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Regional conformational flexibility couples substrate specificity and scissile phosphate diester selectivity in human flap endonuclease 1.


ABSTRACT: Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5'-terminus/flap through the arch and recognition of a single nucleotide 3'-flap by the ?2-?3 loop. Using NMR spectroscopy, we show that the solution conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and ?2-?3 loop are disordered without substrate. Disorder within the arch explains how 5'-flaps can pass under it. NMR and single-molecule FRET data show a shift in the conformational ensemble in the arch and loop region upon addition of DNA. Furthermore, the addition of divalent metal ions to the active site of the hFEN1-DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1-DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.

SUBMITTER: Bennet IA 

PROVIDER: S-EPMC6009646 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Regional conformational flexibility couples substrate specificity and scissile phosphate diester selectivity in human flap endonuclease 1.

Bennet Ian A IA   Finger L David LD   Baxter Nicola J NJ   Ambrose Benjamin B   Hounslow Andrea M AM   Thompson Mark J MJ   Exell Jack C JC   Shahari Nur Nazihah B Md NNBM   Craggs Timothy D TD   Waltho Jonathan P JP   Grasby Jane A JA  

Nucleic acids research 20180601 11


Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5'-terminus/flap through the arch and recognition of a single nucleotide 3'-flap by the α2-α3 loop. Using NMR spectroscopy, we show that the solution conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2-α3 loop are  ...[more]

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