Acidic residues critical for the activity and biological function of yeast DNA polymerase eta.
Rad30 is a member of the newly discovered UmuC/DinB/Rad30 family of DNA polymerases. The N-terminal regions of these proteins are highly homologous, and they contain five conserved motifs, I to V, while their C-terminal regions are quite divergent. We examined the contributions of the C-terminal and N-terminal regions of Rad30 to its activity and biological function. Although deletion of the last 54 amino acids has no effect on DNA polymerase or thymine-thymine (T-T) dimer bypass activity, this C-terminal deletion-containing protein is unable to perform its biological function in vivo. The presence of a bipartite nuclear targeting sequence within this region suggests that at least one function of this portion of Rad30 is nuclear targeting. To identify the active-site residues of Rad30 important for catalysis, we generated mutations of nine acidic residues that are invariant or highly conserved among Rad30 proteins from different eukaryotic species. Mutations of the Asp30 and Glu39 residues present in motif I and of the Asp155 residue present in motif III to alanine completely inactivated the DNA polymerase and T-T dimer bypass activities, and these mutations did not complement the UV sensitivity of the rad30Delta mutation. Mutation of Glu156 in motif III to alanine confers a large reduction in the efficiency of nucleotide incorporation, whereas the remaining five Rad30 mutant proteins retain wild-type levels of DNA polymerase and T-T dimer bypass activities. From these observations, we suggest a role for the Asp30, Glu39, and Asp155 residues in the binding of two metal ions required for the reaction of the incoming deoxynucleoside 5'-triphosphate with the 3'-hydroxyl in the primer terminus, while Glu156 may participate in nucleotide binding.
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061, USA.
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Kondratick
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Washington
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Prakash
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Prakash
D000409:Alanine; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D014466:Ultraviolet Rays; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D000099270:Y-Family DNA Polymerases
D000409:Alanine; D004259:DNA-Directed DNA Polymerase; D009711:Nucleotides; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases