Chapter 16-Molecular Genetics You must know: The structure of DNA The history of the discovery of DNA gained from the work of Replication Replication, transcription and translation Differences between bacterial vs. eukaryotic chromosomes How DNA packaging affects gene expression

Life’s Operating Instructions In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA DNA, the substance of inheritance, is the most celebrated molecule of our time Hereditary information is encoded in DNA and reproduced in all cells of the body This DNA program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits

How do we know DNA the genetic material? Scientific Inquiry Early in the 20th century ID of inheritance was a major challenge Morgan showed that genes are located on chromosomes DNA and protein are candidates for the genetic material Bacterial and viruses helped with this

Evidence that DNA can Transform Bacteria Griffith in 1928 showed the genetic role 2 strains of bacterium One pathogenic, other was not Mixed heat-killed remains with pathogenic with the living, living became pathogenic Transformation

Mixture of heat-killed S cells and living R cells Figure 16.2 EXPERIMENT Mixture of heat-killed S cells and living R cells Heat-killed S cells (control) Living S cells (control) Living R cells (control) RESULTS Figure 16.2 Inquiry: Can a genetic trait be transferred between different bacterial strains? Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells

More evidence In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria Many biologists remained skeptical, mainly because little was known about DNA More evidence for DNA as the genetic material came from studies of viruses that infect bacteria Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research

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

Do you need more? In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2 To determine this, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection They concluded that the injected DNA of the phage provides the genetic information

Additional Evidence It was known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next This evidence of diversity made DNA a more credible candidate for the genetic material

Chargaff’s Rules Base composition of DNA varies between species A and T are equal in number, G and C are equal Basis for these rules was not understood until the discovery of the double helix

Sugar–phosphate backbone Figure 16.5 Sugar–phosphate backbone Nitrogenous bases 5 end Thymine (T) Adenine (A) Cytosine (C) Figure 16.5 The structure of a DNA strand. Phosphate Guanine (G) Sugar (deoxyribose) DNA nucleotide Nitrogenous base 3 end

The Race to discover the structure of DNA DNA was accepted as the genetic matrial But how? Maurice Wilkins and Rosalind Franklin were using x-ray crystallography Rosalind produced the famous plate 51

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

Franklin’s work Enabled Watson to deduce the helical structure of the molecule Width and spacing was calculated The pattern in the photo confirmed that DNA is double helix Thought that Maurice Wilkins “borrowed” Rosie’s plate to show Watson- returned it

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

The proof Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA Franklin had concluded that there were two outer sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior Watson built a model in which the backbones were antiparallel (their subunits run in opposite directions At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray data

Purine  purine: too wide Figure 16.UN01 Purine  purine: too wide Pyrimidine  pyrimidine: too narrow Figure 16.UN01 In-text figure, p. 310 Purine  pyrimidine: width consistent with X-ray data

And there is more Watson and Crick reasoned that the pairing was more specific, dictated by the base structures They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C) The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C

Sugar Sugar Adenine (A) Thymine (T) Sugar Sugar Guanine (G) Figure 16.8 Sugar Sugar Adenine (A) Thymine (T) Figure 16.8 Base pairing in DNA. Sugar Sugar Guanine (G) Cytosine (C)

Form fits function Relationship between structure and function Copying mechanism for genetic material

Base pairing to a Template Strand Each strand acts as a template for building a new strand in replication Parent molecule unwinds Two daughter strands built Base pairing rules

(a) Parent molecule A T C G T A A T G C Figure 16.9-1 Figure 16.9 A model for DNA replication: the basic concept.

(a) Parent molecule (b) Separation of strands A T A T C G C G T A T A Figure 16.9-2 A T A T C G C G T A T A A T A T G C G C (a) Parent molecule (b) Separation of strands Figure 16.9 A model for DNA replication: the basic concept.

(a) Parent molecule (b) Separation of strands (c) Figure 16.9-3 A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of strands (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand Figure 16.9 A model for DNA replication: the basic concept.

