Evidence that DNA can transform bacteria

1. Evidence that DNA can transform bacteria • Frederick Griffith (1928) – Streptococcus pneumoniae bacteria – transformation • Mouse Experiment • Experiment proved that transformation can happen • Avery and colleagues (1944) – announced transformation agent was DNA

2. Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: CONCLUSION EXPERIMENT RESULTS Living S (control) cells Living R (control) cells Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. Figure 16.2 Can the genetic trait of pathogenicity be transferred between bacteria?

3. Evidence that viral DNA can program cells • Alfred Hershey and Martha Chase (1952) – bacteriophages or phages (viruses that infect bacteria) – discovered DNA is the genetic material NOT protein • Blender experiment

4. Phage head Tail Tail fiber DNA 100 nm Bacterial cell Figure 16.3 Viruses infecting a bacterial cell

5. Additional evidence that DNA is the genetic material • Erwin Chargaff (1947) – Chargaff’s rules – The equivalences for any given species between the number of A and T and G and C bases areequal. • Analyzed DNA from different organisms • Humans 30.3% of bases were A’s • E. Coli 26% of bases were A’s

6. Rosalind Franklin – (1950’s) – X-ray diffraction photo of DNA – helped Watson and Crick develop their model of DNA structure

7. (b) (a) Rosalind Franklin Franklin’s X-ray diffraction Photograph of DNA Figure 16.6 Rosalind Franklin and her X-ray diffraction photo of DNA

8. Structure of DNA • Watson & Crick – (1953) – 1 page paper in the British journal Nature “Molecular Structure of Nucleic Acids: A Structure for Deoxynucleic acids”

9. Figure 16.1 Watson and Crick with their DNA model

10. Sugar-phosphate backbone Nitrogenous bases 5 end CH3 O– 5 O H CH2 O P O O 1 4 N O– N H H H H H O 2 3 H Thymine (T) O H H CH2 O O P N O N H O– H N H H H N N H H Adenine (A) H H O H N CH2 O O P H O O– N H N H H H O H Cytosine (C) O 5 H CH2 O N P O O O 1 4 O– H N H Phosphate H H N 2 H 3 DNA nucleotide N H OH N Sugar (deoxyribose) 3 end H H Guanine (G) Figure 16.5 The structure of a DNA strand

11. 5 end G C O OH A T Hydrogen bond P 3 end –O O T A OH O H2C A T 1 nm O O CH2 O C G P O O– –O 3.4 nm O P C G O O H2C O G C T A O CH2 O O G C P O O– –O O P O O H2C O G C T A O CH2 O O P T A O –O O– O P O A T O H2C O A T T A O CH2 OH O O– 3 end P O G C O 0.34 nm 5 end T A (a) Key features of DNA structure (b) Partial chemical structure (c) Space-filling model Figure 16.7 The double helix

12. N O H CH3 N N N N H Sugar N N O Sugar Adenine (A) Thymine (T) H O N H N N N H N Sugar N N N O H Sugar H Cytosine (C) Guanine (G) Figure 16.8 Base pairing in DNA H

13. Purine + Purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width Consistent with X-ray data Unnumbered Figure p. 298

14. DNA Replication Section 16.2 • Semi-conservative model – each of the two daughter molecules will have one old strand, derived from the parent molecules, and one newly made strand

15. A T C G T A A T G C (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. Figure 16.9 A model for DNA replication: the basic concept (layer 1)

16. A A T T C G C G T A T A A A T T G G C C (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. Figure 16.9 A model for DNA replication: the basic concept (layer 2)

17. T A A A T T T A G C C G C C G G A T A T T A A T T A A A T T T A C G G G C C C G (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (b) The first step in replication is separation of the two DNA strands. Figure 16.9 A model for DNA replication: the basic concept (layer 3)

18. T A A A A T A T T T T A G C G C C C C G G G G C A A T T T T A A A A T T T A A A A T A T T T T A C G G G G C G C C C C G (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. (b) The first step in replication is separation of the two DNA strands. Figure 16.9 A model for DNA replication: the basic concept (layer 4)

19. First replication Second replication Parent cell (a) Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. (b) Semiconserva- tive model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple- mentary strand. (c) Dispersive model. Each strand of both daughter mol- ecules contains a mixture of old and newly synthesized DNA. Figure 16.10 Three alternative models of DNA replication

