Acidic residues critical for the activity and biological function of yeast DNA polymerase η

Christine M. Kondratick, M. Todd Washington, Satya Prakash, Louise Prakash

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39 Citations (Scopus)

Abstract

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 rad30Δ 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.

Original languageEnglish (US)
Pages (from-to)2018-2025
Number of pages8
JournalMolecular and Cellular Biology
Volume21
Issue number6
DOIs
StatePublished - Mar 2001

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DNA-Directed DNA Polymerase
Pyrimidine Dimers
Yeasts
Thymine
Mutation
Alanine
Nucleotides
Mutant Proteins
Catalysis
Hydroxyl Radical
Catalytic Domain
Proteins
Metals
Ions
Amino Acids
Rad30 protein

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Acidic residues critical for the activity and biological function of yeast DNA polymerase η. / Kondratick, Christine M.; Washington, M. Todd; Prakash, Satya; Prakash, Louise.

In: Molecular and Cellular Biology, Vol. 21, No. 6, 03.2001, p. 2018-2025.

Research output: Contribution to journalArticle

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