Substrate recognition and regulation of structure-specific nucleases central to DNA replication and repair.

摘要

Flap EndoNuclease-1 (FEN-1), ribonuclease HII (RNase HII) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair. RNase HII is a ribonucleotide-specific endonuclease that cleaves the RNA portion of RNA-DNA/DNA or RNA/DNA duplexes. Structures of RNase HII reveal a novel "cap" domain that is unique to this enzyme family, and is essential for catalysis. Coupled structural, mutational and biochemical results suggest that RNase HII recognizes this substrate by sensing the unique conformation of an RNA/DNA hybrid duplex. These results suggest a broad function for RNase HII compatible with a role in DNA replication or repair. FEN-1 and PCNA are critical components of a central pathway for RNA primer removal and long patch base excision repair. During this process, PCNA coordinates the activities of FEN-1, along with pol delta and DNA ligase to prevent to release of toxic intermediates. Structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results clarify the molecular basis of FEN-1 specificity and PCNA activation. 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 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 explain how PCNA can coordinate enzyme activities within the pathway by promoting exchange of a common kinked DNA intermediate.