Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.739848
Title: Mechanistic studies of 5' nucleases : the FEN superfamily
Author: Shaw, Steven J.
ISNI:       0000 0004 7230 5874
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Abstract:
The flap endonuclease superfamily of 5'-nucleases have roles in DNA replication and repair. They react with DNA via a conserved nucleolytic core, co-ordinating with divalent metal ions to hydrolyse DNA in a structure-specific manner. Two examples are: flap endonuclease 1 (FEN1), which acts in Okazaki fragment maturation (OFM) during DNA replication, and exonuclease 1 (EXO1), which is important in DNA repair pathways. Although FEN1 has been extensively studied in the past, EXO1 has not been thoroughly examined. Therefore, this project focused on better understanding EXO1 by exploring commonalities and differences to FEN1. EXO1 is primarily an exonuclease, removing nucleotides from dsDNA ends. This thesis describes biochemical analyses of the exonucleolytic capabilities of human EXO1 (hEXO1). Establishment of the enzymes substrate specificity, combined with determination of the kinetic parameters, allowed for characterisation of features of its reaction mechanism. In particular, experiments demonstrated that hEXO1 reactions were rate-limited by product release at high substrate concentrations. FEN1 processes endonucleolytic substrates by passing 5'-ssDNA flaps through a helical archway, which is conserved in EXO1. EXO1 also has suggested involvement in OFM; therefore, investigation of whether hEXO1 threads its endonucleolytic substrates was undertaken. Preventing or capturing the threaded state with a biotinylated substrate and streptavidin demonstrated that EXO1 must thread flapped substrates prior to catalysis. Further studies using multiple FEN1 mutants at residues expected to stabilise the threaded state, in combination with substrates with differing 5'-modifications, identified R104 and K132 as important residues for interaction with the +1 phosphate. Arch residue R129 was also identified as being required for efficient catalysis. Biophysical analyses of EXO1 by CD determined that a signal change observed for hFEN1 with substrates containing tandem 2-aminopurines was not produced with hEXO1. However, the DNA base distortion hypothesised to cause the observed shift in FEN1 crystal structures is also present in EXO1 crystals. Finally, multiple N-hydroxyurea inhibitors known to inhibit hFEN1 were shown to be non-specific to FEN1 as biochemical and biophysical techniques demonstrated interaction with and inhibition of hEXO1.
Supervisor: Grasby, Jane A. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.739848  DOI: Not available
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