Molecular Basis of Inheritance Chapter 16 Figure 16.7a, c C T A A T C G GC A C G A T A T AT T A C T A 0.34 nm 3.4 nm (a) Key features of DNA structure G 1 nm G (c) Space-filling model T

(a) Rosalind Franklin Franklin’s X-ray diffraction Photograph of DNA (b) Figure 16.6 a, b DNA Structure Discovery Watson and Crick – 3-D model Used (stole) X-ray crystallography evidence from Rosalind Franklin and Maurice Wilkins DNA – Chemical language that is reproduced in all cells – Directs development of traits

DNA Function Discovery Fredrick Griffith – studied streptococcus pneumoniae – bacteria 2 strains – Disease causing – pathogenic – Harmless – nonpathogenic Killed pathogenic bacteria with heat Mixed killed with nonpathogenic living Some living became pathogenic – Transformed – Inherited in all “offspring” Transformation – change in genotype and phenotype due to the assimilation of external DNA by a cell

DNA Function Discovery 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: Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. EXPERIMENT RESULTS CONCLUSION Living S (control) cells Living R (control) cells Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Mouse diesMouse healthy Mouse dies Living S cells are found in blood sample. Figure 16.2

Genetic Info - DNA or Protein? Bacteriophages - Viruses that infect bacteria Virus are simple and nonliving – DNA or RNA and a protein coat Require a host to reproduce Hershey and Chase – Used radioactive sulfur – tag Protein – Used radioactive phosphorus – tag DNA Centrifuged infected bacteria – found phosphorus in the bacteria Therefore genetic info = DNA not Protein 16_04HersheyChaseExp_A.swf

Genetic Info - DNA or Protein?

Sugar-phosphate backbone Nitrogenous bases 5 end O–O– O P O CH O–O– H H O H H H 3 1 H O CH 3 N O N H Thymine (T) O OP O O–O– CH 2 H H O H H H H N N N H N H H Adenine (A) O O P O O–O– CH 2 H H O H H H H H H H N N N O Cytosine (C) O O P O CH O–O– H O H H 3 1 OH 2 H N N N H O N N H H H H Sugar (deoxyribose) 3 end Phosphate Guanine (G) DNA nucleotide 2 N Figure 16.5 DNA Structure 16_07DNADoubleHelix_A.swf 16_07DNADoubleHelix_A.swf Double Helix Composed of Nucleotides – Sugar – deoxyribose – Phosphate – Base Bases – Purines – “pure AG” Adenine Guanine – Pyrimadines Thymine Cytosine Chargaff’s Discovery – % A = % T – % C = % T A=T C Ξ G

Double Helix Sugar – phosphate backbones – Held together by covalent bonds – Antiparallel – 5’  3’ Interior – bases – Hydrophobic – Held together by hydrogen bonds – Purine must always pair with a pyrimadine – NEVER – purine - purine or pyrimadine - pyrimadine N H O CH 3 N N O N N N NH Sugar Adenine (A) Thymine (T) N N N N Sugar O H N H N H N O H H N Guanine (G) Cytosine (C) Figure 16.8 H

Double Helix

Semi-Conservative Replication Base pairing allows existing DNA strands to serve as templates for new complimentary strands. – Each strand of DNA is ½ old and ½ new. (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. (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. A C T A G A C T A G A C T A G A C T A G T G A T C T G A T C A C T A G A C T A G T G A T C T G A T C T G A T C T G A T C Figure 16.9 a–d

Meselson and Stahl Labeled DNA with heavy isotope 15N Any new DNA would contain 14N When results were centrifuged there was 15N-14N hybrid which confirmed the semi conservative model.

DNA Replication DNA replication begins at origins of replication 16_12OriginsOfReplication_A.swf 16_12OriginsOfReplication_A.swf – Forms a replication “bubble” – prokaryotes – Forms multiple replication “bubbles” with replication forks on either end DNA Polymerases catalyze the elongation of the replication forks Eventually all the bubbles fuse and the entire strand of DNA is copied. Figure a, b

DNA Polymerase 11 different DNA Polymerases in Eukaryotes Add nucleotides to the DNA DNA is antiparallel with 5’  3’ running in opposite directions. 5’ end – free phosphate 3’ end – free OH attached to a deoxyribose New nucleotides are ALWAYS added to the 3’ end by DNA Polymerase III

DNA Polymerase Figure New strandTemplate strand 5 end 3 end Sugar A T Base C G G C A C T P P P OH P P 5 end 3 end 5 end A T C G G C A C T 3 end Pyrophosphate 2 P OH Phosphate

Leading vs. Lagging Strand Leading strand – DNA Polymerase III moves with the replication fork synthesizing a new complimentary strand of DNA Lagging strand – DNA Pol III moves away or against the replication fork and therefore synthesizes fragments of a new complementary strand of DNA – Okazaki fragments DNA Ligase – joins the Okazaki fragments together

Primers DNA Polymerase III cannot start the process of polynucleotide synthesis – can only add nucleotides to an existing chain Primers start the initial chain – 5-10 nucleotides long – Can be RNA or DNA – Primase an RNA polymerase starts the primer DNA Polymerase I – removes primer and replaces with DNA after new strand has been created.

Other Proteins and Enzymes Involved in DNA Replication Helicase – untwists or unwinds the DNA and creates the replication fork – Breaks hydrogen bonds Topoisomerase – relieves tighter twisting in the existing twisted DNA caused by the helicase Single-Strand Binding Protein – stabilize unpaired DNA strands until new complementary stands are added. – Keeps double helix from rewinding

Proofreading and Repair DNA polymerase self corrects mismatched base pairs – mismatch repair Nuclease – cuts out “bad” or mutated DNA errors are only in in a billion DNA Polymerase and DNA Ligase add the correct DNA – nucleotide excision repair

Telomeres Ends of DNA sequences TTAGGG is a telomere in humans 100 to 1000 repetitions Protects the DNA strand Shorten with each round of DNA replication – somatic cells Contributes to aging Telomerase adds telomeres to gametes

Summary 16_09DNAReplicatOverview_A.swf 16_09DNAReplicatOverview_A.swf

DNA Replication Enzymes Enzyme / ProteinFunction(s) Helicase Topoisomerase Single-strand Binding Protein Primase DNA Polymerase III DNA Polymerase I DNA Ligase Nuclease