The Watson-Crick Proposal. DNA Replication. Semiconservative DNA replication

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1 Cell and Molecular Biology The Watson-Crick Proposal DNA Replication DNA strands are complementary Nucleotides are lined up on templates according to base pair rules Kanokporn Boonsirichai Molecular Biology of the Cell Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Semiconservative DNA replication Figure 6-8 Essential Cell Biology ( Garland Science 2010)

2 Replicating DNA Phosphodiester Bond DNA Polymerase DNA Polymerase The first nucleotide polymerizing enzyme, DNA polymerase, was discovered in The free nucleotides that serve as substrates for this enzyme were found to be deoxyribonucleoside triphosphates, and their polymerization into DNA required a singlestranded DNA template.

3 Antiparallel Nature of DNA DNA Replication Fork Sugar/phosphate backbone runs in opposite directions (Crick) One strand runs 5 to 3, while the other runs 3 to 5 DNA polymerase only adds nucleotides at the free 3 end, forming new DNA strands in the 5 to 3 direction only DNA Replication DNA Replication Origin of replication ( bubbles ): beginning of replication Replication fork: Y -shaped region where new strands of DNA are elongating Helicase:catalyzes the untwisting of the DNA at the replication fork DNA polymerase:catalyzes the elongation of new DNA

4 DNA Replication Leading strand: synthesis toward the replication fork (only in a 5 to 3 direction from the 3 to 5 master strand) Lagging strand: synthesis away from the replication fork (Okazaki fragments); joined by DNA ligase(must wait for 3 end to open; again in a 5 to 3 direction) Initiation: Primer (short RNA sequence~w/primaseenzyme), begins the replication process Figure 6-12 Essential Cell Biology ( Garland Science 2010) DNA Replication Requires Several Proofreading Mechanisms The fidelity of copying DNA during replication is such that only about 1 mistake is made for every 10 9 nucleotides copied DNA Proofreading: before forming the covalent bond High affinity of the correct nucleotide for the moving DNA polymerase (H-bonding with the template). DNA polymerase undergoes conformational changes upon nucleotide binding causing incorrectly bound nucleotide to dissociate.

5 Upon Covalent Bonding For some rare instances, an incorrect nucleotide is added to the growing chain. Exonucleolytic Proofreading DNA polymerase requires a base-paired 3 -OH end of a primer strandon which to add further nucleotides. DNA polymerase deals with a mismatched primer strand at a separate catalytic site. This 3 -to-5 proofreading exonucleaseclips off unpaired residues at the primer terminus RNA primers are removed by RNaseH DNA Primase DNA ligase catalyzes the formation of phosphodiester bond with the help of an ATP

6 DNA Helicase DNA helicaseunwinds double-stranded DNA Single-stranded DNA Binding Proteins Sliding clamp keeps DNA polymerase on the DNA

7 The Replication Machine Topoisomerase I DNA topoisomerase prevents DNA tangling

8 Topoisomerase II How to replicate chromosome ends Figure 6-18 Essential Cell Biology ( Garland Science 2010) DNA is constantly damaged by endogenous and exogenous agents DNA REPAIR

9 DNA Repair DNA is the only biomolecule that is specifically repaired. All others are replaced. >100 genes participate in various aspects of DNA repair, even in organisms with very small genomes. DNA repair mechanisms promote genomic stability and prevent cancer. Many, perhaps most, cancers are at least partially attributable to defects in DNA repair. Table 6-1 Essential Cell Biology ( Garland Science 2010) Base losses Types of DNA Damage Types of DNA Damage Base modifications Base modifications Deamination Chemical modification Photodamage Deamination Chemical modification Photodamage

10 Types of DNA damage Replication errors Inter-strand crosslinks DNA-proteins crosslinks Strand breaks DNA Repair Strategies Direct reversal (repairs base modification) Photolyase, methyltransferase, oxidative demethylase Excision repair Base excision, nucleotide excision, transcription coupled excision repair, mismatch repair Lesion avoidance Translesion synthesis, DNA recombination Double strand break repair Homologous recombination, non-homologous end joining Nucleotide Excision Repair (NER) Remove bulky lesions 1.Transcriptioncoupled pathway Signaled by stalled RNA polymerae Base Excision Repair (BER) Removes altered nucleotides generated by reactive chemicals. 2.Global genomic pathway

11 Mismatch Repair (MMR) Removes mismatched bases that were incorporated by DNA polymerase Double-strand Breakage (DSB) Repair Causes of double-strand breaks (DSBs) Ionizing radiation: X-ray, gamma ray, alpha particles, beta particles, neutrons Chemicals: free radicals, chemotherapeutic agents DSB repair pathways Non-homologous end joining (NHEJ) Homologous recombination Figure 6-22 Essential Cell Biology ( Garland Science 2010) Homologous recombination Figure 6-29 Essential Cell Biology ( Garland Science 2010)