Structural basis of nuclear import of flap endonuclease 1 (FEN1)

Abstract

Flap endonuclease 1 (FEN1) is a member of the nuclease family and is structurally conserved from bacteriophages to humans. This protein is involved in multiple DNA-processing pathways, including Okazaki fragment maturation, stalled replication-fork rescue, telomere maintenance, long-patch base-excision repair and apoptotic DNA fragmentation. FEN1 has three functional motifs that are responsible for its nuclease, PCNA-interaction and nuclear localization activities, respectively. It has been shown that the C-terminal nuclear localization sequence (NLS) facilitates nuclear localization of the enzyme during the S phase of the cell cycle and in response to DNA damage. To determine the structural basis of the recognition of FEN1 by the nuclear import receptor importin , the crystal structure of the complex of importin with a peptide corresponding to the FEN1 NLS was solved. Structural studies confirmed the binding of the FEN1 NLS as a classical bipartite NLS; however, in contrast to the previously proposed 354KRKX 8KKK367 sequence, it is the 354KRX 10KKAK369 sequence that binds to importin . This result explains the incomplete inhibition of localization that was observed on mutating residues 365KKK367. Acidic and polar residues in the X 10 linker region close to the basic clusters play an important role in binding to importin . These results suggest that the basic residues in the N-terminal basic cluster of bipartite NLSs may play roles that are more critical than those of the many basic residues in the C - terminal basic cluster. © 2012 International Union of Crystallography. Printed in Singapore - all rights reserved.