DNA Controls Lifes Processes

DNA occurs in different forms

Prokaryotes
1-2 Chromosomes
Circular, small Occur singly (haploid) Carry essential genes

Eukaryotes
Intranuclear chromosomes
Linear Usually in duplicate pairs (diploid)

One or more plasmids

Carries other genes beneficial to the cell

Extranuclear DNA in organelles

Circular, small Replicate independently of the cell

Chromosomes are supercoiled DNA

Levels of Organization in a Eukaryotic Chromosome

DNA + Histone = spooled DNA

Every gene has a specific address (locus) on a chromosome or plasmid.

Genes

DNA differs from RNA

The Role of DNA

Transcription Translation

DNA is the molecule of heredity

Genes are passed from parent to offspring (replication)

DNA encodes the instructions for making proteins

DNA is first coverted into RNA (transcription) RNA is translated into protein (translation)

History of DNA Discovery

Griffith Avery (1939)
Showed proteins are not the molecules of heredity.

Hershey Chase (1944)

Showed DNA is the molecule of heredity.

Watson & Crick (1953)

Described the structure of DNA.

Griffith-Avery Experiments (1939): Transformation

Heat-killed diseasecausing strain of S. pneumoniae Mixture of heat-killed and live non-disease-causing strains of S. pneumoniae

Live diseasecausing strain of S. pneumoniae

Live non-diseasecausing strain of S. pneumoniae

HersheyChase Experiments (1944)

DNA is the molecule of heredity

Watson & Crick (1953)

Described the structure of DNA
Double helix = Two strands of DNA paired together. Paired nucleotide bases in the middle, sugarphosphate backbone outside.
Adenine Thymine Guanine Cytosine

The Structure of DNA

5 4 3 2 1

How Replication Starts

The chromosome unwinds. The two DNA strands separate in the middle. New DNA is made according to the bases on the original strands.

DNA Replication
Strands unwind and separate. New bases are added according to the basepairing rule.

DNA Replication is semi-conservative (Meselson & Stahl)

Parent strands separate. New (daughter) strands are synthesized according to the bases of the parent strands. Results in two half-old, half-new molecules.

Ribose Sugar

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Phosphate (PO4) P

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P A

Nitrogenous organic base

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P A Nucleotide

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P A P C Phosphodiester bond (Links 2 nucleotides)

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P A P C

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P A P C

G
P T

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P A P C

G
P T

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P A P C

G
P T

P G
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P A P C

G
P T

Hydrogen bonds

P P G C

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P A P C

G
P T

P P G C

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P A P C

G
T

P P G C

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P A P C

P P P P P

G
T

P G C

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P A C G
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P P

G
T

P P G C

P C

P P P P P P P A C G P T C C G

G
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P G

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P P A C P G P G P P P P C A T C C G G

G
T

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P P A C P G T P G P P P P C A T C C G G

G
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G
A C

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P P A C P G T P G P P P P C A T C C G G

G
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A C T

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A A
C

P P A C P G T P G P P P P C A T C C G G

G
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G
A C T

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P P A C P G T P G P P P P C A T C C G G

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G T A C A T

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P A C T A P G C G P G C P P P P T C A T G C T P A T C C G G

G
T

G
G T A

C
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P A C C T A P G G P G C P P P P T C A T P A G

G
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G G T
A

P C
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G
A G T

Watson, Crick, Wilkins and Franklins Model

DNA Replication: the Players

Parent DNA, Daughter DNA Helicase DNA Polymerases DNA Ligase The Nucleotides
Adenine = Thymine Guanine Cytosine

Deoxyribose sugar

Act I: Unwinding

Act II: Synthesis

12.15 DNA Replication Takes Many Steps Slide number: 2

1 Helicase binds to origin and separates strands.

3 1 5 3

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12.15 DNA Replication Takes Many Steps Slide number: 3

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining.

3 5 3

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12.15 DNA Replication Takes Many Steps Slide number: 4

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 3 5 3 5 3 3

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12.15 DNA Replication Takes Many Steps Slide number: 5

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 3 3 5 3 5 3 5

5 3

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12.15 DNA Replication Takes Many Steps Slide number: 6

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 3 Overall direction of replication 3 3 4 5 3 5

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12.15 DNA Replication Takes Many Steps Slide number: 7

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 3 Overall direction of replication 3 3 4 5 3 5

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12.15 DNA Replication Takes Many Steps Slide number: 8

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 3 Overall direction of replication 3 3 4 5 3 5 5

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12.15 DNA Replication Takes Many Steps Slide number: 9

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 3 Overall direction of replication 3 3 4 5 3 5 5

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12.15 DNA Replication Takes Many Steps Slide number: 10

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 3 Overall direction of replication 3 3 5 5 3 5 5

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12.15 DNA Replication Takes Many Steps Slide number: 11

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 3 5 3 5 Overall direction of replication

6 3 3 5

6 Leading (continuous) strand synthesis continues in a 5 to 3 direction.

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12.15 DNA Replication Takes Many Steps Slide number: 12

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 3 5 7 3 5 3 5 Overall direction of replication 3 3 5

6 Leading (continuous) strand synthesis continues in a 5 to 3 direction. 7 Discontinuous synthesis produces Okazaki fragments on the 5 to 3 template.

Okazaki fragment

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12.15 DNA Replication Takes Many Steps Slide number: 13

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 3 5 7 35 3 5 Overall direction of replication 3 3 5

6 Leading (continuous) strand synthesis continues in a 5 to 3 direction. 7 Discontinuous synthesis produces Okazaki fragments on the 5 to 3 template.

