Novel thermophilic polymerase III holoenzyme

Abstract

The present invention relates to gene and amino acid sequences encoding DNA polymerase III holoenzyme subunits and structural genes from thermophilic organisms. In particular, the present invention provides DNA polymerase III holoenzyme subunits and accessory proteins of T. thermophilus . The present invention also provides antibodies, primers, probes, and other reagents useful to identify DNA polymerase III molecules.

What is claimed is:  
     
         1 . An isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (uvrD helicase) 68.  
     
     
         2 . The polypeptide of  claim 1  wherein said polypeptide has the amino acid sequence of SEQ ID NO: 68.  
     
     
         3 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide of  claim 1 .  
     
     
         4 . The isolated polynucleotide molecule of  claim 3  comprising a nucleic acid having the sequence of SEQ ID NO: 67.  
     
     
         5 . A vector comprising an isolated polynucleotide of  claim 3 .  
     
     
         6 . A host cell comprising a vector of  claim 5 .  
     
     
         7 . The isolated polypeptide of  claim 1  wherein said polypeptide is a uvrD helicase from a thermophilic organism.  
     
     
         8 . The isolated polypeptide of  claim 7  wherein said thermophilic organism is  Thermus thermophilus.    
     
     
         9 . An isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (DNA-G Primase) 72.  
     
     
         10 . The polypeptide of  claim 9  wherein said polypeptide has the amino acid sequence of SEQ ID NO: 72.  
     
     
         11 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide of  claim 9 .  
     
     
         12 . The isolated polynucleotide molecule of  claim 11  comprising a nucleotide sequence having the sequence of SEQ ID NO: 71.  
     
     
         13 . A vector comprising an isolated polynucleotide of  claim 11 .  
     
     
         14 . A host cell comprising a vector of  claim 13 .  
     
     
         15 . The isolated polypeptide of  claim 9  wherein said polypeptide is a DNA G primase from a thermophilic organism.  
     
     
         16 . The isolated polypeptide of  claim 15  wherein said thermophilic organism is  Thermus thermophilus.    
     
     
         17 . An isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (priA helicase) 76.  
     
     
         18 . The polypeptide of  claim 17  wherein said polypeptide has the amino acid sequence of SEQ ID NO: 76.  
     
     
         19 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide of  claim 17 .  
     
     
         20 . The isolated polynucleotide molecule of  claim 19  comprising a nucleotide sequence having the sequence of SEQ ID NO: 75.  
     
     
         21 . A vector comprising an isolated polynucleotide of  claim 19 .  
     
     
         22 . A host cell comprising a vector of  claim 21 .  
     
     
         23 . The isolated polypeptide of  claim 17  wherein said polypeptide is a priA helicase from a thermophilic organism.  
     
     
         24 . The isolated polypeptide of  claim 23  wherein said thermophilic organism is  Thermus thermophilus.    
     
     
         25 . An isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (delta subunit) 10.  
     
     
         26 . The polypeptide of  claim 25  wherein said polypeptide has the amino acid sequence of SEQ ID NO: 10.  
     
     
         27 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide of  claim 25 .  
     
     
         28 . The isolated polynucleotide molecule of  claim 27  comprising a nucleotide sequence having the sequence of SEQ ID NO: 9.  
     
     
         29 . A vector comprising an isolated polynucleotide of  claim 27 .  
     
     
         30 . A host cell comprising a vector of  claim 29 .  
     
     
         31 . The isolated polypeptide of  claim 25  wherein said polypeptide is a delta subunit from a thermophilic organism.  
     
     
         32 . The isolated polypeptide of  claim 31  wherein said thermophilic organism is  Thermus thermophilus.    
     
     
         33 . An isolated antibody molecule, where in said antibody specifically binds to at least one antigenic determinant on a polypeptide of  claim 25 .  
     
     
         34 . An isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (delta prime subunit) 17.  
     
     
         35 . The polypeptide of  claim 34  wherein said polypeptide has the amino acid sequence of SEQ ID NO: 17.  
     
     
         36 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide of  claim 34 .  
     
     
         37 . The isolated polynucleotide molecule of  claim 36  comprising a nucleotide sequence having the sequence of SEQ ID NO: 16.  
     
     
         38 . A vector comprising an isolated polynucleotide of  claim 36 .  
     
     
         39 . A host cell comprising a vector of  claim 38 .  
     
     
         40 . The isolated polypeptide of  claim 34  wherein said polypeptide is a delta prime subunit from a thermophilic organism.  
     
     
         41 . The isolated polypeptide of  claim 40  wherein said thermophilic organism is  Thermus thermophilus.    
     
     
         42 . An isolated antibody molecule, wherein said antibody specifically binds to at least one antigenic determinant on a polypeptide of  claim 37 .  
     
