Bacillus stearothermophilus tau, delta, and delta prime polymerase subunits and use thereof

Abstract

The present invention relates to an isolated DNA molecule from a thermophilic bacterium which encodes a DNA polymerase III-type enzyme subunit. Also encompassed by the present invention are host cells and expression system including the heterologous DNA molecule of the present invention, as well as isolated replication enzyme subunits encoded by such DNA molecules. Also disclosed is a method of producing a recombinant thermostable DNA polymerase III-type enzyme, or subunit thereof, from a thermophilic bacterium, which is carried out by transforming a host cell with at least one heterologous DNA molecule of the present invention under conditions suitable for expression of the DNA polymerase III-type enzyme, or subunit thereof, and then isolating the DNA polymerase III-type enzyme, or subunit thereof.

1 . An isolated  Bacillus  delta or delta prime or tau subunit of a DNA polymerase III-type enzyme, the isolated delta or delta prime or tau subunit: 
 (i) comprising the amino acid sequence of SEQ ID NO: 178 or 180 or 182; or    (ii) being encoded by a nucleic acid molecule hybridizing to the complement of SEQ ID NO: 177 or 179 or 181 under hybridization conditions comprising at most about 0.9M sodium citrate buffer at a temperature of at least about 37° C.    
     
     
         2 . The isolated  Bacillus  delta or delta prime or tau subunit according to  claim 1  wherein the  Bacillus  species is  Bacillus stearothermophilus.    
     
     
         3 . The isolated  Bacillus  delta or delta prime or tau subunit according to  claim 1  wherein the delta subunit comprises the amino acid sequence of SEQ ID NO: 178.  
     
     
         4 . The isolated  Bacillus  delta or delta prime or tau subunit according to  claim 1  wherein the delta prime subunit comprises the amino acid sequence of SEQ ID NO: 180.  
     
     
         5 . The isolated  Bacillus  delta or delta prime or tau subunit according to  claim 1  wherein the tau subunit comprises the amino acid sequence of SEQ ID NO: 182.  
     
     
         6 . The isolated  Bacillus  delta or delta prime or tau subunit according to  claim 1  wherein the delta or delta prime or tau subunit is encoded by a nucleic acid molecule that hybridizes to the complement of SEQ ID NO: 177 or 179 or 181 under hybridization conditions comprising at most about 0.9M sodium citrate buffer at a temperature of at least about 37° C.  
     
     
         7 . The isolated  Bacillus  delta or delta prime or tau subunit according to  claim 1  wherein the subunit is purified.  
     
     
         8 . A clamp loader complex comprising at least one of the  Bacillus  delta or delta prime or tau subunits according to  claim 1 .  
     
     
         9 . A clamp loader complex comprising the  Bacillus  delta, delta prime, and tau subunits according to  claim 1 .  
     
     
         10 . A DNA polymerase III-type enzyme complex comprising the clamp loader according to  claim 8 .  
     
     
         11 . A DNA polymerase III-type enzyme complex comprising the clamp loader according to  claim 9 .  
     
     
         12 . A kit comprising: 
 a container that contains therein either a deoxynucleoside triphosphate or a dideoxynucleoside triphosphate; and    a container that contains therein the DNA polymerase III-type enzyme complex according to  claim 10 .    
     
     
         13 . A kit comprising: 
 a container that contains therein either a deoxynucleoside triphosphate or a dideoxynucleoside triphosphate; and    a container that contains therein the DNA polymerase III-type enzyme complex according to  claim 11.