The screening of expression and purification conditions for replicative DNA polymerase associated B-subunits, assignment of the exonuclease activity to the C-terminus of archaeal pol D DP1 subunit.
The B-subunits of replicative DNA polymerases belong to the superfamily of calcineurin-like phosphoesterases and are conserved from Archaea to humans. Recently we and others have shown that the B-subunit (DP1) of the archaeal family D DNA polymerase is responsible for proofreading 3'-5' exonuclease activity. The similarity of B-subunit sequences implies a common fold, but since the key catalytic and metal binding residues of the phosphoesterase domain are disrupted in the eukaryotic B-subunits, their common function has not been identified. To study the structure and activities of B-subunits in more detail, we expressed 13 different recombinant B-subunits in Escherichia coli. We found that the solubility of a protein could be predicted from the calculated GRAVY score. These scores were useful for the selection of proteins for successful expression. We optimized the expression and purification of Methanocaldococcus (Methanococcus) jannaschii DP1 of DNA polymerase D (MjaDP1) and show that the protein co-purifies with a thermostable nuclease activity. Truncation of the protein indicates that the N-terminus (aa 1-134) is not needed for catalysis. The C-terminal part of the protein containing both the calcineurin-like phosphoesterase domain and the OB-fold is sufficient for the nuclease activity.
Jokela
Maarit
M
Biocenter Oulu and Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014, Oulu, Finland.
Raki
Mari
M
Heikkinen
Kaisa
K
Sepponen
Katri
K
Eskelinen
Anitta
A
Syväoja
Juhani E
JE
D001105:Archaea / Q000235:genetics / Q000378:metabolism*; D019641:DNA, Archaeal; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism*; D015751:Genes, pol; D006801:Humans; D046911:Macromolecular Substances; D017017:Methanococcus / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D019641:DNA, Archaeal; D046911:Macromolecular Substances; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases