Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair.
Flap EndoNuclease-1 (FEN-1) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair. To clarify the molecular basis of FEN-1 specificity and PCNA activation, we report here structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results. FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity. Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular beta sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity. The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites.
Chapados
Brian R
BR
Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92122, USA.
Hosfield
David J
DJ
Han
Seungil
S
Qiu
Junzhuan
J
Yelent
Biana
B
Shen
Binghui
B
Tainer
John A
JA
D019609:Archaeoglobus; D001665:Binding Sites / Q000502:physiology; D020134:Catalytic Domain / Q000502:physiology; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D045585:Flap Endonucleases / Q000737:chemistry* / Q000378:metabolism*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008968:Molecular Conformation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity / Q000502:physiology
D046911:Macromolecular Substances; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D045585:Flap Endonucleases