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

DNA i

Flash Overview

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