Ch. 16 Warm-Up 1. Draw and label a nucleotide. 2. What is the complementary DNA strand to: DNA: A T C C G T A T G A A C 3. Explain the semiconservative model of DNA replication.

Ch. 16 Warm-Up 4. Chargaff’s Rules: If cytosine makes up 22% of the nucleotides, then adenine would make up ___ % ?

THE MOLECULAR BASIS OF INHERITANCE Chapter 16

What you must know The structure of DNA. The major steps to replication. The difference between replication, transcription, and translation. The general differences between the bacterial chromosome and eukaryotic chromosomes. How DNA is packaged into a chromosome.

16.1 Problem: Is the genetic material of organisms made of DNA or proteins?

Frederick Griffith (1928) Transformation

Frederick Griffith (1928) Conclusion: living R bacteria transformed into deadly S bacteria by unknown, heritable substance Oswald Avery, et al. (1944) Discovered that the transforming agent was DNA

Hershey and Chase (1952) Bacteriophages (bacteria – eaters): T2 virus infects bacteria; (Has two parts protein and DNA) But which part is the transforming agent????? Tagged Protein = Radioactive S Tagged DNA = Radioactive P

Hershey and Chase (1952) Mixed radioactive sulfur phages with bacteria – incorporated into their protein Mixed radioactive phosphorous phages with bacteria – incorporated into their DNA Conclusion: DNA entered infected bacteria  DNA must be the genetic material!

Edwin Chargaff (1947) Known DNA is a polymer of nucleotides with three parts Chargaff’s Rules: DNA composition varies between species Ratios: %A = %T and %G = %C

Rosalind Franklin (1950’s) Worked with Maurice Wilkins X-ray crystallography = images of DNA Provided measurements on chemistry of DNA

James Watson & Francis Crick (1953) Discovered the double helix by building models to conform to Franklin’s X-ray data and Chargaff’s Rules.

Structure of DNA DNA = double helix “Backbone” = sugar + phosphate “Rungs” = nitrogenous bases

Structure of DNA Nitrogenous Bases Pairing: Adenine (A) Guanine (G) Thymine (T) Cytosine (C) Pairing: purine + pyrimidine A = T G Ξ C Purine – 2 Rings Pyrimidine – 1 ring

Structure of DNA Hydrogen bonds between base pairs of the two strands hold the molecule together like a zipper.

Antiparallel: one strand (5’ 3’), other strand runs in opposite, upside-down direction (3’  5’) Phosphate group The 5' and 3' mean "five prime" and "three prime", which indicate the carbon numbers in the DNA's sugar backbone. The 5' carbon has a phosphate group attached to it and the 3' carbon a hydroxyl group. This asymmetry gives a DNA strand a "direction" Hydroxyl group

DNA Comparison Double-stranded Circular One chromosome In cytoplasm 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

16.2 Problem: How does DNA replicate?

Replication: Making DNA from existing DNA 3 alternative models of DNA replication

Meselson & Stahl

Replication is semiconservative

DNA Replication Video

Bacteria Replication Begins at single site - origin of replication Proteins that initiate replication recognize the sequence and sep. two strands “Bubble” Both directions 500 nucleotides per second

Eukaryotic Replication: Helicase: unwinds DNA at many origins of replication Initiation proteins separate 2 strands  forms replication bubble Proceeds in both directions Primase: puts down RNA primer to start replication, short stretch DNA polymerase III: adds complimentary bases to leading strand (new DNA is made 5’  3’) Lagging strand grows in 3’5’ direction by the addition of Okazaki fragments DNA polymerase I: replaces RNA primers with DNA DNA ligase: seals fragments together

1. Helicase unwinds DNA at origins of replication and creates replication forks

2. Primase adds RNA primer

3. DNA polymerase III adds nucleotides in 5’3’ direction on leading strand 50 nucleotides per second in humans

Leading strand vs. Lagging strand

Okazaki Fragments: Short segments of DNA that grow 5’3’ that are added onto the Lagging Strand DNA Ligase: seals together fragments

Replication http://media.pearsoncmg.com/bc/bc_0media_b io/bioflix/bioflix.htm?10adnarep http://www.hhmi.org/biointeractive/dna- replication-advanced-detail

Problem: Who bothers proofreading these days?

Proofreading and Repair Eukaryotic error: 1 in 10 billion DNA polymerases proofread as bases added Mismatch repair: special enzymes fix incorrect pairings A mutation to one of these “Special Enzymes” could lead to cancer Cell is under constant surveillance Trying to repair DNA changes before they become mutations Nucleotide excision repair: Nucleases cut damaged DNA and fill the gaps

Problem: If you can only add bases from the 3’->5’ end Problem: If you can only add bases from the 3’->5’ end. What happens when the primer gets the to 5’ end?

Problem at the 5’ End DNA polymerase only adds nucleotides to 3’ end No way to complete 5’ ends Leads to shorter and shorter DNA strands with staggered ends!

So what’s a guy to do?

Telomeres: repeated units of short nucleotide sequences (TTAGGG) at ends of DNA (don’t code for anything) Telomeres “cap” ends of DNA to postpone erosion of genes at ends – about 100 to 1000 times – buffer zone Telomerase: enzyme that adds to telomeres Germ cells Aging Cancer cells have high telomerase activity Video Telomeres stained orange at the ends of mouse chromosomes

DNA Replication review

BioFlix: DNA Replication http://media.pearsoncmg.com/bc/bc_0media_b io/bioflix/bioflix.htm?8apdnarep

Separation of DNA Strands What enzyme unzips the strands?

Leading

Lagging

DNA Extraction Strawberry Bonanza