Welcome to Class 18 Introductory Biochemistry

Lecture 18: Outline and Objectives DNA Replication DNA polymerase the enzymatic reaction proofreading and accuracy DNA synthesis origins & initiation the replication fork leading & lagging strand synthesis termination DNA Repair Mutations Mechanisms mismatch repair base excision repair nucleotide excision repair direct repair

Replication: DNA  DNA Semiconservative model hybrid duplex of old and new strand Conservative model duplex of only old or only newly synthesized DNA Figure 1-31

Replication is semiconservative Meselson-Stahl Experiment: Figure 25-2 Conclusion: DNA synthesis is semi-conservative

DNA synthesis is performed by DNA polymerases Requires: template strand to copy primer strand with 3’ OH dNTP substrates Catalyzes: nucleophilic attack by 3’ OH phosphodiester bond formation 5’→3’ synthesis

DNA synthesis is always 5’ → 3’ basepairing directs choice of dNTP Primer strand 5'GTCA Template strand 3'CAGTCAG Figure 25-5

Accuracy or fidelity in replication Base pair geometry is important Figure 25-6

All DNA polymerases have a 3’→5’ exonuclease activity Which polymerase replicates the genome? Pol I is the only polymerase with a 5’→3’ exonuclease activity All DNA polymerases have a 3’→5’ exonuclease activity

Accuracy is essential 3’ → 5’ exonuclease activity is for proofreading DNA polymerases insert one incorrect nucleotide for every 104 to 105 correct ones. Proofreading improves 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 25-7

DNA Replication has Three Major Stages Initiation Elongation Termination Figure 25-3 Replication Initiates at Origins

Relication of a circular chromosome Check this one could be figure 25-3 but it is not the same info being displayed Tritium labeling experiments show that both strands are replicated at the same time. Figure 25-3

The E. Coli chromosome The origin Figure 25-1

Initiation of replication requires specific sequences and proteins DUE = DNA unwinding element (contains high amount AT) Figure 25-10

Model for initiation of replication Figure 25-11

Elongation: Priming RNA primers primase 5’ 3’ 3’ 5’ Origin 5’ 5’ 3’ 3’

Elongation: Polymerization 5’ 5’ 3’ 3’ 5’ 5’ Origin DNA polymerase III DNA Polymerases Synthesize Only 5’→3’ 5’ How are the other strands copied? 5’ 3’ 3’ 5’ 5’ Origin

Elongation: Polymerization 5’ 5’ 3’ 3’ 5’ 5’ Leading Strands Origin 5’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ Lagging Strands Origin

Elongation: DNA replication is semidiscontinuous Leading strand synthesis is continuous (and in the direction of fork movement) Lagging strand synthesis is discontinuous (and opposite to fork movement) Figure 25-4

Elongation: Lagging strand synthesis Figure 25-12

Elongation: Removal of RNA primers 5’→3’ exo activity 5’→3’ polymerase activity Figure 25-15

Elongation: Removal of RNA primers by Pol I 5’→3’ exo activity removes RNA 5’→3’ polymerase activity fills in with DNA RNA replaced with DNA Figure 25-8

DNA ligase: sealing the nick Phosphodiester bond formation adenylylation of enzyme activation of 5’ phosphate nucleophilic attack by 3’ OH Figure 25-16

Steps in elongation Leading Strand Both Lagging Strand  For the leading strand- The primosome synthesizes an RNA primer at the origin  For the lagging strand- The primosome synthesizes an RNA primer for each Okazaki fragment  dNTPs are added by DNA Polymerase III  as the replication fork moves, DnaB helicase unwinds the DNA SSB stabilizes the single strands DNA gyrase relieves the strain caused by unwinding  RNA primers are removed by DNA polymerase I and the nicks are closed by DNA ligase

Elongation: Overview Figure 25-12

The DNA pol III enzyme Polymerase activity Polymerase activity Increases processivity Figure 25-10 (5th edition)

The DNA pol III clamp loader Figure 25-14

DNA synthesis on the leading and lagging strands Figure 25-13

DNA Replication has Three Major Stages Initiation Elongation Termination Figure 25-3 Replication initiates at origins

Termination of replication Replication forks stop at the terminus region Figure 25-17

Termination: the final stages Figure 25-18

Replication in eukaryotes is both similar and more complex E. coli Humans chromosome(s) circular linear length 1.36 mm 100 mm (avg.) replication rate ~50 nt/sec ~5 nt/sec The replication rate is slower, and the chromosomes are longer - - How does this work?

