A SINGLE SIDE-CHAIN PREVENTS ESCHERICHIA-COLI DNA-POLYMERASE-I (KLENOW FRAGMENT) FROM INCORPORATING RIBONUCLEOTIDES

Although nucleic acid polymerases from different families shaw strikin g similarities in structure, they maintain stringent specificity for t he sugar structure of the incoming nucleoside triphosphate, The Klenow fragment of E. coli DNA polymerase I selects its natural substrates, deoxynucleotides, over ribonucleotides by several thousand fold. Analy sis of mutant Klenow fragment-derivatives indicates that discriminatio n is provided by the Glu-710 side chain which sterically blacks the 2' -OH of an incoming rNTP. A nearby aromatic side chain, at position 762 , plays an important role in constraining the nucleotide so that the G lu-710 ''steric gate'' fan be fully effective. Even with the E710A mut ation, which is extremely permissive for addition of a single ribonucl eotide to a DNA primer, Klenow fragment does not efficiently synthesiz e pure RNA, indicating that additional barriers prevent the incorporat ion of successive ribonucleotides.