Aquifex aeolicus delta prime polymerase subunit 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  Aquifex  delta prime subunit of a DNA polymerase III-type enzyme, the isolated delta prime subunit: 
 (i) comprising the amino acid sequence of SEQ ID NO: 126; or    (ii) being encoded by a nucleic acid molecule hybridizing to the complement of SEQ ID NO: 125 under hybridization conditions comprising at most about 0.9M sodium citrate buffer at a temperature of at least about 37° C.    
     
     
         2 . The isolated  Aquifex  delta prime subunit according to  claim 1  wherein the  Aquifex  species is  Aquifex aeolicus.    
     
     
         3 . The isolated  Aquifex  delta prime subunit according to  claim 2  wherein the delta prime subunit comprises the amino acid sequence of SEQ ID NO: 126.  
     
     
         4 . The isolated  Aquifex  delta prime subunit according to  claim 1  wherein the delta prime subunit is encoded by a nucleic acid molecule that hybridizes to the complement of SEQ ID NO: 125 under hybridization conditions comprising at most about 0.9M sodium citrate buffer at a temperature of at least about 37° C.  
     
     
         5 . The isolated  Aquifex  delta prime subunit according to  claim 1  wherein the delta prime subunit is purified.  
     
     
         6 . A clamp loader complex comprising the  Aquifex  delta prime subunit according to  claim 1 .  
     
     
         7 . A DNA polymerase III-type enzyme complex comprising the clamp loader according to  claim 6 .  
     
     
         8 . 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 7.