1. Chapter 2: DNA Synthesis (Replication) Required reading: Stryer’s Biochemistry 5 th edition p. 127-128, 750-754, 759-766, 768-773 (or Stryer’s Biochemistry 4 th edition p. 88-93, 799-809, 982-986, 809-814) Normal recitation time: Thursdays 11-12, B-185 PWB Exam: Wed., December 1 Proposed recitation time for this exam: Tue, Nov. 30, 11-12

2. DNA Polymerization Reaction

4. NC E. coli DNA Polymerase I Klenow Fragment Polymerase 3'  5‘ Nucl. 5'  3' Nucl.

5. Typical Polymerase Structure: E. Coli Pol I thumb palm fingers exonuclease polymerase

6. Polymerase with bound DNA

7. Mechanism of phosphoryl transfer

8. Polymerase fidelity mechanisms 1.Watson-Crick base pairing between the incoming dNTP and the corresponding base in the template strand. 2. H-bond formation between the minor groove of the new base pair and the amino acids in the polymerase active site. 3. Proofreading mechanism via 3' exonuclease that excises incorrectly added nucleotides.

9. 1. Correct Watson-Crick base pairing between the incoming dNTP and the corresponding base in the template strand induces conformational change required for polymerization reaction: Thumb Fingers

10. 2. H-bond formation between the minor groove of the new base pair and amino acids in the polymerase active site:

11. All Watson-Crick base pairs contain two H- bond acceptors at the same sites of the minor groove HN N O O N N N N NH 2 AT GC NH N N O 2 N N N H 2 N O N N O 2 N N N 2 NH 2 O C:G HN N O O N N N N 2 T:A

12. 3. 3’-Exonuclease Proofreading function of DNA polymerases excises incorrectly added nucleotides.

13. Fidelity of DNA Polymerization: Absolutely Essential!! Error Probability = Polymerization error (10 -4 ) X 3'  5' Nuclease error (10 -3 ) = 10 -7 (1 in 10,000,000 nt)

14. DNA Polymerization Has Three Stages 1) Initiation 2) Priming 3) Processive Synthesis

15. Problems to overcome: DNA Polymerization 1.The two strands must be separated, and local DNA over-winding must be relaxed. The single stranded DNA must be prevented from re-annealing and protected from degradation by cellular nucleases. 2. Both antiparallel strands must be synthesized simultaneously in the 5’  3’ direction. 3’ 5’ 3. A primer strand is required.

16. DNA Polymerization: Initiation DNA replication begins at a specific site. Example: oriC site from E. coli. 245 bp out of 4,000,000 bp contains a tandem array of three 13-mers; GATCTNTTNTTTT Synthesis takes place in both directions from the origin (two replication forks)

17. E. coli replication origin GATC common motif in oriC AT bp are common to facilitate duplex unwinding

18. DNA Polymerization: Initiation DNA replication begins at a specific site. Example: oriC site from E. coli. 245 bp out of 4,000,000 bp contains atandem array of three 13-mers; GATCTNTTNTTTT GATC common motif in oriC AT bp are common to facilitate duplex unwinding Synthesis takes place in both directions from the origin (two replication forks)

20. EnzymeFunction dnaA recognize replication origin and melts DNA duplex at several sites Helicase (dnaB) unwinding of ds DNA DNA gyrase generates (-) supercoiling SSB stabilize unwound ssDNA Primase (dnaG)an RNA polymerase, generates primers for DNA Pol Enzymes involved in the initiation of DNA Polymerization

21. Crystal structure of bacterial DNA helicase Stryer Fig. 27.16

22. DNA helicase: proposed mechanism Stryer Fig. 27.17 B1 A1

23. Problems to overcome: DNA Polymerization 1.The two strands must be separated, and local DNA over-winding must be relaxed. The single stranded DNA must be prevented from re-annealing and protected from degradation by cellular nucleases. 2. Both antiparallel strands must be synthesized simultaneously in the 5’  3’ direction. 3’ 5’ 3. A primer strand is required. (overall direction)

24. Lagging strand is synthesized in short fragments (1000-2000 nucleotides long) using multiple primers 3’ 5’

25. Problems to overcome: DNA Polymerization 1.The two strands must be separated, and local DNA over-winding must be relaxed. The single stranded DNA must be prevented from re-annealing and protected from degradation by cellular nucleases. 2. Both antiparallel strands must be synthesized simultaneously in the 5’  3’ direction. 3’ 5’ 3. A primer strand is required.

26. A short stretch of RNA is used as a primer for DNA synthesis ( dnaG)

27. What is the function of RNA priming? DNA polymerase tests the correctness of the preceding base pair before forming a new phosphodiester bond de novo synthesis does not allow proofreading of the first nucleotide Low fidelity RNA primer is later replaced with DNA

28. Lagging strand synthesis in E. coli 3'5' Primase 3' 5' DNA Pol III 3' 5' RNA primer 3' 5' 3' DNA Ligase 5' Template DNA 5' 3' Okazaki fragment 5'3' 5' 3'5' New DNA 3' 5' 3' DNA Pol I 3'

29. DNA Synthesis 3' 5' SSB 5' DNA Pol I DNA Ligase Helicase Gyrase 3' 5' DNA Pol III Primase

31. E. coli DNA Polymerase III Processive DNA Synthesis The bulk of DNA synthesis in E. coli is carried out by the DNA polymerase III holoenzyme. Extremely high processivity: once it combines with the DNA and starts polymerization, it does not come off until finished. Tremendous catalytic potential: up to 2000 nucleotides/sec. Low error rate (high fidelity) 1 error per 10,000,000 nucleotides Complex composition (10 types of subunits) and large size (900 kd)

32. E. coli Pol III: an asymmetrical dimer Polymerase Stryer Fig. 27.30 clamp loader Sliding clamp 3'-5' exonuclease

33.  2 sliding clamp is important for processivity of Pol III

34. Stryer Fig. 27.33 Lagging strand loops to enable the simultaneous replication of both DNA strands by dimeric DNA Pol III

35. DNA Ligase seals the nicks OH P O -OO O- O P O O DNA Ligase + (ATP or NAD+) AMP + PPi Forms phosphodiester bonds between 3’ OH and 5’ phosphate Requires double-stranded DNA Activates 5’phosphate to nucleophilic attack by trans-esterification with activated AMP

36. DNA Ligase -mechanism 1.E + ATP  E-AMP + PPi OH + DNA-3' P O AMP-O O O- 5'-DNA OP O O O- DNA-3' 5'-DNA + AMP-OH 3. 2. E-AMP + P-5’-DNA  P O AMP-O O O- 5'-DNA (+)H 2 N P O O(-) O OH O Ade ENZYME OH

37. DNA Synthesis in bacteria: Take Home Message 1) DNA synthesis is carried out by DNA polymerases with high fidelity. 2) DNA synthesis is characterized by initiation, priming, and processive synthesis steps and proceeds in the 5’  3’ direction. 3) Both strands are synthesized simultaneously by the multisubunit polymerase enzyme (Pol III). One strand is made continuously (leading strand), while the other one is made in fragments (lagging strand). 4) Pol I removes the RNA primers and fills the resulting gaps, and the nicks are sealed by DNA ligase