7.1 DNA Structure & Replication

Review: Draw a nucleotide

Review: State the name of the 4 bases in DNA. Which are purines Review: State the name of the 4 bases in DNA. Which are purines? Pyrimidines?    Adenine     Guanine     Thymine  Cytosine Adenine and guanine are purines (double ring bases) Thymine and cytosine are pyrimidines (single ring bases)

 Review: Outline how the DNA nucleotides are linked together by covalent bonds into a single strand Nucleotides are linked into a single strand via a condensation reaction The phosphate group (attached to the 5'-C of the sugar) joins with the hydroxyl (OH) group attached to the 3'-C of the sugar This results in a phosphodiester bond between the two nucleotides and the formation of a water molecule Successive condensation reactions between nucleotides results in the formation of a long single strand

Review: Explain how double helix is formed using complementary base pairing & h bonds Two polynucleotide chains of DNA are held together by hydrogen bonds between complementary base pairs Adenine pairs with thymine (A=T) via two hydrogen bonds Guanine pairs with cytosine (G=C) via three hydrogen bonds In order for bases to be facing each other and thus able to pair, the two strands must run in opposite directions (i.e. they are anti-parallel) As the polynucleotide chain lengthens, the atoms that make up the molecule will arrange themselves in an optimal energy configuration  This position of least resistance results in the double-stranded DNA twisting to form a double helix with approximately 10 - 15 bases per twist

 Review: Draw and label a simple diagram of the molecular structure of DNA https://www.youtube.com/watch?v=kTH13oI8BSI

Skill: Analysis of the results of the Hershey-Chase experiment providing evidence that DNA is the genetic material Late 1800’s – scientists knew chromosomes had role in heredity Knew chromo’s made of protein and nucleic acid Until 1940’s – thought protein was the genetic material (because it has more variety due to 20 different amino acids & specificity of function) 1952 - Alfred Hershey & Martha Chase wanted to know if genetic material of viruses was protein or DNA 1950’s – scientists knew viruses are infectious agents that inject their genetic material & non-genetic portion of virus remains outside host cell; infected cell makes large numbers of new viruses & bursts Hershey & Chase worked with T2 bacteriophage because very simple structure Bacteriophage = virus that infects & replicates within a bacterium!

Hershey-Chase experiment

Nature of science: Making careful observations: Rosalind Franklin’s X-ray diffraction provided crucial evidence that DNA is a double helix. Watson & Crick: discovered double helix structure (1953) Chargaff: biochemist who researched base ratios in DNA – “Chargaff’s Ratios” (1950) Rosalind Franklin: biophysicist who researched structure of DNA by X-ray diffraction (1950) Franklin’s contributions: (rigorous experimental techniques & diligent observation) Improved resolution of a camera to make more detailed measurements of X-ray diffraction patterns Produced high quality samples of DNA Obtained sharpest x-ray diffraction images of DNA ever made! (“the most beautiful x-ray photographs of any substance ever taken”) Calculated dimensions of DNA helix Note: James Watson was shown the best diffraction pattern and calculations, used them with Crick to build DNA model, published results, together with Wilkins won Nobel Prize in 1962. Franklin died in 1958 so never awarded Nobel Prize.

Rosalind Franklin’s x-ray diffraction photograph of DNA (“photo 51” by Gosling)

Application: Rosalind Franklin and Maurice Wilkins’ investigation of DNA structure by X-ray diffraction. X-ray diffraction = scattering of x-rays Franklin’s deductions: Cross in center indicated helical shape Angle of cross shape showed steepness of angle Distance between horizontal bars showed turns of helix to be 3.4 nm apart Distance between middle of diffraction pattern & top showed there was a repeating structure within DNA, with distance of 0.34 nm between repeats (turned out to be distance between base pairs)

DBQ’s due tomorrow! P 349 & 353

Understanding: DNA structure suggested a mechanism for DNA replication Linus Pauling – molecular models Rosalind Franklin – X-ray diffraction patterns of DNA Erwin Chargaff – base composition ratios in DNA Watson & Crick’s 1st DNA model: sugar-phosphate strands wrapped around each other, with bases facing outwards Franklin again: “Wait! Bases hydrophobic compared to S-P backbone, so likely in center; & DNA helix is tightly packed!” So…Watson & Crick’s 2nd+ DNA models: bases on inside, strands not too far apart, pyrimidine paired with purine, and bases upside down in relation to each other; A has surplus - charge and T has surplus + charge, so pairing electrically compatible; C & G forms 3 H bonds, which enhances stability All this suggests COMPLEMENTARY BASE PAIRING, which then suggests the mechanism for replication: “SEMI-CONSERVATIVE REPLICATION”!

