Nucleic acid encoding Bacillus stearothermophilus delta prime polymerase subunit

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 DNA molecule from a  Bacillus  species encoding a delta prime subunit of a DNA polymerase III-type enzyme, the isolated DNA molecule either: 
 (i) comprising a nucleotide sequence of SEQ ID NO: 179;    (ii) encoding an amino acid sequence of SEQ ID NO: 180; or    (iii) hybridizing to the complement of SEQ ID NO: 179 under hybridization conditions comprising at most about 0.9M sodium citrate buffer at a temperature of at least about 37° C.    
     
     
         2 . The isolated DNA molecule according to  claim 1 , wherein the  Bacillus  species is  Bacillus stearothermophilus.    
     
     
         3 . The isolated DNA molecule according to  claim 1 , wherein the DNA molecule encodes an amino acid sequence of SEQ ID NO: 180.  
     
     
         4 . The isolated DNA molecule according to  claim 1 , wherein the DNA molecule comprises a nucleotide sequence of SEQ ID NO: 179.  
     
     
         5 . The isolated DNA molecule according to  claim 1 , wherein the DNA molecule hybridizes to the complement of SEQ ID NO: 179 under hybridization conditions comprising at most about 0.9M sodium citrate buffer at a temperature of at least about 37° C.  
     
     
         6 . An expression system comprising an expression vector into which is inserted a heterologous DNA molecule according to  claim 1 .  
     
     
         7 . A host cell comprising a heterologous DNA molecule according to  claim 1 .  
     
     
         8 . A method of producing a recombinant thermostable delta prime subunit of a DNA polymerase III-type enzyme from a  Bacillus  species, said method comprising: 
 transforming a host cell with the heterologous DNA molecule according to  claim 1  under conditions suitable for expression of the delta prime subunit, and    isolating the delta prime subunit.    
     
     
         9 . An isolated DNA molecule from  Bacillus stearothermophilus  encoding a delta prime subunit of a DNA polymerase III enzyme, wherein the delta prime subunit is capable of forming a portion of a clamp loader that can cooperate with a DNA polymerase to form a DNA polymerase III-like particle.