CHAPTER 16MOLECULAR BASIS OF INHERITANCE

DNA as genetic material?

• Deducted that DNA is the genetic material• Initially worked by studying bacteria & the

viruses that infected them• 1928 – Frederick Griffiths – studied

bacterial transformation using Streptococcus pneumonia

• Used a pathogenic (disease causing) strain & nonpathogenic strain

• Killed pathogenic strain w/ heat & mixed with living nonpathogenic strain

• Some of the nonpathogenic transformed to pathogenic• Oswald-Avery – deducted that the transforming agent

was DNA

Hershey – Chase Experiment

• Used viruses that infect bacteria –bacteriophage or phage

• T2 is common virus of E. coli –bacteria of mammal intestines

• Radioactively tagged sulfur in proteins & the phosphorous in DNA of the T2 phage

• Infected E.Coli with T2 phage• Radioactive sulfur in supernate – deducted that

protein did not enter bacteria• Radioactive phosphorous in cells – deducted

that DNA enter cells

T2 Phage

Review ofDNA Structure

• Erwin Chargaff –studied the nitrogen bases in the DNA of many species

• Noticed that amount of adenine equals thymine and amount of cytosine equals guanine

• Chargaff’s rule

Building a Structural Model of DNA

• Rosalind Franklin & Maurice Wilkins –used X - ray crystallography to uncover the double helix of DNA

• Sugar – phosphate backbone on outside• Watson & Crick – purine paired with a

pyrimidine (purine to purine would make helix too wide)

• Side groups on each nitrogen base forms a hydrogen bond between them

Watson & Cricks Hypothesis of DNA Replication

• Each strand serves as a template for the new strand

Origin of DNA Replication

1. Replication begins at specific sites where the two parental strands separate & form replication bubbles

2. Bubbles expand laterally, as DNA replication proceeds in both directions

3. Replication bubbles fuse & synthesis of daughter strands is complete

Antiparallel Elongation

• Enzyme DNA polymerase adds new nucleotides to the template strands

• Two strands of DNA run antiparallel(opposite directions)

• DNA polyermase III adds nucleotides in the 5’ to 3’ direction

• Leading strand – new strand made in 5’ to 3’direction

• Lagging strand – strandproduce by nucleotides being added in the direction away from the replication fork ; synthesis occurs with a series of segments called Okazaki fragments

• DNA Ligase – enzyme that joins the sugar –phosphate backbones of Okazaki fragments to form new DNA strand

Priming DNA Synthesis

• DNA polymerases cannot initiate synthesis of a polynucleotide (can only add nucleotides to the 3’ end)

• Primer – initial nucleotide chain; short & consist of either DNA or RNA ; initiate DNA replication

• Primase – enzyme that can start a stretch of RNA from scratch

1. Primase joins RNA nucleotide to primer2. DNA polymerase III adds DNA

nucleotides to 3’ end of primer3. Continues adding DNA nucleotides4. DNA polymerase I replaces the RNA

nucleotide with DNA versions

Proteins For Leading & Lagging Strands

Corrects “overwinding”ahead of replication fork

Topiosomerase

Stabilizes single-stranded DNA until used as a template

Single-strand binding protein

Unwinds double helix at replication fork

Helicase

FUNCTIONPROTEIN

DNA Ligase

DNA pol I

DNA pol III

Primase

Protein

Joins the 3’ end of DNA that replaces the primer to rest of leading strand

Removes primer from 5’ end of leading strand & replaces it with DNA

Continuously synthesizes the leading strand, adding on to primer

Synthesizes a single RNA primer at 5’ end of leading strand

Function for Leading Strand

Joins Okazaki fragments

Removes the primer from the 5’end of each fragment & replaces it with DNA, adding on to 3’ end of adjacent fragment

Elongates each Okazaki fragment, adding on to its primer

Synthesizes an RNA primer at the 5’ end of each Okazaki fragment

Function for Lagging Strand

Summary of DNA replication1. Helicase unwinds double helix2. Single-stranded binding proteins stabilize template strands3. Leading strand is synthesized continuously in 5’-3’ direction by

DNA pol III4. Primase begins synthesis fo RNA primer for 5th Okazaki fragment5. DNA pol III completes synthesis of fourth fragment; when it

reaches RNA primer on third fragment it will break off & move toreplication fork, and add DNA nucleotides to 3’ end of fifth fragment

6. DNA pol I removes primer from 5’ end of second fragment, replacing it with DNA nucleotides that it adds one by one to 3’ end of third fragment. Replacement of last RNA nucleotide with DNA leaves the sugar-phosphate backbone with a free 3’ end

7. DNA ligase binds 3’ end of the second fragment to the 5’ end of the first fragment

1

2 3

4

5 6

7

Proofreading & Repairing DNA

• DNA polymerases proofread nucleotides & immediately replace any incorrect pairing

• Some mismatched nucleotides evade proofreading or occur after DNA synthesis is complete – damaged

• Mismatch repair – cells use special enzymes to fix incorrect nucleotide pairs

• 130 repairing enzymes identified in humans to date

Nucleotide Excision Repair

• Thymine dimer – type of damage caused by UV radiation

• Buckling interrupts DNA replication

Replicating Ends of DNA Molecules

• Since only adds to 3’ end, no way to complete 5’ ends

• Even if Okazaki fragment is started with a an RNA primer, it can not be replaced with DNA when removed

• Results in shorter DNA molecules• Problem exists only in eukaryotes due to

linear DNA• Prokaryote DNA is circular

• Telomeres – nucleotide sequences at ends of DNA molecules

• Consist of multiple repetitions of a short sequence• TTAGGG – human telomere• Telomeres prevent the erosion of genes near the ends of

DNA molecules• Somatic cells of older individuals have shorter telomeres

– related to aging?• In germ cells the erosion of telomeres can lead to loss of

essential genes• Telomerase – enzyme that catalyzes the lengthening of

telomeres; restores the original length