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

Question generated from Griffith

• What was the heritable agent, protein or

DNA?

1944 Avery –

further studied

transformation by

destroying lipids,

CHO, and

proteins

Conclusions of Avery, et. al

• DNA is heritable agent.

• Results, unfortunately, were not enough to

sway the opinion of most scientists.

– How could one molecule cause so much

variation?

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

Video of Hershey –

Chase Expt

Significance of Hershey &

Chase Expt.• Unambiguously proved that DNA is the

agent of heredity, not proteins.

1953 Watson and Crick – DNA

Model

1962 Nobel Prize awarded to

Watson and Crick and Wilkins

** Conclusion: Now that we

know DNA’s structure, we can

figure out what it does and how it

does it.

DNA Replication

Models of DNA Replication

Semi-Conservative Model

(1950s - Meselson and Stahl)

• Meselson & Stahl Experiment

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

Overview of DNA

Replication

• DNA Replication Fork

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

• Many multicellular

organisms do not have

telomerases that elongate

telomeres (humans have

them in some cells)

• So, telomeres = limiting

factor in life span of certain

tissues

• Older individuals typically

have shorter telomeres

Homework…

• Watch Bozeman Videos #27

• DNA & RNA – Parts 1 and 2 (2 videos)