Regional conformational flexibility couples substrate specificity and scissile phosphate diester selectivity in human flap endonuclease 1.
Nucleic Acids Res
; 46(11): 5618-5633, 2018 06 20.
Article
em En
| MEDLINE
| ID: mdl-29718417
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 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 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Endonucleases Flap
Limite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Reino Unido