Eukaryotic chromosomes are long and linear multiple origins of replication Multiple origins of replication are necessary to replicate large chromosomes Chromosomes must be replicated only once per cell cycle

Initiation of eukaryotic DNA replication requires two steps (helicase) 2. Coordinate Activation 1. Formation of the Pre-RC CDK enzymes off CDK enzymes on pre-RC: pre-replicative complex Figure 25-19

Eukaryotic chromosomes are long and linear multiple origins of replication linear chromosomes present a problem...

The ends of linear chromosomes present a replication problem 3’ 5’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ Maybe figure 25-30 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3' 3’ 5’ Not replicated!

Eukaryotic chromosomes are long and linear multiple origins of replication Telomeres are repeated sequences e.g., 5’- (TxGy)n (x, y = 14) 3’- (AxCy)n which stabilize the ends of linear chromosomes linear chromosomes present a problem...

Telomerase adds telomeres to chromosome ends 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

Reverse transcription: RNA-dependent DNA synthesis Figure 26-31

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

Mutations Can be caused by: Mistakes in replication DNA Damage

Deamination A good reason for having T instead of U in DNA Spontaneously, ~ 100/day Deaminating agents induce these conversions at high levels Spontaneously, ~ 1/day Figure 8-30a

Deaminating agents metabolized to Nitrous Acid (HNO2), a strong deaminating agent Figure 8-32a

Depurination can occur: spontaneously, through the action of alkylating agents N7 alkylation increases depurination hydrolysis Figure 8-30

UV irradiation is another source of DNA damage Generates a block to replication Defects in repair of this lesion lead to Xeroderma pigmentosum Figure 8-31

Alkylating agents Figure 8-32b

Alkylation can change base-pairing properties Figure 25-27a cannot pair with C

DNA damage can result in mutations DNA damage on one strand can be repaired using information from the other strand Mistake is replicated Mutation! Figure 25-27b

DNA Repair Is necessary to repair DNA damage Four Major Mechanisms: 1. Mismatch Repair 2. Base Excision Repair 3. Nucleotide Excision Repair 4. Direct Repair

Mismatch repair allows correction of replication errors parental strand is marked a window of opportunity N6 methyl-A still pairs with T Figure 25-21 Methylation distinguishes between template and newly synthesized strands

Mismatch repair mismatch MutL-MutS binds to mismatch MutL-MutS + MutH “finds” Me site The methylated site could be 1,000 bp from the mismatch site (either 5’ or 3’). MutH cleaves unmodified strand Figure 25-22

Mismatch repair Exonuclease activity (5’→3’ or 3’→5’) degrades DNA from Me past mismatch DNA Polymerase III replaces DNA (copies methylated strand) End Result: DNA containing mismatch is resynthesized Figure 25-23

Base excision repair cleaves N-glycosyl bond removes sugar damaged base cleaves N-glycosyl bond removes sugar nick is sealed Different glycosylase for each base lesion Figure 25-24

Nucleotide-excision repair Excinuclease: excision endonuclease makes 2 cuts excises the damaged DNA Used for removal of large bulky lesions (i.e. pyrimidine dimers) Figure 25-25

Direct repair does not remove base or nucleotide Repairs the defect directly BUT it’s expensive "Suicide Enzyme" Cost = one protein inactivated per repair p. 1033

Direct repair of alkylated bases by AlkB in E. coli Oxidative demethylation by an a-ketoglutarate-iron dependent dioxygenase Figure 25-28

Information Pathways Coming up: transcription!