Welcome to Class 18! Lecture 18: Outline and Objectives. Replication is semiconservative! Replication: DNA DNA! Introductory Biochemistry!

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1 Lecture 18: Outline and Objectives Welcome to Class 18! Introductory Biochemistry! l DNA Replication! l DNA polymerase! l the enzymatic reaction! l proofreading and accuracy! l DNA synthesis! l origins & initiation! l the replication fork! l leading & lagging strand synthesis! l termination! l DNA Repair! l Mutations! l Mechanisms! l mismatch repair! l base excision repair! l nucleotide excision repair! l direct repair! Replication: DNA DNA! Replication is semiconservative! Meselson-Stahl! Experiment:! Semiconservative model! hybrid duplex of old and new strand!! Conservative model! duplex of only old or only newly synthesized DNA! Figure 1-31! Conclusion: DNA synthesis is semi-conservative! Figure 25-2! 1!

2 DNA synthesis is performed by DNA polymerases! DNA synthesis is always 5ʹ Requires:! template strand to copy! primer strand with 3ʹ OH! dntp substrates! Catalyzes:! nucleophilic attack by 3ʹ OH! phosphodiester bond formation! 5ʹ 3ʹ synthesis! basepairing directs choice of dntp! Primer strand!5'gtca! Template strand!3'cagtcag! Accuracy or fidelity in replication! Figure 25-5! DNA polymerases! Base pair geometry! is important! Which polymerase replicates the genome?! Pol I is the only polymerase with a 5ʹ 3ʹ exonuclease activity! Figure 25-6! All DNA polymerases have a 3ʹ 5ʹ exonuclease activity! 2!

3 Accuracy is essential! DNA Replication has Three Major Stages! Initiation! 3ʹ 5ʹ exonuclease activity is for! proofreading! Elongation! Termination! DNA polymerases insert one incorrect nucleotide for every 104 to 105 correct ones.! Proofreading improve the inherent accuracy of the polymerization reaction by 100- to 1000-fold.! In combination, one net error for every 106 to 108 bases added.! Figure 25-3! Figure 25-7! Replication Initiates at Origins! The E. Coli chromosome! Relication of a circular chromosome! The origin! Tritium labeling experiments show that both strands! are replicated at the same time.! Figure 25-3! Figure 25-1! 3!

4 Initiation of replication requires specific sequences and proteins! DUE = DNA unwinding element (contains high amount AT)! Model for initiation of replication! Figure 25-10! Figure 25-11! Elongation: Polymerization! Elongation: Priming! primase Origin! Origin! DNA polymerase III DNA Polymerases Synthesize Only 5ʹ 3ʹ! Origin! RNA primers! Origin! How are the other strands copied?! 4!

5 Elongation: Polymerization! Elongation: DNA replication is semidiscontinuous! Origin! Leading Strands! Leading strand synthesis! is continuous! (and in the direction of fork movement)! Lagging Strands! Origin! Lagging strand synthesis! is discontinuous! (and opposite to fork movement)! Figure 25-4! Elongation: Lagging strand synthesis! Elongation: Removal of RNA primers! 5ʹ 3ʹ exo activity! 5ʹ 3ʹ polymerase activity! Figure 25-12! Figure 25-15! 5!

6 Elongation: Removal of RNA primers by Pol I! DNA ligase: sealing the nick! Phosphodiester bond formation! ➀ adenylylation of enzyme! ➁ activation of 5ʹ phosphate! ➂ nucleophilic attack by 3ʹ OH! 5ʹ 3ʹ exo activity! removes RNA! 5ʹ 3ʹ polymerase activity! fills in with DNA! RNA replaced with DNA! Figure 25-8! Steps in elongation! Leading Strand! Both! ① For the leading strand-! The primosome synthesizes! an RNA primer at the origin! Figure 25-16! Elongation: Overview! Lagging Strand! ① For the lagging strand-! The primosome synthesizes! an RNA primer for each! Okazaki fragment! ② dntps are added by! DNA Polymerase III! ③ as the replication fork moves,! DnaB helicase unwinds the DNA! SSB stabilizes the single strands! DNA gyrase relieves the strain caused!by unwinding! ④ RNA primers are removed by DNA polymerase I and the nicks are closed by DNA ligase! Figure 25-12! 6!

7 The DNA pol III enzyme! The DNA pol III clamp loader! Polymerase activity! Polymerase activity! Increases processivity! Figure 25-14! Figure 25-10(5th edition)! DNA synthesis on the leading and lagging strands! DNA Replication has Three Major Stages! Initiation! Elongation! Termination! Figure 25-3! Figure 25-13! Replication Initiates at Origins! 7!

8 Termination of replication! Termination: the final stages! Replication forks stop at the terminus region! Figure 25-17! Figure 25-18! Replication in Eukaryotes is Both Similar and More Complex! Eukaryotic chromosomes are long and linear!!!!e. coli!!humans! chromosome(s)!!circular!!linear!! length!!!1.36 mm!!100 mm (avg.)!! replication rate!!~50 nt/sec!~5 nt/sec! multiple origins of replication! The replication rate is slower, and the chromosomes are longer - -! Multiple origins of replication are necessary to replicate large chromosomes!! Chromosomes must be replicated only once per cell cycle! How does this work?! 8!

