Chapter 16 DNA The Molecular Basis of Inheritance

DNA molecule responsible for all cell activities and contains the genetic code Genetic Code method cells use to store the program that is passed from one generation to another

DISCOVERY OF THE GENETIC CODE 1928: Frederick Griffith Transformation

1928: Frederick Griffith 1. Grew 2 strains of bacteria on plates - smooth colonies- caused disease (virulent) - rough edge colonies-did not cause disease (avirulent) 2. Injected into mice Results: - smooth colonies: died - rought colonies: lived Conclusion: bacteria didn’t produce a toxin to kill mice Experiment 1

1. Injected mice with heat killed virulent strain 2. Injected mice with non -virulent strain + heat killed virulent strain Results: - heat killed: lived - mixed strains: mice developed pneumonia Conclusion: heat killed virulent strain passed disease causing abilities to non virulent strain Experiment 2

After Experiment Cultured bacteria from dead mice and they grew virulent strain. Griffith hypothesized that a factor was transferred from heat killed cells to live cells. TRANSFORMATION

1944: Avery (et al) 1. Repeated Griffith’s experiment with same results. - result: transformation occurred 2. Did a second experiment using enzymes that would destroy RNA. - result: transformation occurred 3. Did third experiment using enzymes that would destroy DNA. - result: no transformation CONCLUSION DNA was transforming factor

1952: Hershey / Chase - studied how viruses (bacteriophage) affect bacteria. Bacteriophage Virus composed of DNA core and protein coat that infect bacteria

Hershey Chase Experiment 1. They labeled virus protein coat with radioactive sulfur 2. They labeled virus DNA with radioactive phosphorous Result observed that bacteria had phosphorous *** virus injected bacterial cells with its phosphorous labeled DNA*** Conclusion DNA carried genetic code since bacteria made new DNA. animation

DISCOVERY OF STRUCTURE OF DNA

Early 1950’s: Rosalind Franklin (English) x ray crystallography evidence: X pattern showed that fibers of DNA twisted and molecules are spaced at regular intevals on length fiber. Maurice Wilkins: x ray diffraction, worked with Franklin

Same time period: Chargaff (American biochemist) Chargaff’s Rule:

1953 Watson (American) & Crick (English) **double helix model** won Nobel prize in 1962 Discovered the double helix by building models to conform to Franklin’s X-ray data and Chargaff’s Rules

DNA - double strand of nucleotides - may have 1000’s of nucleotides in 1 strand (very long molecule) - bases join up in specific (complementary) pairs: • complementary pairs (base pairing rules) 1 purine bonds with 1 pyrimidine on one rung of the ladder connected by a weak H bond C-G A–T Order of nucleotides not important, proper complementary bases must be paired.

STRUCTURE OF DNA Backbone Phosphate + Deoxyribose sugar (5 C) Rungs 4 Nitrogenous bases - Purines Adenine Guanine A G - Pyrimidines Thymine Cytosine T C D bases attached to sugar E. bases attached to each other by weak H bond

Nucleotide Structure Purines Sugar Base Phosphate Pyrimidines

DNA makes up Chromosomes (chromatin packing)

DNA Comparison Prokaryotic DNA Eukaryotic DNA • Double-stranded • Circular • One chromosome • In cytoplasm • No histones • Supercoiled DNA • Double-stranded • Linear • Usually 1+ chromosomes • In nucleus • DNA wrapped around histones (proteins) • Forms chromatin

REPLICATION OF DNA Process of duplication of DNA - Before cell can divide a new copy of DNA must be made for the new cell - Semiconservative replication: each strand acts as a template (pattern) for new strand to be made End Result: one old strand, one new daughter strand

DNA REPLICATION

Models of DNA Replication

Discovery of Replication Model Meselson and Stahl • Cultured E coli with heavy isotope 15 N for many generations. • Transferred to light isotope 14 N • Sampled after first and second replication • Removed bacteria and extracted DNA • Centrifuged to separate different density DNA

Meselson and Stahl • Compared results to models of replication • 1 st replication: Hybrid DNA (14 and 15) -Eliminated conservative model • 2 nd replication: Light and hybrid DNA - Eliminated dispersive model - Supported semi-conservative model of replicaiton.

