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Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε

To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644...

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Detalles Bibliográficos
Autores principales: Pursell, Zachary F., Isoz, Isabelle, Lundström, Else-Britt, Johansson, Erik, Kunkel, Thomas A.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1888828/
https://www.ncbi.nlm.nih.gov/pubmed/17452367
http://dx.doi.org/10.1093/nar/gkm132
Descripción
Sumario:To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol ε synthesize DNA with high fidelity, but M644F Pol ε has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol ε. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol α (L868M/F) and Pol δ (L612M), these data indicate that the active site location occupied by Met644 in Pol ε is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol ε is distinct from that of L868M/F Pol α or L612M Pol δ, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo.