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Chapter 16Chapter 16
Molecular Basis of Inheritance
(DNA structure and Replication)
Helicase Enzyme
What is the genetic material? DNA or protein?
The Amazing Race
1928 Griffith – transformation of pneumonia bacterium
1944 Avery – further studied
transformation by destroying lipids,
CHO, and proteins
1947 Chargaff –
• Quantified purines and pyrimidines
• Suggested base pairing rules (A=T, C=G)
1950 Wilkins and Franklin – DNA X-rays
(a) Rosalind Franklin (b) Franklin’s X-ray diffraction
photograph of DNA
1952 Hershey and Chase – bacteriophages – incorporation of
radioactive viral DNA in new phages
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive protein
Radioactive DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive protein
Radioactive DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
Empty protein shell
Phage DNA
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive protein
Radioactive DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
Empty protein shell
Phage DNA
Centrifuge
Centrifuge
Pellet
Pellet (bacterial cells and contents)
Radioactivity (phage protein) in liquid
Radioactivity (phage DNA) in pellet
1953 Watson and Crick – DNA Model
1962 Nobel Prize awarded to Watson and Crick and Wilkins
** Conclusion: DNA = Genetic Material, not Protein
Models of DNA Replication
Semi-Conservative Model(1950s - Meselson and Stahl)
Fun DNA Replication Facts
• 6 billion bases in human cell = 2 hours of replication time
• 500 nucleotides added per second
• Accurate (errors only 1 in 10,000 base pairs)
Anti-Parallel
Structure of DNA
Mechanism of Replication Step 1
• Origins of Replication = Special site(s) on DNA w/Specific sequence of nucleotides where replication begins– Prokaryotic Cells =
1 site (circular DNA)– Eukaryotic Cells =
several sites (strands)
Steps 2 - 5• Helicase: (enzyme) unwinds
DNA helix forming a “Y” shaped replication fork on DNA
• Replication occurs in two directions, forming a replication bubble
• To keep strands separate, DNA binding proteins attach to each strand of DNA
• Topoisomerases: enzymes that work w/helicase to prevent “knots” during unwinding.
Step 6 - Priming
• Priming = due to physical limitation of DNA Polymerase, which can only add DNA nucleotides to an existing chain
• RNA primase – initiates DNA replication at Origin of Replication by adding short segments of RNA nucleotides.
• Later these RNA segments are replaced by DNA nucleotides by DNA Pol.
Step 7• DNA Pol. = enzyme that
elongates new DNA strand by adding proper nucleotides that base-pair with parental DNA template
• DNA Pol. can only add nucleotides to the 3’ end of new DNA, so replication occurs from a 5’ to 3’ direction
• Leading vs. Lagging Strand results
Leading vs. Lagging Strand
• Leading Strand: strand that can elongate continuously as the replication for progresses
• Lagging Strand: strand that cannot elongate continuously and moves away from replication fork.
• Short Okazaki fragments are added from a 5’ to 3’ direction, as replication fork progresses.
3’5’
3’ 5’
5’
3’
3’5’
Step 8• DNA Ligase = enzyme that “ligates” or covalently
bonds the Sugar-Phosphate backbone of the short Okazaki fragments together
• Primers are required prior to EACH Okazaki fragment
Step 10: Fixing Errors
• DNA Pol. Proofreads as it elongates
• Special enzymes fix a mismatch nucleotide pairs
• Excision Repair:– Nuclease: Enzyme
that cuts damaged segment
– DNA Pol. Fills in gap with new nucleotide
Mutations
• Thymine Dimers (covalent bonding btwn Thymine bases) –often caused by over-exposure to UV rays DNA buckeling skin cancer results, unless corrected by excision repair
• Substitutions: incorrect pairing of nucleotides• Insertions and Deletions: an extra or missing
nucleotide causes “frameshift” mutations (when nucleotides are displaced one position)
Problems with Replication
• Since DNA Polymerase can only add to a 3’ end of a growing chain, the gap from the initial 5’ end can not be filled
• Therefore DNA gets shorter and shorter after each round of replication
Solution?• Bacteria have circular DNA
(not a problem)• Ends of some eukaryotic
chromosomes have telomeres at the ends (repeating nucleotide sequence that do not code for any genes)
• Telomeres can get shorter w/o compromising genes
• Telomerase = enzyme that elongates telomeres since telomeres will shorten
Telomerases are not in most organisms
• Most multicellular organisms do not have telomerases that elongate telomeres (humans don’t have them)
• So, telomeres = limiting factor in life span of certain tissues
• Older individuals typically have shorter telomeres