A useful strategy to construct DNA polymerases with different properties by using genetic resources from environmental DNA.
DNA polymerases synthesize new DNA strands according to the template DNA, using deoxynucleotide triphosphates during DNA replication and repair, and are essential to maintain genome integrity in DNA metabolism. In addition, these enzymes are widely used for genetic engineering techniques, including dideoxy-sequencing, PCR, DNA labeling, mutagenesis, and other in vitro experiments. Thermostable DNA polymerases are especially useful for PCR and cycle-sequencing. We propose a powerful strategy using environmental DNA as a genetic resource to investigate the structure-function relationships of the family B DNA polymerases. The region corresponding to the active center of the DNA polymerizing reaction in the structural gene of P. furiosus DNA polymerase I (PolBI) was substituted by PCR fragments amplified from DNAs within soil samples from various locations in Japan. The chimeric pol genes were constructed within the PolBI expression plasmid. The chimeric enzymes thus produced revealed DNA polymerase activities with different properties.
Matsukawa
Hiroaki
H
Department of Genetic Resources Technology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.
Yamagami
Takeshi
T
Kawarabayasi
Yutaka
Y
Miyashita
Yukiko
Y
Takahashi
Masaaki
M
Ishino
Yoshizumi
Y
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D019843:Archaeal Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D004256:DNA Polymerase I