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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Calcium-Binding Proteins / biosynthesis
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Calcium-Binding Proteins / chemistry*
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Calcium-Binding Proteins / genetics
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DNA Repair
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Deoxyadenine Nucleotides / biosynthesis
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Deoxyadenine Nucleotides / chemistry*
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Deoxyadenine Nucleotides / genetics
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Deoxyribonucleotides / biosynthesis
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Deoxyribonucleotides / chemistry*
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Deoxyribonucleotides / genetics
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Eye Proteins / biosynthesis
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Eye Proteins / chemistry*
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Eye Proteins / genetics
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Genetic Complementation Test
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Humans
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Kinetics
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Membrane Proteins / biosynthesis
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Membrane Proteins / chemistry*
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Membrane Proteins / genetics
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Membrane Transport Proteins / biosynthesis
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Membrane Transport Proteins / chemistry*
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Membrane Transport Proteins / genetics
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Mice
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Phenotype
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Pyrophosphatases / biosynthesis
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Pyrophosphatases / chemistry*
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Pyrophosphatases / genetics
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Saccharomyces cerevisiae Proteins / biosynthesis
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / genetics
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Sequence Homology, Amino Acid
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Substrate Specificity / genetics
Substances
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Calcium-Binding Proteins
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Deoxyadenine Nucleotides
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Deoxyribonucleotides
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Eye Proteins
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Membrane Proteins
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Membrane Transport Proteins
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PITPNC1 protein, human
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Pitpnm protein, mouse
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Saccharomyces cerevisiae Proteins
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6-N-hydroxylaminopurine deoxynucleoside triphosphate
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Pyrophosphatases