strand-displacement products of these polymerases. This suggests that a series of 1 nt 3'-flap with 5'-variable length-flap configurations were generated as transient intermediates, in which the length of the 5'-flaps depended on the displacement distance of the downstream strand by phPol B or D. Therefore, phFEN-1 might act successively on displaced 5'-variable flaps.
authors: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1 Central 6-9, Tsukuba, Ibaraki, 305-8566, Japan, eriko-matsui@aist.go.jp.
authors: Eriko
authors: E
authors: Matsui
authors: Yuji
authors: Y
authors: Urushibata
authors: Junko
authors: J
authors: Abe
authors: Ikuo
authors: I
authors: Matsui
mesh_terms: D019843:Archaeal Proteins / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D019641:DNA, Archaeal / Q000378:metabolism*; D045585:Flap Endonucleases / Q000378:metabolism*; D043902:Pyrococcus horikoshii / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D045585:Flap Endonucleases