1. Presented by Dr. Ujwalkumar Trivedi Head Department of Biotechnology Marwadi University Rajkot (Gujarat)

2. Gregor Mendel (1866): discovered that inherited traits are determined by discrete units, or 'genes,’ - passed on from the parents. Freidrich Miescher(1868): discovered DNA Isolated something new from the nuclei of eukaryotic cells Later called DNA!!!

3. Thomas Hunt Morgan (1910): ◦ Worked with fruit flies  Specifically eye color of these flies! ◦ Discovered genes are located (linked) on chromosomes

4.  Fredrick Griffith (1928): ◦ Studied effects of virulent (virus-causing) bacteria vs. nonvirulent bacteria injected into mice ◦ Used transformation:  Inserted foreign DNA and changed protein/ trait ◦ believed that the transforming agent was an inheritance molecule.

7. Oswald Avery, Colin MacLeod, & Maclyn McCarty (1944):  Reported that “transforming agent” in Griffith's experiment was DNA.  Also u sed the Pneumococcus bacteria and test tubes (NOT mice)

9. Alfred Hershey & Martha Chase (1952) ◦ Confirmed DNA was genetic material ◦ Used bacteriophages (viruses) ◦ HYPOTHESIZED  DNA, not protein, is the hereditary material

12. Glycosidic Bond

20. How does DNA replicate? ConservativeSemi-ConservativeDispersive Hypotheses:

21. 1.Bacteria cultured in medium containing a heavy isotope of Nitrogen ( 15 N) Meselson-Stahl Experiment

22. 2.Bacteria transferred to a medium containing elemental Nitrogen ( 14 N) Meselson-Stahl Experiment

23. 3.DNA sample centrifuged after First replication

24. Meselson-Stahl Experiment 4.DNA sample centrifuged after Second replication

25. Rate of Mutation in Genomes Bacteria: 3bp/10 10bp Humans: 1bp/10 10bp Rate of DNA replication Prokaryotes: 1000bp/sec Eukaryotes: 50bp/secs

27. DnaA Protein

29. DomainModel organism(s) Helicase a Direction of movement BacteriaEscherichia coliDnaB5′ → 3′ ArchaeaSulfolobus solfataricus MCM3′ → 5′ EukaryaSaccharomyces cerevisiae Drosophila melanogaster Xenopus laevis Mcm2–73′ → 5′

32. Nobel Prize Physiology or medicine 1959

36. Proof reading is mediated by 3’to 5’ exonuclease activity of DNA polymerase

39. Prokaryote:1000bp Eukaryote: 100bp Reiji Okazaki (1968)

43. Prokaryotic family A polymerases include the DNA polymerase I (Pol I) enzyme, which is encoded by the polA gene and ubiquitous among prokaryotes. This repair polymerase is involved in excision repair with both 3'–5' and 5'–3' exonuclease activity and processing of Okazaki fragments generated during lagging strand synthesis. Cells lacking Pol I have been found suggesting Pol I activity can be replaced by the other four polymerases. Pol I adds ~15- 20 nucleotides per second, thus showing poor processivity. Instead, Pol I starts adding nucleotides at the RNA primer:template junction known as the origin of replication (ori).

45. DNA polymerase II, a family B polymerase, is a polB gene product also known as DnaA. Pol II has 3'–5' exonuclease activity and participates in DNA repair, replication restart to bypass lesions, and its cell presence can jump from ~30-50 copies per cell to ~200–300 during SOS induction. Pol II is also thought to be a backup to Pol III as it can interact with holoenzyme proteins and assume a moderate level of processivity. The main role of Pol II is thought to be the ability to direct polymerase activity at the replication fork and helped stalled Pol III bypass terminal mismatches.

46. DNA polymerase III holoenzyme is the primary enzyme involved in DNA replication in E. coli and belongs to family C polymerases. It consists of three assemblies: the pol III core, the beta sliding clamp processivity factor, and the clamp-loading complex. The core consists of three subunits: α, the polymerase activity hub, ɛ, exonucleolytic proofreader, and θ, which may act as a stabilizer for ɛ. The holoenzyme contains two cores, one for each strand, the lagging and leading. The beta sliding clamp processivity factor is also present in duplicate, one for each core, to create a clamp that encloses DNA allowing for high processivity. The third assembly is a seven- subunit (τ2γδδ′χψ) clamp loader complex. Recent research has classified Family C polymerases as a subcategory of Family X with no eukaryotic equivalents.

47. The replisome is composed of the following: 2 DNA Pol III enzymes, each comprising α, ε and θ subunits. (It has been proven that there is a third copy of Pol III at the replisome. [1] ) [1] the α subunit (encoded by the dnaE gene) has the polymerase activity.dnaE the ε subunit (dnaQ) has 3'→5' exonuclease activity.dnaQ the θ subunit (holE) stimulates the ε subunit's proofreading.holE 2 β units (dnaN) which act as sliding DNA clamps, they keep the polymerase bound to the DNA.dnaNDNA clamps 2 τ units (dnaX) which acts to dimerize two of the core enzymes (α, ε, and θ subunits).dnaX 1 γ unit (also dnaX) which acts as a clamp loader for the lagging strand Okazaki fragments, helping the two β subunits to form a unit and bind to DNA. The γ unit is made up of 5 γ subunits which include 3 γ subunits, 1 δ subunit (holA), and 1 δ' subunit (holB). The δ is involved in copying of the lagging strand.Okazaki fragmentsholAholB Χ (holC) and Ψ (holD) which form a 1:1 complex and bind to γ or τ. X can also mediate the switch from RNA primer to DNA. [2]holCholD [2]

48. Mutant viable? Yes! Yes! No Function repair replication + DNA pol IV: mutagenesis + DNA pol V: error-prone repair

49. Type IV and Type V are involved in DNA repair