20. New strand Template strand 3’ end 5’ end 3’ end 5’ end Sugar A T A T Base Phosphate C G C G G G C C A T A T OH P P P P 3’ end P Pyrophosphate C C OH 2 P Nucleoside triphosphate 5’ end 5’ end Figure 16.13 Incorporation of a nucleotide into a DNA strand

21. Origin of replication Parental (template) strand 0.25 µm Daughter (new) strand 1 Replication begins at specific sites where the two parental strands separate and form replication bubbles. Bubble Replication fork 2 The bubbles expand laterally, as DNA replication proceeds in both directions. 3 Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete. Two daughter DNA molecules (a) In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. (b) In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). Figure 16.12 Origins of replication in eukaryotes

22. DNA pol Ill elongates DNA strands only in the 5 3 direction. One new strand, the leading strand, can elongate continuously 5 3 as the replication fork progresses. 2 3 1 4 3 5 Parental DNA 5 The other new strand, the lagging strand must grow in an overall 3 5 direction by addition of short segments, Okazaki fragments, that grow 5 3 (numbered here in the order they were made). 3 Okazaki fragments 2 3 1 5 DNA pol III Template strand DNA ligase joins Okazaki fragments by forming a bond between their free ends. This results in a continuous strand. Leading strand Lagging strand 3 1 2 Template strand DNA ligase Overall direction of replication Figure 16.14 Synthesis of leading and lagging strands during DNA replication

23. 6 7 1 5 2 4 3 3 5 3 5 Templatestrand Primase joins RNA nucleotides into a primer. DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. RNA primer 3 5 3 1 5 After reaching the next RNA primer (not shown), DNA pol III falls off. Okazakifragment 3 3 5 1 5 After the second fragment is primed. DNA pol III adds DNAnucleotides until it reaches the first primer and falls off. 5 3 3 5 2 1 DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2. 5 3 3 5 2 1 DNA ligase forms a bond between the newest DNAand the adjacent DNA of fragment 1. The lagging strand in this region is nowcomplete. 5 3 3 5 2 1 Overall direction of replication Figure 16.15 Synthesis of the lagging strand

24. Table 16.1 Bacterial DNA replication proteins and their functions

25. Overall direction of replication Lagging strand Leading strand Helicase unwinds the parental double helix. Origin of replication 1 Molecules of single- strand binding protein stabilize the unwound template strands. The leading strand is synthesized continuously in the 5 3 direction by DNA pol III. 2 3 Leading strand Lagging strand OVERVIEW DNA pol III Leading strand 5 Replication fork DNA ligase DNA pol I 3 Primase 2 Parental DNA Lagging strand DNA pol III 1 Primer 3 Primase begins synthesis of RNA primer for fifth Okazaki fragment. 4 3 5 4 DNA pol I removes the primer from the 5 end of the second fragment, replacing it with DNA nucleotides that it adds one by one to the 3’ end of the third fragment. The replacement of the last RNA nucleotide with DNA leaves the sugar- phosphate backbone with a free 3 end. DNA pol III is completing synthesis of the fourth fragment, when it reaches the RNA primer on the third fragment, it will dissociate, move to the replication fork, and add DNA nucleotides to the 3 endof the fifth fragment primer. DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment. 5 6 7 Figure 16.16 A summary of bacterial DNA replication

26. Proofreading and repairing DNA • Errors do occur • 1 in 10 billion nucleotides on entire DNA • 1 in 100,000 for incoming nucleotides • Proofreading is done by DNA pol III as it attaches new nucleotides • Mismatch repair cells use special enzymes to fix mismatched nucleotides • A-C for example

27. A thymine dimer distorts the DNA molecule. 1 3 4 2 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease Repair synthesis by a DNA polymerase fills in the missing nucleotides. DNA polymerase DNA ligase DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. Figure 16.17 Nucleotide excision repair of DNA damage

28. Telomeres • Repeated units of bases • TTAGGG in humans • Do NOT contain genes • They protect the genes from being eroded (getting shorter and shorter) through DNA replication rounds

29. 5 End of parental DNA strands Leading strand Lagging strand 3 Last fragment Previous fragment RNA primer 5 Lagging strand 3 Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase Removal of primers and replacement with DNA where a 3 end is available 5 3 Second round of replication 5 3 New leading strand New lagging strand 5 3 Further rounds of replication Shorter and shorter daughter molecules Figure 16.18 Shortening of the ends of linear DNA molecules

30. 1 µm Figure 16.19 Telomeres