Chapter 11 DNA: The Carrier of Genetic Information

Experiments in DNA ???Protein as the genetic material 20 AA – many different combinations = unique properties Genes control protein synthesis DNA and RNA – only 4 nucleotides = dull

Experiments in DNA Frederick Griffith – 1928 – Bacteria – pneumococcus – 2 strains – (S) smooth strain – virulent (lethal) Mice – pneumonia - death – (R) rough strain – avirulent Mice survive – Heat killed (S) strain Mice survive – Heat killed (S) + live (R) Mice died Found living (S) in dead mice

Griffith continued –  transformation - type of permanent genetic change where the properties of 1 strain of dead cells are conferred on a different strain of living cells – “transforming principle” was transferred from dead to living cells

Fig Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Living S cells RESULTS EXPERIMENT

Avery, MacLeod, McCarty – Identified Griffith’s transforming principle as DNA – Live (R) + purified DNA from (S)  R cells transformed – R + (S) DNA  die – R = (S) protein  live – DNA responsible for transformation – Really?

Hershey and Chase – 1952 – Bacteriophages – Radioactive labels Viral protein – sulfur Viral DNA - phosphorus – infect bacteria, agitate in blender, centrifuge – Found Sulfur sample – all radioactivity in supernatant (not cells) Phosphorus sample – radioactivity in pellet (inside cells) – SO – bacteriophages inject DNA into bacteria, leaving protein on outside – DNA = hereditary material

Fig Bacterial cell Phage head Tail sheath Tail fiber DNA 100 nm

Fig EXPERIMENT Phage DNA Bacterial cell Radioactive protein Radioactive DNA Batch 1: radioactive sulfur ( 35 S) Batch 2: radioactive phosphorus ( 32 P) Empty protein shell Phage DNA Centrifuge Pellet Pellet (bacterial cells and contents) Radioactivity (phage protein) in liquid Radioactivity (phage DNA) in pellet

Rosalind Franklin (in lab of Wilkins) – X-ray diffraction on crystals of purified DNA – (X-ray crystallography) – Determine distance between atoms of molecules arranged in a regular, repeating crystalline structure Helix structure Nucleotide bases like rungs on ladder

Fig (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

James Watson and Francis Crick – 1953 – Model for DNA structure = double helixdouble helix – DNA now widely accepted as genetic material – Took all available info on DNA and put together – Showed – DNA can carry info for proteins Serve as own template for replication

Structure of DNA Nucleotides – Deoxyribose – Phosphate – Nitrogenous base (ATCG) Purines – adenine, guanine – 2 rings Pyrimidines – thymine, cytosine – 1 ring – covalent bonds link = sugar-phosphate backbone 3’ C of sugar bonded to 5’ phosphate = phophodiester linkage 5’ end – 5’ C attached to phosphate 3’ end – 3’ C attached to hydroxyl

Chargaff # purines = # pyrimidines – #A = #T – #C = # G Each cross rung of ladder – 1 purine + 1 pyrimidine

Fig. 16-UN1 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data

Hydrogen bonding between N bases A-T = 2 H bonds G-C = 3 H bonds Complementary base pairs # possible sequences virtually unlimited  many genes, much info

Fig Cytosine (C) Adenine (A)Thymine (T) Guanine (G)

Fig Sugar–phosphate backbone 5 end Nitrogenous bases Thymine (T) Adenine (A) Cytosine (C) Guanine (G) DNA nucleotide Sugar (deoxyribose) 3 end Phosphate

Fig. 16-7a Hydrogen bond 3 end 5 end 3.4 nm 0.34 nm 3 end 5 end (b) Partial chemical structure(a) Key features of DNA structure 1 nm

DNA Replication Semiconservative – each strand of DNA is template to make opposite new strand Meselson and Stahl – E. coli and isotopes of N – 15N – heavy/dense; 14N “normal” – Bacteria with 15N in DNA replicated with medium having 14N – Centrifuge  – Supports semiconservative model Explains how mutagens can be passed on

Fig a EXPERIMENT RESULTS Bacteria cultured in medium containing 15 N Bacteria transferred to medium containing 14 N DNA sample centrifuged after 20 min (after first application) DNA sample centrifuged after 20 min (after second replication) Less dense More dense

Fig A T G C TA TA G C (a) Parent molecule AT GC T A T A GC (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand (b) Separation of strands A T G C TA TA G C A T G C T A T A G C

Fig Parent cell First replication Second replication (a) Conservative model (b) Semiconserva- tive model (c) Dispersive model

Steps of DNA Replication 1. DNA helicase – 2. Helix-destabilizing proteins – 3. Topoisomerases – 4. RNA primer – 5. DNA polymerase – 6. Origin of replication – – Leading strand Leading strand – Lagging strand Lagging strand 7. DNA Ligase

Leading Strand

Fig b 0.25 µm Origin of replicationDouble-stranded DNA molecule Parental (template) strand Daughter (new) strand Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes

Fig A C T G G G GC CC C C A A A T T T New strand 5 end Template strand 3 end 5 end 3 end 5 end 3 end Base Sugar Phosphate Nucleoside triphosphate Pyrophosphate DNA polymerase

Fig a Overview Leading strand Lagging strand Origin of replication Primer Overall directions of replication

Fig Overview Origin of replication Leading strand Lagging strand Overall directions of replication Leading strand Lagging strand Helicase Parental DNA DNA pol III PrimerPrimase DNA ligase DNA pol III DNA pol I Single- strand binding protein

Telomeres Telomeres – caps end of chromosome; short non-coding sequences repeated many times Cell can divide many times before losing crucial info Lagging strand is discontinuous, so DNA polymerase unable to complete replication, leaving small part unreplicated  small part lost with each cycle

Fig Ends of parental DNA strands Leading strand Lagging strand Last fragment Previous fragment Parental strand RNA primer Removal of primers and replacement with DNA where a 3 end is available Second round of replication New leading strand New lagging strand Further rounds of replication Shorter and shorter daughter molecules