Substrate Specificity of RdgB Protein, a Deoxyribonucleoside Triphosphate Pyrophosphohydrolase

Nicholas E. Burgis,Richard P. Cunningham

doi:10.1074/jbc.m608708200

Nicholas E. Burgis, Richard P. Cunningham

Open Access

https://doi.org/10.1074/jbc.m608708200

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Abstract

We have previously reported the identification of a DNA repair system in Escherichia coli for the prevention of the stable incorporation of noncanonical purine dNTPs into DNA. We hypothesized that the RdgB protein is active on 2'-deoxy-N-6-hydroxylaminopurine triphosphate (dHAPTP) as well as deoxyinosine triphosphate. Here we show that RdgB protein and RdgB homologs from Saccharomyces cerevisiae, mouse, and human all possess deoxyribonucleoside triphosphate pyrophosphohydrolase activity and that all four RdgB homologs have high specificity for dHAPTP and deoxyinosine triphosphate compared with the four canonical dNTPs and several other noncanonical (d)NTPs. Kinetic analysis reveals that the major source of the substrate specificity lies in changes in K(m) for the various substrates. The expression of these enzymes in E. coli complements defects that are caused by the incorporation of HAP and an endogenous noncanonical purine into DNA. Our data support a preemptive role for the RdgB homologs in excluding endogenous and exogenous modified purine dNTPs from incorporation into DNA.

Highlights

Phohydrolase and that it may play a role in detoxifying 2Ј-deoxy-HAP triphosphate
The data we present strongly suggest that the RdgB homologs have a primary role in excluding noncanonical purines from deoxyribonucleoside triphosphate pools, including deoxy-N-6hydroxylaminopurine triphosphate (dHAPTP)
Rescue of the extreme HAP-sensitive phenotype by RdgB homologs was demonstrated by constructing plasmids with the E. coli, yeast, mouse, and human homologs of RdgB cloned into a pBR322-based plasmid so that expression was driven by the E. coli endonuclease IV promoter

Summary

EXPERIMENTAL PROCEDURES

E. coli Growth Conditions—The medium used for growth was TY (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl/ liter at pH 7.2). The mouse rdgB coding sequence was amplified and cloned by reverse transcription-PCR. The amplified human rdgB coding sequence was gel-purified, digested with EcoRI and HindIII, and cloned into EcoRI/HindIII-digested pcDNA3.1Zeoϩ. Recombinant yeast, mouse, and human homologs were expressed in a similar manner in NEB195 cells (BL21 (DE3) ⌬rdgB). Analysis of cell lysate on an SDS-PAGE gel showed major bands with apparent molecular masses consistent with the predicted molecular masses of 23.9 kDa, 23.7 kDa, and 23.2 kDa for the His6-tagged recombinant yeast, mouse, and human proteins, respectively. The elutions were performed with Buffer B containing 100 mM NaCl. The eluants were analyzed by SDS gel electrophoresis and fractions displaying the highest degree of purity were pooled and dialyzed (1:250 (v/v)) overnight in appropriate buffer. Water suppression during the NMR experiment was accomplished using the water gate pulse sequence supplemented with excitation sculpting [23]

RESULTS

Homolog Substrate

DISCUSSION