Biomaterial Database

dNTP binding site in rat DNA polymerase beta revealed by controlled proteolysis and azido photoprobe cross-linking. 1996

D K Srivastava, and R K Evans, and A Kumar, and W A Beard, and S H Wilson

Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1068, USA.

Mild proteolysis of rat DNA polymerase beta (beta-pol) generates an N-terminal 8 kDa domain and a C-terminal 31 kDa domain; the 31 kDa domain is degraded to 6 and 27 kDa fragments by further proteolysis [Kumar, A., Widen, S.G., Williams, K.R., Kedar, P., Karpel, R.L., & Wilson S.H. (1990) J. Biol. Chem. 265, 2124-2131]. In the present study, we found that more vigorous trypsin digestion of the 27 kDa fragment of beta-pol produces 10 and 12 kDa subdomains. Thus, rat beta-pol has four distinct proteolytic fragments of 8, 6, 10, and 12 kDa, extending from the N-terminus to the C-terminus, respectively. To map the location of the dNTP binding site(s), intact beta-pol was photoaffinity labeled with 8-azido-ATP or 5-azido-dUTP in presence or absence of competitor dNTP (dATP). The labeled enzyme was subjected to controlled proteolysis, and the resulting labeled peptides were separated and sequenced. Competition with dATP showed that three regions of beta-pol in solution combine to form the dNTP binding pocket as follows: residues 4-40 of the 8 kDa domain; residues 142-206 of the 10 kDa subdomain; and residues 263-280 of the 12 kDa subdomain (alpha-helices M and N). These results are discussed in light of the recent crystal structure of dATP bound to rat beta-pol.

UI	MeSH Term	Description	Entries
D008958	Models, Molecular	Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures.	Molecular Models,Model, Molecular,Molecular Model
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D010446	Peptide Fragments	Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.	Peptide Fragment,Fragment, Peptide,Fragments, Peptide
D010782	Photolysis	Chemical bond cleavage reactions resulting from absorption of radiant energy.	Photodegradation
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D003432	Cross-Linking Reagents	Reagents with two reactive groups, usually at opposite ends of the molecule, that are capable of reacting with and thereby forming bridges between side chains of amino acids in proteins; the locations of naturally reactive areas within proteins can thereby be identified; may also be used for other macromolecules, like glycoproteins, nucleic acids, or other.	Bifunctional Reagent,Bifunctional Reagents,Cross Linking Reagent,Crosslinking Reagent,Cross Linking Reagents,Crosslinking Reagents,Linking Reagent, Cross,Linking Reagents, Cross,Reagent, Bifunctional,Reagent, Cross Linking,Reagent, Crosslinking,Reagents, Bifunctional,Reagents, Cross Linking,Reagents, Cross-Linking,Reagents, Crosslinking
D003854	Deoxyribonucleotides	A purine or pyrimidine base bonded to a DEOXYRIBOSE containing a bond to a phosphate group.	Deoxyribonucleotide
D004256	DNA Polymerase I	A DNA-dependent DNA polymerase characterized in prokaryotes and may be present in higher organisms. It has both 3'-5' and 5'-3' exonuclease activity, but cannot use native double-stranded DNA as template-primer. It is not inhibited by sulfhydryl reagents and is active in both DNA synthesis and repair.	DNA Polymerase alpha,DNA-Dependent DNA Polymerase I,Klenow Fragment,DNA Pol I,DNA Dependent DNA Polymerase I,Polymerase alpha, DNA
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs