Denatured toops (single-stranded Denaturation mapp ng the denatured loops are reproducible and thus can be used as points of reference DNA replication starts at specific- origins)
Denatured loops (single-stranded) Nondenatured DNA (double-stranded) Denaturation mapping: the denatured loops are reproducible and thus can be used as points of reference (DNA replication starts at specific origins)
6. The chemistry of DNA polymerization was revealed by in vitro studies using a DNA polymerase purified from E coli Arthur Kornberg purified DNA polymerase I, a monomeric 103 kDa protein, from E coli in 1955 and revealed the major features of the dna synthesis process DNA polymerase I was found to catalyze DNA polymerization in vitro in the presence of a single stranded DNA template, a preexisting primer with a free 3-oh group and the dNTPs
6. The chemistry of DNA polymerization was revealed by in vitro studies using a DNA polymerase purified from E. coli • Arthur Kornberg purified DNA polymerase I, a monomeric 103 kDa protein, from E. coli in 1955 and revealed the major features of the DNA synthesis process. • DNA polymerase I was found to catalyze DNA polymerization in vitro in the presence of a singlestranded DNA template, a preexisting primer with a free 3`-OH group and the dNTPs
The fundamental reaction for dNa synthesis is a nucleophilic attack by the 3-OH group of the growing strand on the 5-a-phosphorus of a incoming dNTP selected via base-pairing: the newly synthesized dna is al ways extended in the 5 to 3 direction
• The fundamental reaction for DNA synthesis is a nucleophilic attack by the 3`-OH group of the growing strand on the 5`-a-phosphorus of a incoming dNTP selected via base-pairing: the newly synthesized DNA is always extended in the 5` to 3` direction
Incoming O deoxynucleoside 5-triphosphate O-P-O O=P-0 0=P-O O=P-0 5 Growing O=P-0 OH OH DNA strand OH primer G T Template DNA strand 35 De
7. DNA polymerase I has 3.5, as well as 53 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 polymerization 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 has 3` 5`, as well as 5` 3` 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 polymerization 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