7. DNA polymerase I is a 103-KDa trifunctional protein having one polymerase and two exonuclease activities The 3-5 exonuclease activity was found to be able to remove mismatched base pairs, thus to proofread the newly incorporated nucleotides (increasing the accuracy by 100 to 1000 fold) a"sliding back model was proposed for DNA polymerase I to proofread mismatched base pairs The enzyme can be cleaved into two parts by mild protease treatment: the small fragment contains the 5 to 3 exonuclease activity and the large(called the Klenow fragment) contains the rest two activities
7. DNA polymerase I is a 103-kDa trifunctional protein having one polymerase and two exonuclease activities • The 3` 5` exonuclease activity was found to be able to remove mismatched base pairs, thus to proofread the newly incorporated nucleotides (increasing the accuracy by 100 to 1000 fold). • A “sliding back” model was proposed for DNA polymerase I to proofread mismatched base pairs. • The enzyme can be cleaved into two parts by mild protease treatment: the small fragment contains the 5’ to 3’ exonuclease activity and the large (called the Klenow fragment) contains the rest two activities
The 5 to 3 exonuclease activity is unique for dNa polymerase I, enabling it to catalyze the nick translation process: an RNA or DNA strand paired to a dna template is simultaneously degraded and replaced; an activity used for both DNA repair and the removal of rna primers in DNa replication (clean-up functions ), also for incorporating radioisotope-labeled dNTPs into a DNa probe(in vitro labeling
• The 5` to 3` exonuclease activity is unique for DNA polymerase I, enabling it to catalyze the nick translation process: an RNA or DNA strand paired to a DNA template is simultaneously degraded and replaced; an activity used for both DNA repair and the removal of RNA primers in DNA replication (clean-up functions), also for incorporating radioisotope-labeled dNTPs into a DNA probe (in vitro labeling)
DNA polymerase I DNA polymerase DNA polymease I is ctive site 3-5(proofreading) proposed to slide back exonuclease active site to proofread a mismatched base pair using its 3 to 5 5-0100OH OH 3 Cis a rare tautomeric form of cytosine(C") exonuclease activity that pairs with A and Holelotoletolo is incorporated into the growing strand Before the polymerase moves on, the cytosine The mispaired undergoes a taut A OMM shift from C to C. The too o 0ONM nucleotide is removed Colololelot new nucleotide is now mispaired. DNA polymerase slides forward and resumes its The mispaired 3-OH polymerization activity end of the growing 11 strand blocks further 良 elongation. DNA -OH o000010000OH MOMM polymerase slides back CoLotolotooro to position the mispaired base in the exonuclease o00000o010L0 active site
DNA polymease I is proposed to slide back to proofread a mismatched base pair using its 3` to 5` exonuclease activity
Protease cleavage 35 kDa small Large fragment 68 KDa fragment (Klenow fragment 5′→3 3 5 Polymerase Exonuclease Exonuclease DNA polymerase I has three enzymatic activities in a single polypeptide chain, which can be cleaved into two functional parts by mild protease treatment
DNA polymerase I has three enzymatic activities in a single polypeptide chain, which can be cleaved into two functional parts by mild protease treatment. Protease cleavage 35 kDa 68 kDa
Structure of the Klenow fragment ferase of DNA polymease d onal he 3 to 5 exonuclease omaid
The 3` to 5` exonuclease domain The polymerase domain Structure of the Klenow fragment of DNA polymease I