Okazaki fragment

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12.15 DNA Replication Takes Many Steps Slide number: 14

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 3 3 3 5

3 5

35

3 5

6 Leading (continuous) strand synthesis continues in a 5 to 3 direction. 7 Discontinuous synthesis produces Okazaki fragments on the 5 to 3 template.

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12.15 DNA Replication Takes Many Steps Slide number: 15

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 3 3 3 5

3 5

35

3 5

6 Leading (continuous) strand synthesis continues in a 5 to 3 direction. 7 Discontinuous synthesis produces Okazaki fragments on the 5 to 3 template.

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12.15 DNA Replication Takes Many Steps Slide number: 16

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 3 3 3 5

3 5

6 Leading (continuous) strand synthesis continues in a 5 to 3 direction. 7 Discontinuous synthesis produces Okazaki fragments on the 5 to 3 template. 8 DNA polymerase I removes RNA primers, replaces them with DNA.

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12.15 DNA Replication Takes Many Steps Slide number: 17

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 3 3 3 5

3 5

6 Leading (continuous) strand synthesis continues in a 5 to 3 direction. 7 Discontinuous synthesis produces Okazaki fragments on the 5 to 3 template. 8 DNA polymerase I remove RNA primers, replaces them with DNA. 9 DNA ligase repairs nick in sugar phsophate backbone.
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12.15 DNA Replication Takes Many Steps Slide number: 18

1 Helicase binds to origin and separates strands. 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template. 4 DNA polymerase III adds DNA nucleotides to the RNA primer. 5 DNA polymerase proofreading activity checks and replaces incorrect bases just added. 5 3 3 3 5

3 5

6 Leading (continuous) strand synthesis continues in a 5 to 3 direction. 7 Discontinuous synthesis produces Okazaki fragments on the 5 to 3 template. 8 DNA polymerase I remove RNA primers, replaces them with DNA. 9 DNA ligase repairs nick in sugar phsophate backbone.
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Summary of DNA Synthesis

Prokaryotic v. Eukaryotic Replication

Telomeres & Telomerase

Telomeres are repeating DNA sequences at the ends of chromosomes (~15,000 bp)
Prevent chromosomes from losing pair sequences at their ends Prevent chromosomes from fusing to each other Lost on lagging strand during replication (25200 bp/division)
Once a critical length is reached, the DNA can no longer replicate and the cell will undergo apoptosis.

Telomeres & Telomerase

Telomerase is an enzyme that elongates the lagging strand by adding bases to the ends
Found in fetal cells, adult germ cells, and tumor cells Generally absent in somatic cells

What does this mean for a cell?

Shortening telomeres = cellular aging Telomerase stimulates the cell to continually grow and divide = immortal cancer in somatic cells

Act III: Proofreading

DNA Pol sometimes makes mispairing mistakes in replication Mispairing mistakes are usually corrected by DNA Pol, but it isn't infallible.
DNA Pol usually misses, or leaves, one mistake uncorrected for every 109 to 1012 bases it reads.

In addition to the mistakes of DNA Pol, chemicals (e.g., thalidomide, uranium) and UV also cause changes in DNA (mutations).

Correcting Mutations
Excision repair
"bad" or mutated DNA is cut out and another DNA Pol will re-synthesize DNA based on the parent strand. Corrects about 99% of mispairings missed by DNA Pol.

Deficiencies in DNA repair mechanisms can result in several diseases

Xeroderma pigmentosum: extreme susceptibility to UV (skin, eye, and tongue cancers prevalent) Ataxia: neurological disorders; immunological deficiencies; high susceptibility to lymphomas

Application of DNA Replication: Polymerase Chain Reaction (Ch 19.2)

In PCR, a gene of interest is identified, isolated, and amplified to create many identical copies Steps
1.

2. 3. 4. 5.

Create a reaction mix of DNA bases, a DNA sample containing the gene of interest, primers, and Taq polymerase Heat mix to 95C to separate the strands of the double helix Cool to 50-60C to allow primers to anneal Heat to 72C to allow Taq Pol to synthesize new DNA Repeat Steps 2-4 as many times as necessary to get desired amplification, usually ~30

Result: one cycle doubles the amount of DNA; n cycles = 2n DNA molecules

At a Minimum, You Should Be Able To:

What are the four nucleotides of DNA? What are their molecular parts? What kind of bond holds two DNA chains together in a double helix? Explain the pairing relationships between the bases. Explain semiconservative replication. Explain the process of DNA replication. How is DNA replication different between prokaryotes and eukaryotes? Ch 14: TYU #1, 2, 3; AC #2b

You Should Be Able To:

Name the three molecular parts of a nucleotide in DNA. Name the four different kinds of nitrogen-containing bases that occur in the nucleotides of DNA. What kind of bond holds two DNA chains together in a bouble helix? Which nucleotide base pairs with adenine? Which pairs with guanine? When regions of a double helix are unwound during DNA replication, do the two unwound strands join back together again after a new DNA molecule has formed? The four bases in DNA may differ greatly in relative amounts from one species to the next -- yet the relative amounts are always the same among members of a single species. How does base pairing explain these twin properties -- the unity and diversity -- of DNA molecules? State the Central Dogma Rule of Molecular Biology. Are the proteins specified by eukaryotic DNA assembled on the DNA molecule? If so, state how. If not, tell where they are assembled, and on which molecules.

Summary of DNA Synthesis