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1、Major Contents of Lecture 2,Enzymes and other proteins in DNA replication system DNA polymerase I, II, and III in prokaryotes Five types of DNA polymerases in eukaryotes Helicase, DnaA, DnaC, SSB, primase, ligase, topoisomerase in prokaryotes,DNA-pol I isolated from E. coli in 1956.,There are three
2、kinds of DNA Polymerases in prokaryotes.,Klenow fragment,A-F segments (323 AA): the small fragment, having 53exonuclease activity G-R segments (604 AA): the large fragment called Klenow fragment, having DNA polymerase and 35exonuclease activity, a useful tool enzyme,35 exonuclease activity excise mi
3、smatched nuleotides,53 exonuclease activity cut primer or excise mutated segment,？,Exonuclease Activities of DNA Pol.I,Two metal ions participate in the active site of DNA polymerase.,Klenow Fragment Polymerization center,Base Selection of DNA polymerases,Two residues of DNA polymerases interact wit
4、h minor groove by two hydrogen bonds to watson-crick base pair in the active site.,Base Selection of DNA polymerases,Only the proper shaped base pairs will readily fit the tight pocket.,DNA-pol of E. coli,DNA-pol III,The core enzyme 5 3 polymerase 3 5 exonuclease base selection heterodimerization su
5、bunits “clamp” -complex,The 2 serve as a sliding DNA clamp.,DNA-pol in procaryotes,DNA-pol in Eukaryotes,DNA-pol : elongation,DNA-pol III Helicase,DNA-pol : initiate replication and synthesize primers,DnaG, primase,DNA-pol : replication with a low fidelity,DNA-pol : DNA replication in mitochondria,D
6、NA-pol : proofreading and gap filling,DNA-pol I,SOS Repair DNA-pol II,Helicase in E. coli,DnaB It opens the double strand DNA with ATP consumption. It needs the help from DnaA and DnaC. DnaA - recognize the origin DnaC - transport and assist DnaB,PcrA contains four domains A1, A2, B1, B2. ATP bindin
7、g to A1 closes the cleft. Then ATP hydrolysis pushes B1 and B2 towards the double stranded part and the cleft open up again.,Helicases PcrA hydrolyze ATP to power the strand separation.,The Hexameric Helicase from Bacteriophage T7,SSB protein (Single strand DNA binding protein, SSB) may bind and sta
8、blize DNA in the single-strand form. Prevent the dsDNA formation. Protect the vulnerable ssDNA from nucleases.,Primases catalyze the synthesis of RNA primer.,DnaG in E Coli Using free NTPs to synthesize short RNA primers.,DNA Ligases seal the break.,Necessary for Replication Repair Recombination,Top
9、oisomerases prepare DNA duplex ready for unwinding by phosphodiester bond cutting and ligation.,Also called -protein in prokaryotes It cuts a phosphoester bond on one DNA strand (i.e., a single-strand break), rotates the broken DNA freely around the other strand to release the constraint, and then r
10、eseals the cut. No need of ATP Relax supercoiled structures.,Topoisomerase I (topo I),It is named gyrase in prokaryotes. It cuts phosphoester bonds on both strands (i.e., a double-strand break) of dsDNA, introduce the negative supercoil, and then forms the phosphoester bonds again. It can change dsD
11、NA into the negative-supercoil state with consumption of ATP. Introduce negative supercoils.,Topoisomerase II (topo II),Right or Wrong,DNA polymerase III has activities of polymerase and 53 exonuclease. Formation of 35 phosphodiester bonds can be catalyzed by DNA polymerase, helicase, ligase, and to
12、poisomerase. Enzymes essential for DNA replication in prokaryotes include DNA polymerase III, helicase, SSB, DNA polymerase I, ligase, and so on. Topological changes of parent DNA duplex during DNA replication consume energy from ATP hydrolysis.,Question,Which structure do DNA molecules mostly keep
13、in living cells? B-double helix A-double helix Z-double helix Negative supercoils Positive supercoils,Terms and Concepts,Klenow fragment,Questions,Please summarize activities and functions of three types of prokaryotic DNA polymerases in a table. Please list functions of five important eukaryotic DNA polymerase and names of their corresponding prokaryotic DNA polymerases. Which enzymes and non-enzyme proteins cooperate to replicate DNA in E. coli?,