Steps of Replication 1. Enzyme DNA helicase attaches to DNA molecule at origins of replication and breaks H bonds so strand unwinds - single strand binding proteins bind to unpaired bases to keep them from re-binding - topoisomerase relieves additional strain on forward DNA by breaking and rejoining DNA strands - replication forks: two areas on either end of the DNA where double helix separates - forms replication bubble: “bubble” under electron microscope

2. enzyme RNA Primase lays down an RNA primer on DNA strand - RNA primer segment signals beginning of replication 3. Enzyme DNA polymerase III moves along each of DNA strand adds complementary bases of nucleotides floating freely in nucleus A. DNA polymerase III begins synthesis at RNA primer segment B. DNA polymerase I replaces RNA primers with DNA nucleotides

DNA Directionality antiparallel strands 5’ 3’ 3’ 5’

- Directionality: DNA polymerase III reads the template in the 3’ to 5’ direction Daughter DNA strand (since it is complementary) must be synthesized in the 5’ to 3’ direction Strands are antiparallel.

But if there exist no DNA polymerases capable of polymerizing DNA in the 3' to 5' direction, how could this be?

Discontinuous synthesis - synthesis only occurs when a large amount of single strand DNA is present - daughter DNA is then synthesized in 5’ to 3’ direction - leading and lagging strands: - leading strand – continuously synthesized DNA strand - lagging strand - delayed, fragmented, daughter DNA - Okazaki fragmentsdiscontinuous fragmented DNA segments

D. DNA ligase stitches together Okazaki fragments into a single, unfragmented daughter molecule E. enzyme chops off RNA primer and replaces it with DNA

3. DNA polymerase III catalyzes formation of H bonds between nucleotides of template and newly arriving nucleotides which will form daughter DNA 4. Once all DNA is copied, daughter DNA detaches Animation DNA Replication Fork Okazaki fragment animation

End Replication Problem -On one end, RNA primer cannot be replaced with DNA because it is a 5’ (DNA polymerase III can only read from 3’ to 5’) -Causes daughter DNA’s to be shorter with each replication (cell division)

Solution to End Replication Problem telomeres: repeated units of non-coding short nucleotide sequences (TTAGGG) at ends of DNA - become shorter with repeated cell divisions - once telomeres are gone, coding sections of chrom. are lost and cell does not have enough DNA to function ***telomere theory of aging*** - telomerase: special enzyme that contains an RNA template molecule so that telomeres can be added back on to DNA (rebuilds telomeres) ** found in: Cancer cells - immortal in culture Stem cells ** not found in most differentiated cells

Telomeres and Telomerase

Speed of Replication • Multiple replication forks- replication occurs simultaneously on many points of the DNA molecule • Would take 16 days to replicate 1 strand from one end to the other on a fruit fly DNA without multiple forks • Actually takes ~ 3 minutes / 6000 sites replicate at one time • Human chromosome replicated in about 8 hours with multiple replication forks working together

Accuracy and Repair • DNA polymerases proofread as bases are added - can remove damaged nucleotides and replace with new ones for accurate replication • Mismatch repair: special enzymes fix incorrect pairings • Nucleotide excision repair: • Nucleases cut damaged DNA • DNA polymerase and ligase fill in gaps RNA does not have this abilityreason RNA viruses mutate so much

Nucleotide Excision Repair Errors: Pairing errors: 1 in 100, 000 nucleotides Complete DNA: 1 in 10 billion nucleotides

Importance of DNA 1. Controls formation of all substances in the cell by the genetic code 2. Directs the synthesis of specific strands of m RNA to make proteins RNA (Ribonucleic acid) Another nucleic acid takes orders from DNA Used in protein synthesis