TY - JOUR
T1 - Construction and expression of a chimeric gene encoding human terminal deoxynucleotidyltransferase and DNA polymerase β
AU - Quintana-Hau, Juan D.
AU - Uribe-Luna, Sonia
AU - Espinosa-Lara, Mercedes
AU - Maldonado-Rodriguez, Rogelio
AU - Logsdon, Naomi
AU - Beattie, Kenneth L.
N1 - Funding Information:
This researchw ass upportedb y Grant GM25530f rom the National Instituteso f Health and by a grant from the Stateo f TexasH igher EducationC oordinatingB oard AdvancedT echnologyP rogram.The authorst hank Dr. Ross Andersonf or informationa bout TdT recombinant DNAs and assays.
PY - 1995/10/3
Y1 - 1995/10/3
N2 - A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Polβ and TdT to investigate the region of Polβ required for template utilization. Site-directed mutagenesis and recombinant DNA procedures were used for construction of a gene encoding a chimeric form of the two enzymes and termed TDT::POLB, in which the DNA region encoding amino acids (aa) 154-212 of TdT was replaced by the corresponding region encoding aa 1-60 of Polβ. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of Polβ represents most of the N-terminal domain of the enzyme possessing single-stranded DNA (ssDNA)-binding activity, it was hypothesized that the chimeric protein, unlike TdT, might possess template-dependent DNA polymerase activity. The chimeric gene product was produced in Escherichia coli, purified and subjected to preliminary enzymological characterization. The finding that the chimeric TdT::Polβ protein possessed significant template-dependent polymerase activity suggests that aa 1-60 of Polβ are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Polβ possesses ssDNA-binding activity [Kumar ., J. Biol. Chem. 265 (1990a) 2124-2131; Kumar ., Biochemistry 29 (1990b) 7156-7159; Prasad ., J. Biol. Chem. 268 (1993) 22746-22755].
AB - A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Polβ and TdT to investigate the region of Polβ required for template utilization. Site-directed mutagenesis and recombinant DNA procedures were used for construction of a gene encoding a chimeric form of the two enzymes and termed TDT::POLB, in which the DNA region encoding amino acids (aa) 154-212 of TdT was replaced by the corresponding region encoding aa 1-60 of Polβ. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of Polβ represents most of the N-terminal domain of the enzyme possessing single-stranded DNA (ssDNA)-binding activity, it was hypothesized that the chimeric protein, unlike TdT, might possess template-dependent DNA polymerase activity. The chimeric gene product was produced in Escherichia coli, purified and subjected to preliminary enzymological characterization. The finding that the chimeric TdT::Polβ protein possessed significant template-dependent polymerase activity suggests that aa 1-60 of Polβ are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Polβ possesses ssDNA-binding activity [Kumar ., J. Biol. Chem. 265 (1990a) 2124-2131; Kumar ., Biochemistry 29 (1990b) 7156-7159; Prasad ., J. Biol. Chem. 268 (1993) 22746-22755].
KW - DNA polymerizing enzyme
KW - domain substitution
KW - protein engineering
KW - recombinant DNA
UR - http://www.scopus.com/inward/record.url?scp=0028972365&partnerID=8YFLogxK
U2 - 10.1016/0378-1119(95)00291-D
DO - 10.1016/0378-1119(95)00291-D
M3 - Artículo
SN - 0378-1119
VL - 163
SP - 289
EP - 294
JO - Gene
JF - Gene
IS - 2
ER -