MCB Lecture 5 – DNA Metabolism, DNA Sequencing, and PCR
 Meselson-Stahl Experiment – Experiment that proved that DNA is semi-conservative. Added heavy DNA parent molecule to solution of nucleotides. DNA replicated, and the daughter DNA was all hybrid. The second generation was 50% hybrid and 50% light.
 In what direction is new DNA synthesized? 5’  3’ direction.
 What do DNA Polymerases need to start? Primer (RNA
Polymerases do not need a primer).
 What type of reaction does DNA Polymerase use to elongate the chain? Nucleophillic attack, transesterification reaction.
 Processive Enzymes – DNA Polymerases are processive enzymes, which means they can carry out many reactions before dissociating. (much faster)
 Distributive Enzymes – Catalyze one reaction and then dissociate.
 Structure of DNA Polymerase addition to chain? o Sliding clamp + Clamp Loader + ATP is added to the template strand. DNA Polymerase binds, ADP+P are given off, and clamp loader releases.
 How often do polymerases make mistakes? 1/10,000-1/100,000
(10^5)
 How does DNA Polymerase III recognize incorrect base pairing?
Weaker H-bonding of mispairs can be recognized and the DNA kinks, so it cannot physically fit through the enzyme for it to continue.
 How much does DNA Polymerase III improve fidelity of replication? Factor of 100-1000 (10^7)
 In which direction does DNA Polymerase III exonuclease fix mispairs? 3’  5’ direction.
 oriC – the origin of replication in Bacteria. 245 base pairs with two repeat structures. o Two repeat sequences:
 Tandem array of three 13-bp sequence (A-T Rich)
 Four 9-bp sequences (dispersed). o Mechanism:
 DnaA binds to the 9bp repeats that are dispersed.
 This opens up the DNA at the 13 bp repeat (with HU protein and ATP)

 DnaC protein associates with DnaA.
 DnaB is able to bind to three 13- bp sequence
 DnaB can unwind the DNA (like Helicase) and initiate replication.
 Methylation at the Origin of Replication: o Fully methylated – initiation of replication occurs o Hemi-methylated – resistant to methylation
 DNA Helicase – unwinds double-stranded DNA. Requires a lot of energy. They induce positive supercoiling (resolved by topoisomerases)
 What is protecting single-stranded DNA from helicases in: o Eukaryotes - RPA o Bactiera – SSB
 What are primers made up of? RNA (provide free 3’-OH to which nucleotides are added)
 What makes Primers? RNA Polymerase Primase
 Primosome – a complex of Primase and Helicase
 How many bases are Okazaki Fragments in: o Eukaryotes? 100-200bp o Prokaryotes? 1000-2000bp
 Trombone Model of Replication – Lagging strand forms a loop so that both strands of DNA work in 5’  3’ concert.
 Replisome – two DNA Polymerases working together as part of a single complex o DNA Polymerase III in Bacteria o DNA Polymerase Delta ( δ) in Eukaryotes
 DNA Polymerase I – binds to “nick” where DNA Polymerase III released fragment Degrades primer in 5’  3’ direction by exonuclease activity. Then, completes synthesis in 5’  3’ direction. Then, proofreads in 3’  5’ direction.
 DNA Ligase – closes the final “nick”
 Does DNA Polymerase I have high or low processivity? Low.
 Origin of Replication in Eukaryotes – o ORC – DnaA – binds to origin of replication o Cdc6 – DnaC – required for MCM binding – known as matchmaker factor
 CDK (Cyclin dependent kinase) regulates the concentration

o MCM – DnaB – has helicase activity to start replication.
 What two molecules form a complex for synthesis of the primer in eukaryotes? Primase + DNA Polymerase a.
 What is the eukaryotic primer made of? 5’ ribonucleotides and 3’ deoxyribonucleotides.
 Eukaryotic: o Sliding Clamp – PCNA o Clamp Loader – PCNA

 Replicative Cell Senescence – Human Chromosomes become shorter by losing telomeric repeats at every replication cycle because once the RNA Primers are removed, there is no free –OH group to complete the strand. They can usually double 40 times before dying. o Telomerase – adds telomeric repeats to the chromosome ends at the germ cell stage, but the gene is repressed in somatic cells. (if it is lifted, cancer can have unlimited growth)
 Consists of a protein and RNA (template to recognize telomeric DNA)
 What is the common reason for DNA Polymerase to mismatch?
Slippage when there are multiple tandem repeats.

 MutS/MutL Dimer Mismatch Repair: o The dimer first recognizes the mismatch. It knows which strand is the new strand because it is the unmethylated strand. o MutH cleaves the unmethylated strand in the vicinity of the mismatch. o Exonucleases, SSBs, and helicases degrade the cut strand. o The gap is filled by DNA Polymerase III. o Nick sealed by DNA Ligase.
 High-Fidelity DNA Synthesis o 5’  3 polymerization – 1 in 10^5 o 3’  5’ exonucleolytic proofreading – 1 in 10^2 o Strand-directed mismatch repair - 1 in 10^2 o Total: 1 in 10^9 (billion)
 CpG o Frequenctly methylated C in human DNA o Deamination of methylated C  T o Deamination of C  U
 Uracil DNA glycosylase o Sometimes the G is removed instead of the T during repair.
 Exinucleases – o Cuts damanged strand on both sides (endonucleases). o In bacteria, 12 bp long strand o In humans, 29 bp long strand o Cut the piece in two places. o Region is unwound by: XPB, XPD (helicases) o Damanged strand is filled by DNA Polymerase and completed by DNA Ligase.
 Diseases of Nucleotide Excision Repair: o Xeroderma Pigmentosum (XP) – autosomal recessive, has 2-
50000x higher risk for malignant melanoma. o Cockayne Syndrome – autosomal recessive, no increased cancer risk.
 Double Stranded Breaks: o Caused by oxygen radicals (x-rays) and DNA replication. o No template strand. o Ku protein binds to double stranded breaks o Joined by special DNA Ligase IV.