9 Initiation of Eukaryotic DNA Replication Requires Two Steps! Eukaryotic chromosomes are long and linear! multiple origins of replication! (helicase)! 1. Formation of the Pre-RC! 2. Coordinate Activation! linear chromosomes present a problem...! Figure 25-19! The ends of linear chromosomes present a replication problem! Eukaryotic chromosomes are long and linear! multiple origins of replication! Telomeres are repeated sequences! e.g.,!5ʹ - (T x G y ) n (x, y = 1 4)!!3ʹ - (A x C y ) n! which stabilize the ends of linear chromosomes! linear chromosomes present a problem...! Not replicated!! 9!

10 Telomerase adds telomeres! to chromosome ends! Reverse transcription:! RNA-dependent DNA synthesis! Telomerase synthesizes DNA from an RNA template! (a reverse transcriptase)! The template is an RNA molecule that is part of the enzyme! Telomerase is an RNP enzyme! Figure 26-38! Figure 26-31! Lecture 18: Outline and Objectives ü DNA Replication! ü DNA polymerase! ü the enzymatic reaction! ü proofreading and accuracy! ü DNA synthesis! ü origins & initiation! ü the replication fork! ü leading & lagging strand synthesis! ü termination! Ø DNA Repair! l Mutations! l Mechanisms! l mismatch repair! l base excision repair! l nucleotide excision repair! l direct repair! DNA repair! Mutation: a permanent change in the DNA sequence! Mutations can be:! silent no effect on gene function! deleterious impairs gene function! advantageous enhances gene function! Mutations can lead to:! genetic diversity! cancer in somatic cells! birth defects in germ cells! 10!

11 Mutations! Mutations! Can be caused by:! Mistakes in replication! DNA Damage! Deamination! Deaminating agents! Spontaneously,! ~ 100/day! A good reason for having T instead of U in DNA! Spontaneously,! ~ 1/day! Deaminating agents! induce these conversions! at high levels! Figure 8-30a! metabolized to Nitrous Acid (HNO 2 ) a strong deaminating agent! Figure 8-32a! 11!

12 UV irradiation is another source of DNA damage! Generates a block to replication! can occur:! spontaneously,! through the action of alkylating agents! N 7 alkylation increases depurination! Depurination! hydrolysis! Defects in repair of this lesion lead to Xeroderma pigmentosum! Figure 8-31! Figure 8-30! Alkylating agents! Alkylation can change base-pairing properties! Figure 8-32b! cannot pair with C! Figure 25-27a! 12!

13 DNA damage can result in mutations! DNA Repair! DNA Damage! Is necessary to repair DNA damage! Four Major Mechanisms:! DNA damage on one strand can be repaired using information from the other strand! 1. Mismatch Repair! Mistake is! replicated! 2. Base Excision Repair! 3. Nucleotide Excision Repair! 4. Direct Repair! Mutation!! Figure 25-27b! Factors involved in DNA repair:! Mismatch repair allows correction of replication errors! Four Major Mechanisms:! 1. Mismatch Repair! 2. Base Excision Repair! parental strand! is marked! 3. Nucleotide Excision Repair! 4. Direct Repair! a window! of opportunity! N6 methyl-a! still pairs with T! Figure 25-21! Methylation distinguishes! between template and! newly synthesized strands! 13!

14 Mismatch repair! Mismatch repair! Exonuclease activity mismatch! (5ʹ 3ʹ or 3ʹ 5ʹ ) degrades DNA from Me past mismatch! MutL-MutS! binds to! mismatch! MutL-MutS + MutH! finds Me site! MutH cleaves! unmodified strand! DNA Polymerase III replaces DNA! (copies methylated strand)! The methylated site! could be 1,000 bp! from the mismatch! site (either 5ʹ or 3ʹ ).! Figure 25-22! Base excision repair! End Result:! DNA containing mismatch is resynthesized! Figure 25-23! Base excision repair! damaged base! cleaves N-glycosyl! bond! removes! sugar! nick is sealed! Different glycosylase for each base lesion! Figure 25-24! Two types of BER! 14!

15 Nucleotide-excision repair! Direct repair does not remove base or nucleotide! Excinuclease:! excision endonuclease! makes 2 cuts! excises the damaged DNA! Repairs the defect directly BUT it s expensive! "Suicide Enzyme"! Used for removal of large bulky lesion (i.e. pyrimidine dimer)! Figure 25-25! Cost = one protein inactivated per repair! p. 1033! Information Pathways! Factors involved in DNA repair:! Four Major Mechanisms:! 1. Mismatch Repair! 2. Base Excision Repair! 3. Nucleotide Excision Repair! 4. Direct Repair! Coming up:! transcription!! 15!