Substrate specificity of RdgB protein, a deoxyribonucleoside triphosphate pyrophosphohydrolase.
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.
Burgis
Nicholas E
NE
Department of Biological Sciences, University at Albany, State University of New York, Albany, New York 12222, USA.
Cunningham
Richard P
RP
D000818:Animals; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004260:DNA Repair; D003838:Deoxyadenine Nucleotides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D003854:Deoxyribonucleotides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005136:Eye Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D010641:Phenotype; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics
D002135:Calcium-Binding Proteins; D003838:Deoxyadenine Nucleotides; D003854:Deoxyribonucleotides; D005136:Eye Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C107847:PITPNC1 protein, human; C485161:Pitpnm protein, mouse; D029701:Saccharomyces cerevisiae Proteins; C047890:6-N-hydroxylaminopurine deoxynucleoside triphosphate; D011755:Pyrophosphatases