THERMOSTABLE BST DNA-POLYMERASE-I LACKS A 3'-]5'-PROOFREADING EXONUCLEASE ACTIVITY

A thermostable DNA polymerase, the Bst DNA polymerase I, from Bacillus stearothermophilus N3468 was prepared to near-homogeneity. The domina nt species of the Bsi DNA polymerase I preparation sized about 97 kDa when analyzed on SDS polyacrylamide gels. The Bst pol A gene that code s for Bst polymerase I was cloned and sequenced. Comparative sequence analysis showed that all three conserved 3'-->5' exonuclease motifs fo und in E. coli DNA polymerase I were missing in Bst DNA polymerase I. This cast doubt on the existence of a 3'-->5' exonuclease function in that enzyme. Four biochemical assays were used to measure exonuclease activities of Bst DNA polymerase I, testing both full-length Bst polym erase I and the Bst large fragment which lacks the N-terminal 5'-->3' exonuclease domain. These exonuclease assays demonstrated thar Bst DNA polymerase I only contained a double-strand dependent 5'-->3' exonucl ease activity but lacked any detectable 3'-->5' proofreading exonuclea se activity. The lack of 3'-->5' exonuclease Function in a variety of thermostable repair DNA polymerases may reflect enhancement of thermos tability al the expense of proofreading activity.