title: Two modes of FEN1 binding to PCNA regulated by DNA.
abstract: The FEN1 nuclease functions during Okazaki fragment maturation in the eukaryotic cell. Like many other proliferating cell nuclear antigen (PCNA)-binding proteins, FEN1 interacts with the interdomain connector loop (IDCL) of PCNA, and PCNA greatly stimulates FEN1 activity. A yeast IDCL mutant pcna-79 (IL126,128AA) failed to interact with FEN-1, but, surprisingly, pcna-79 was still very active in stimulating FEN1 activity. In contrast, a C-terminal mutant pcna-90 (PK252,253AA) showed wild-type binding to FEN1 in solution, but poorly stimulated FEN1 activity. When PCNA was loaded onto a DNA substrate coupled to magnetic beads, it stabilized retention of FEN1 on the DNA. In this DNA-dependent binding assay, pcna-79 also stabilized retention of FEN1, but pcna-90 was inactive. Therefore, in the absence of DNA, FEN1 interacts with PCNA mainly through the IDCL. However, when PCNA encircles the DNA, the C-terminal domain of PCNA rather than its IDCL is important for binding FEN1. An FF-->GA mutation in the PCNA-interaction domain of FEN1 severely decreased both modes of interaction with PCNA and resulted in replication and repair defects in vivo.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA.
authors.forename: X V
authors.initials: XV
authors.lastname: Gomes
authors.forename: P M
authors.initials: PM
authors.lastname: Burgers
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V