Understanding: Nucleosomes help to supercoil DNA! Eukaryotic DNA is associated with proteins, whereas prokaryotic DNA is “naked”. Nucleosome = 8 histone proteins, 2 wraps of DNA coiled around, short section of “linker” DNA to connect nucleosomes, another H1 histone to bind DNA to core Function of nucleosome = “supercoiling” of DNA 50,000 µm DNA fits into 5 µm space 30nm fiber facilitates further packing

30 nm fibre

Skill: Utilization of molecular visualization software to analyse the association between protein and DNA within a nucleosome http://www.rcsb.org/pdb/home/home.do Rotate molecule to see 2 copies of each histone. Note the approx. 150 bp of DNA wrapped twice around octamer core. Note N-terminal tail projecting from histone core for each protein. Chemical modification of this tail is involved in regulating gene expression. Visualize positively charged amino acids on nucleosome core. Suggest how they play a role in association of the protein core with the negatively charged DNA.

Leading Strand Lagging Strand Understanding: DNA replication is continuous on the leading strand & discontinuous on the lagging strand Leading Strand Lagging Strand Antiparallel to lagging strand Made in a continuous fashion Nucleotides added in a 5’ to 3’ direction TOWARDS the replication fork Only one RNA primer added Antiparallel to leading strand Made in fragments (Okazaki) Nucleotides added in a 5’ to 3’ direction AWAY from the replication fork Several RNA primers added (one at the beginning of each O. fragment)

Understanding: DNA replication is carried out by a complex system of enzymes

Understanding: DNA polymerases can only add nucleotides to the 3’ end of a primer

Replication Bubble (Replication occurs in both directions away from the origin)

Prokaryotes vs eukaryotes http://highered. mheducation

Understanding: Some regions of DNA do not code for proteins but have other important functions. Coding sequences = portions of DNA that code for the production of polypeptides Non-coding sequences = portions of DNA that DO NOT code for polypeptides, but have other functions Used as a guide to produce tRNA and rRNA Regulate gene expression by allowing it (“enhancers”) or stopping it (“silencers”) Makes up majority of eukaryotic genome Can be repetitive sequences (~60% of DNA in humans) Moderately repetitive sequences or Highly repetitive sequences (“satellite DNA”) Can occur at the telomeres (ends) and the centromere Telomeres protect gene expression because enzymes can’t replicate DNA all the way to the ends; without telomeres, important genes at the end would not get expressed!

Application: Tandem repeats are used in DNA profiling VNTR = variable number of tandem repeats = short DNA sequence which varies between individuals in number of times the sequence is repeated Serves as genetic signature Tandem = having 2 things arranged one right after the other (e.g. tandem bicycle) Each variety can be inherited as an allele VNTR analysis is basis of DNA profiling (for criminal investigations, genealogy, paternity) Locus = physical location of an allele DNA profile Cut DNA at specific loci with same restriction enzyme Separate the DNA fragments by size through gel electrophoresis Compare 2 individual’s profiles

DNA profiling using VNTRs

Criminal

Paternity

DNA profiling for genealogical investigations Deduce paternal lineage by analyzing short tandem repeats from Y-chromosome Deduce maternal lineage by analyzing mitochondrial DNA variations in single nucleotides at specific “hypervariable regions” Great article about “Mitochondrial Eve”: http://www.bradshawfoundation.com/journey/eve.html

Application: Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing. https://www.youtube.com/watch?v=e2G5zx-OJIw Dideoxyribonucleic acids = STOP replication as soon as they’re added instead of a regular nucleotide Labeled with fluorescent markers Fragments separated by gel electrophoresis Sequence of bases analyzed by comparing color of fluorescence with length of fragment “Sanger Sequencing Method”