Semiconservative Model Watson and Crick’s semiconservative model predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand

Meselson and Stahl Supported the semiconservative model Labeled nucleotides of old strands with heavy isotopes of nitrogen New nucleotides labeled with a lighter isotope

Bacteria cultured in medium with 15N (heavy isotope) 2 Figure 16.11 EXPERIMENT 1 Bacteria cultured in medium with 15N (heavy isotope) 2 Bacteria transferred to medium with 14N (lighter isotope) RESULTS 3 DNA sample centrifuged after first replication 4 DNA sample centrifuged after second replication Less dense More dense CONCLUSION Predictions: First replication Second replication Conservative model Figure 16.11 Inquiry: Does DNA replication follow the conservative, semiconservative, or dispersive model? Semiconservative model Dispersive model

Structure of DNA Prokaryotic DNA Eukaryotic DNA One double stranded circular DNA molecule Small amount of protein Linear DNA Protein packaged as chromatin 4 levels of packaging

Nucleosome- packaging- 1 10-nm fiber, basic unit DNA histone form beads on a string 8 histone molecules with the aa tail projecting outward Histone H1 (different from the rest) binds DNA to the next histone

Why do we need all this packaging? 1. more tightly packaged, less accessible to transcription enzymes 2. reduces gene expression 3. Interphase- chromatin is the highly extended form or euchromatin which can undergo transcription 4. More condensed is heterochromatin- generally not transcribed 5. Barr bodies are heterochromatin

Packaging 2- 30nm fiber String of nucleosomes coils to form chromatin fiber that is 30 nm Interphase is when this occurs Histone 3 &4

Packaging -3 300 nm fiber Looped domains Formed to a scaffold of non histone proteins

Packaging -4 1,400nm Maximally folding compact chromosome Metaphase 2 chromatids

Chromosome with Euchromatin and Heterochromatin

DNA Replication S phase Mitosis/Meiosis Fast and accurate More than a dozen enzymes are involved

6 points of DNA Replication 1. Begins at sites-Origins of Replication Bubbles will eventually fuse

Replication Fork or bubble 2. Initiation proteins bind Separate strands DNA replication proceeds in both directions along the DNA strand until molecule is copied DNA strand is short RNA called primer, enzymes primase does this Enzymes Helicase SS binding proteins Topoisomerase Relieves strand by breaking, swiveling and rewind the DNA

DNA Polymerase 3. Catalyzes elongation of new DNA at the replication Fork Bacterial uses different polymerases, III and I Adds DNA nucleotide to primer which is RNA, done by dATP, exergonic reaction

Adding nucleotides 4. DNA polymerase adds nucleotides to the strand one at a time in the 5’-3’ direction Bonding of A-T, C-G Basically purine to pyrimidine Keeps the symmetry to the molecule

Antiparallel elongation 5 Leading and Lagging strands 5. leading toward fork is continuous Lagging 3’-5’-away from fork is discontinuous Antiparallel A-T C-G Purine to pyrimidine

Okazaki Fragments 6.Lagging is in pieces Starts as discontinous Sealed by DNA ligase Forms continuous strand

DNA Accuracy Dependent on the specificity of base pairing A-T, C-G Mismatch Repair Enzymes that cut out nuclease New sequence is DNA poly and DNA ligase Cancer cells accumulate errors Nucleotide excision repair system

Of importance DNA repair enzymes on skin after exposure to UV rays Thymine bases, or thymine dimers cause DNA to buckle, interferes with replication Xeroderma pigmentosum

Telomeres- ase-Blackburn Replicating the ends TTAGGG- does not code for anything noncoding repeating sequence 100-1000x repeated Telomerase Enzyme that stimulates ends to lengthen in germ cells Zygotes only have maximum Somatic cells do not have this at birth

Lagging strand template 3 5 DNA pol III Lagging strand 3 5 Figure 16.18 DNA pol III Parental DNA Leading strand 5 5 3 3 3 5 3 5 Connecting protein Helicase Lagging strand template 3 5 Figure 16.18 A current model of the DNA replication complex. DNA pol III Lagging strand 3 5

Bozeman Links Structure of DNA https://www.youtube.com/results?search_query=bozeman+dna+structure Replication https://www.youtube.com/results?search_query=bozeman+dna+replication