分子生物学英文课件：lecture3 the replication of DNA

1、Chapter 8 DNA Replication,Study objectives,The Chemistry of DNA Synthesis The Mechanism of DNA Polymerase The Specialization of DNA Polymerases The Replication Fork DNA Synthesis at the Replication Fork Initiation of DNA Replication Binding and Unwinding Finishing Replication,Process,Regulation at I

2、nitiation Incorrect base pair dramatically lowers the rate of catalysis (kinetic selectivity,Distinguishing different dNTPs: kinetic selectivity,Incorrect base pair reduces the rate of catalysis dramatically , resulting in the DNA pol preferentially adding correctly base paired dNTPs,Distinguishing

3、between rNTP and dNTP by steric exclusion of rNTPs from the active site,In the DNA pol, the nucleotide-binding pocket is too small to allow the presence of a 2-OH on the incoming nucleotide. This space is occupied by two amino acids,How to reduce the discrimination of DNA pol between dNTPs and rNTPs

4、,DNA Pol resemble a hand that grips the primer-template junction,Schematic of DNA pol bound to a primer:template junction,A similar view of the T7 DNA pol bound to DNA,Figure 8-8,Thumb,Fingers,Palm,DNA Polymerase-palm domain,Contains two catalytic sites, one for addition of dNTPs and one for removal

5、 of the mispaired dNTP. The polymerization（聚合） site: (1) binds to two metal ions that alter the chemical environment around the catalytic site and lead to the catalysis. (2) Monitors the accuracy of base-pairing for the most recently added nucleotides by forming extensive hydrogen bond contacts with

6、 minor groove of the newly synthesized DNA. Exonuclease(外切酶) site/proof reading site (See proofreading 校阅,Figure 8-6,Figure 8-7,Metal ion B: interacts with the triphosphates of the incoming dNTP to neutralize their negative charge,Metal ion A：interacts with the 3-OH, resulting in reduced association

7、 between the O and H, which leaves nucleophilic 3O,DNA pol “grips”the template and the incoming dNTP when a correct base pair made A tyrsine makes stacking interactions with the base of the dNTP two charged residues associate with the triphosphate,DNA Polymerase-finger domain,Binds to the incoming d

8、NTP, encloses the correct paired dNTP to the position for catalysis Bends the template to expose the only nucleotide at the template that ready for forming base pair with the incoming nucleotide Stabilization of the pyrophosphate(焦磷酸,DNA Polymerase-thumb domain,Not directly involved in catalysis Int

9、eracts with the synthesized DNA to maintain correct position of the primer and the active site, and to maintain a strong association between DNA Pol and its substrate,DNA Pol are processive enzymes,Processivity: characteristic of enzymes that operate on polymeric substrates. The processivity of DNA

10、Pol is the average number of nucleotides added each time(1000 nt/sec) the enzyme binds a primer:template junction (varying from a few to 50,000 nucleotides). The rate of DNA synthesis is closely related to the polymerase processivity, because the rate-limiting step is the initial binding of polymera

11、se to the primer-template junction,Figure 8-9,Exonucleases proofread newly synthesized DNA,The occasional flicking of the bases into “wrong” tautomeric form results in incorrect base pair and mis-incorporation of dNTP. (10-5 mistake) The mismatched dNMP is removed by proofreading exonuclease, a part

12、 of the DNA polymerase,How does the exonucleases work,Kinetic selectivity,When an incorrect nucleotide is incorporated into DNA, the rate of DNA synthesis is reduced because of the incorrect positioning of the 3-OH. At mismatched 3-end, at least 3-4 nts of the primer is single-stranded, increases th

13、e affinity for the exonuclease active site, which removed the mismatched nt,DNA Pols are specialized for different roles in the cell,Each organism has a distinct set of different DNA Pols Different organisms have different DNA Pols A total of 5 different DNA Pol have been reported in E. coli. DNA po

14、l I Discovered by Arthur Kormberg in 1957 Utilized in filling in small DNA segments during replication and repair processes. DNA pol II Serve as an alternative repair polymerase; it can also replicate DNA when the template is damaged. DNA pol III The primary polymerase during normal DNA replication.

15、 DNA pol IV: functions in DNA repair DNA pol V: funtions in DNA repair,Bacterial DNA polymerase III: a distinct polymerase fold,Lamers et al. (2006) Cell 126, 881-92; Bailey et al. (2006) Cell 126, 893-904,Revealed conserved features of the DNA polymerase that copies bacterial genomes. Established a

16、 new model of the elongation complex including binding sites for DNA and interacting proteins,Pol III structure - Model for DNA complex,The replication fork,Solution: the replication fork,DNA polymerase cant replicate a genome! No single stranded template The single strand template is unstable No pr

17、imer No 3-5 polymerase Too slow and distributive,Replication fork summary,DNA polymerase cant replicate a genome. Problem Solution ATP? No single stranded templateHelicase + The ss template is unstableSSB (RPA (euks) - No primerPrimase (+) No 3-5 polymeraseReplication fork Too slow and distributiveS

18、SB and sliding clamp - Sliding clamp cant get onClamp loader (/RFC) + Lagging strand contains RNAPol I 5-3 exo, RNaseH - Lagging strand is nickedDNA ligase + Helicase introduces positive Topoisomerase II + supercoils DNA replication is fast and processive Replication fork is organized around an asym

19、metric, DNA- polymerase III dimer. Both strands made 5-3. “Leading strand” is continuous; “lagging strand” is discontinuous,The replication fork,The junction between the newly separated template strands and the unreplicated duplex DNA,Figure 8-11,Leading strand,Lagging strand,Okazaki fragment,Replic

20、ation fork,Replication fork enzymes extend the range of DNA polymerase substrate,DNA Pol can not accomplish replication without the help of other enzymes The born and death of a RNA primer: primase and RNase H/exonuclease/DNA Pol/ligase The initiation of a new strand of DNA require an RNA primer Pri

21、mase is a specialized RNA polymerase dedicated to making short RNA primers on an ssDNA template. Do not require specific DNA sequence. DNA Pol can extend both RNA and DNA primers annealed to DNA template Dealing the DNA structure (helicase, topoisomerase, SSB,RNA primers must be removed to complete

22、DNA replication,A joint efforts of RNase H, DNA polymerase 3 domains. The Specialization of DNA Polymerases The Replication Fork: the enzyme/proteins required to synthesize the leading and lagging strands. DNA Synthesis at the Replication Fork: Holoenzyme/trombone model to explain how the anti-paral

23、lel template strands are copied/replicated toward the replication fork. Replisome/protein interaction,Summary,Initiation of DNA Replication/binding and unwinding: the replicon model; initiation in bacteria; initiation control in eukaryotes-a link with cell cycle (pre-RC assembly and activiation). Finishing Replication: Finishing in bacteria; Finishing in eukaryotes-the end replication problem and resolution