     
         43 . An isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (beta subunit) 23.  
     
     
         44 . The polypeptide of  claim 43  wherein said polypeptide has the amino acid sequence of SEQ ID NO: 23.  
     
     
         45 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide of  claim 43 .  
     
     
         46 . The isolated polynucleotide molecule of  claim 45  comprising a nucleotide sequence having the sequence of SEQ ID NO: 22.  
     
     
         47 . A vector comprising an isolated polynucleotide of  claim 45 .  
     
     
         48 . A host cell comprising a vector of  claim 47 .  
     
     
         49 . The isolated polypeptide of  claim 43  wherein said polypeptide is a beta subunit from a thermophilic organism.  
     
     
         50 . The isolated polypeptide of  claim 49  wherein said thermophilic organism is  Thermus thermophilus.    
     
     
         51 . An isolated antibody molecule, wherein said antibody specifically binds to at least one antigenic determinant on a polypeptide of  claim 43 .  
     
     
         52 . An isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (ssb protein) 32.  
     
     
         53 . The polypeptide of  claim 52  wherein said polypeptide has the amino acid sequence of SEQ ID NO: 32.  
     
     
         54 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide of  claim 52 .  
     
     
         55 . The isolated polynucleotide molecule of  claim 54  comprising a nucleotide sequence having the sequence of SEQ ID NO: 31.  
     
     
         56 . A vector comprising an isolated polynucleotide of  claim 54 .  
     
     
         57 . A host cell comprising a vector of  claim 56 .  
     
     
         58 . The isolated polypeptide of  claim 52  wherein said polypeptide is an SSB protein from a thermophilic organism.  
     
     
         59 . The isolated polypeptide of  claim 58  wherein said thermophilic organism is  Thermus thermophilus.    
     
     
         60 . An isolated antibody molecule, where in said antibody specifically binds to at least one antigenic determinant on a polypeptide of  claim 55 .  
     
     
         61 . An isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (epsilon 1, dnaQ-1) 37.  
     
     
         62 . The polypeptide of  claim 61  wherein said polypeptide has the amino acid sequence of SEQ ID NO: 37.  
     
     
         63 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide of  claim 61 .  
     
     
         64 . The isolated polynucleotide molecule of  claim 63  comprising a nucleotide sequence having the sequence of SEQ ID NO: 36.  
     
     
         65 . A vector comprising an isolated polynucleotide of  claim 63 .  
     
     
         66 . A host cell comprising a vector of  claim 65 .  
     
     
         67 . The isolated polypeptide of  claim 61  wherein said polypeptide is an epsilon subunit from a thermophilic organism.  
     
     
         68 . The isolated polypeptide of  claim 67  wherein said thermophilic organism is  Thermus thermophilus.    
     
     
         69 . An isolated antibody molecule, where in said antibody specifically binds to at least one antigenic determinant on a polypeptide of  claim 61 .  
     
     
         70 . An isolated polypeptide wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: (dnaQ-2 protein) 82.  
     
     
         71 . The polypeptide of  claim 70  wherein said polypeptide has the amino acid sequence of SEQ ID NO: 82.  
     
     
         72 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide of  claim 70 .  
     
     
         73 . The isolated polynucleotide molecule of  claim 72  comprising a nucleotide sequence having the sequence of SEQ ID NO: 81.  
     
     
         74 . A vector comprising an isolated polynucleotide of  claim 72 .  
     
     
         75 . A host cell comprising a vector of  claim 74 .  
     
     
         76 . The isolated polypeptide of  claim 70  wherein said polypeptide is an epsilon-2 subunit from a thermophilic organism.  
     
     
         77 . The isolated polypeptide of  claim 76  wherein said thermophilic organism is  Thermus thermophilus.    
     
     
         78 . An isolated antibody molecule, where in said antibody specifically binds to at least one antigenic determinant on a polypeptide of  claim 73 .  
     
     
         79 . A method of producing a polypeptide encoded by a nucleotide sequence, wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of one of SEQ ID NOS: 68, 72, 76, 10, 17, 23, 32, 37, and 82, comprising culturing a host cell comprising said nucleotide sequence under conditions such that said polypeptide is expressed, and recovering said polypeptide.  
     
     
         80 . A method of synthesizing DNA which comprises utilizing one or more polypeptides, said one or more polypeptides comprising an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 68, 72, 76, 10, 17, 23, 32, 37 and 82.  
     
     
         81 . The method of  claim 80  further comprising providing in any order: a reaction mixture comprising components comprising template, and nucleotides, and incubating said reaction mixture for a length of time and at a temperature sufficient to obtain DNA synthesis.  
     
     
         82 . The method of  claim 81  wherein said one or more polypeptides further comprises an N-terminal linked peptide or a C-terminal linked peptide.