Partial Functional Deficiency of E160D Flap Endonuclease-1 Mutantin Vitro and in Vivo Is Due to Defective Cleavage of DNA Substrates

Geoffrey Frank,Junzhuan Qiu,Ma Somsouk,Yehua Weng,Lina Somsouk,John P Nolan,Binghui Shen

doi:10.1074/jbc.273.49.33064

Geoffrey Frank, Junzhuan Qiu + Show 5 more

Open Access

https://doi.org/10.1074/jbc.273.49.33064

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Abstract

To assess the roles of the active site residues Glu160 and Asp181 of human FEN-1 nuclease in binding and catalysis of the flap DNA substrate and in vivo biological processes of DNA damage and repair, five different amino acids were replaced at each site through site-directed mutagenesis of the FEN-1 gene. The mutants were then expressed in Escherichia coli and purified using a His-tag. Even though the mutants bind to the flap DNA to different degrees, most of the mutants lost flap nuclease activity with the exception of an E160D mutant. This mutant retained wild type-like binding ability, specificity, and partial catalytic activity. Detailed steady state and pre-steady state kinetic analysis revealed that the functional deficiency of this mutant was due to retardation of the endonucleolytic cleavage. When the mutant enzyme E160D was expressed in yeast, it partially complements the biological functions of the homologous yeast gene, RAD27, and reverses the hyper-temperature lethality and hypersensitivity to methyl methanesulfonate, in a manner corresponding to the in vitro activity.

Highlights

To assess the roles of the active site residues Glu160 and Asp181 of human FEN-1 nuclease in binding and catalysis of the flap DNA substrate and in vivo biological processes of DNA damage and repair, five different amino acids were replaced at each site through sitedirected mutagenesis of the FEN-1 gene
In the polymerase ␤-dependent pathway, FEN-1 is functional without proliferating cell nuclear antigen and replification factor C but requires DNA synthesis, which leads to a flap structure formation
By comparing the two conserved regions of 6 eukaryotic FEN-1/XPG family members, 5 archaebacterial homologs, 6 5Ј-nuclease domains of prokaryotic DNA polymerases, and 4 bacteriophage exonucleases, the amino acid residues that we identified to be essential for DNA substrate binding and cleavage are strictly conserved in this extended family

Summary

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol 273, No 49, Issue of December 4, pp. 33064 –33072, 1998 Printed in U.S.A. Partial Functional Deficiency of E160D Flap Endonuclease-1 Mutant in Vitro and in Vivo Is Due to Defective Cleavage of DNA Substrates*. To assess the roles of the active site residues Glu160 and Asp181 of human FEN-1 nuclease in binding and catalysis of the flap DNA substrate and in vivo biological processes of DNA damage and repair, five different amino acids were replaced at each site through sitedirected mutagenesis of the FEN-1 gene. In the polymerase ␤-dependent pathway, FEN-1 is functional without proliferating cell nuclear antigen and replification factor C but requires DNA synthesis, which leads to a flap structure formation. By comparing the two conserved regions of 6 eukaryotic FEN-1/XPG family members, 5 archaebacterial homologs, 6 5Ј-nuclease domains of prokaryotic DNA polymerases, and 4 bacteriophage exonucleases, the amino acid residues that we identified to be essential for DNA substrate binding and cleavage are strictly conserved in this extended family. Can the hydroxyl group in the original residues be replaced or partially replaced by a hydroxyl group from any other amino acid residues? What is the effect of the carbon chain length, Asp versus Glu?

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION