Dynamic DNA Notes Chapter 16

Science as a Process: Many scientists contributed to current knowledge of DNA structure and function. Here are some highlights.

A. Morgan (remember fruit flies) Determined that genes are on chromosomes Chromosomes are made of DNA and protein Unknown: Is DNA or Protein the genetic material Best candidate at the time: proteins great heterogeneity specificity of function little known about DNA

Griffith (1928) - studied strep pneumoniae bacteria S strain - living bacteria that is pathogenic (able to cause disease in mouse) R strain - mutant form, nonpathogenic Heat Killed S cells - Harmless

B. Griffith (1928) - studied strep pneumoniae bacteria Inject living S cells into mouse - mouse dies Inject living R cells into mouse - mouse lives Inject heat killed S cells into mouse - mouse lives Mix heat killed S cells with living R cells - mouse dies and living S cells are found in it’s blood

Griffith’s conclusion: Transformation: Some portion of dead cells was transformed into living cells Unknown: What is the transforming agent

C. Avery, McCarty, MacLeod (1944) Purified various chemicals from the heat killed bacteria and attempted to transform the living R cells Conclusion: Only DNA was able to transform the R cells into pathogenic cells so DNA must be the transforming agent

D. Hershey and Chase (1952) - Studied bacteriophages (virus that infects a bacteria) - T2 infects the bacteria e. coli They wanted to confirm that DNA is the transforming agent. Viruses are made of DNA (or RNA) surrounded by a protein coat

Tag DNA with a radioactive isotope of some T2 phages with Phosphorous (only found in DNA) Tag the protein of different T2 phages with radioactive sulfur (only found in protein) Allow the T2 tagged with phosphorous to infect e. coli

Allow the T2 tagged with sulfur to infect other e coli. Put in blender, centrifuge and force bacteria to bottom of test tube leaving virus parts on top (they are lighter) Test the bacteria at bottom of tube for radioactivity and the virus parts on top

Conclusion: Only radioactive phosphorus was found in the bacteria and radioactive sulfur remained with the virus parts on top. Therefore, DNA was being injected into the bacteria so DNA is the transforming agent.

E. More Circumstantial evidence for DNA DNA is exactly doubled before mitosis DNA is distributed equally to daughter cells after mitosis Diploid cells have exactly twice as much DNA as haploid gametes DNA varies from one species to the next

DNA has molecular diversity - there are varying amounts of nitrogen bases (amount of A, G, T and C are not equal) Chargaff’s rule - the amount of A is about the same as T, amount of G is same as C

II. Watson and Crick’s Discoveries (1953) Structure of DNA Used as photograph done by X-ray crystallography to help determine the structure of DNA

Watson and Crick DNA is a Double Helix (like a twisted ladder) composed of 2 strands Backbone - sugar-phosphate (sides of ladder) Middle - nitrogen bases (rungs of ladder) Twist every 10 base pairs

Watson and Crick Purines (2 rings) can’t pair together because the double helix would be too wide Pyrimidines (1 ring) can’t pair together because the double helix would be too narrow Adenine pairs with Thymine making 2 hydrogen bonds Cytosine pairs with Guanine making 3 hydrogen bonds (this explains Chargaff’s rule) The bases can be in any order along the chain, resulting in the huge variety of DNA found in different species

B. DNA Replication The structure of DNA (A-T, G-C) also allows for copying mechanism (form fits function) Semiconservative Model for replication Each original strand serves as a template for the replication of a complementary strand.

An Example Original Strand A-T G-C T-A C-G Separates and becomes template A T G C T A C G A C A G T C T C A G

Rockin’ Reproduction… The Details 1. Helicase unwinds the DNA, producing a replication fork! Single-stranded binding proteins prevent the single strands of DNA from recombining. Topoisomerase removes twists and knots that form in the double-stranded template as a result of unwinding induced by helicase.

Rockin’ Reproduction (cont) 2. RNA primase initiates DNA replication at special nucleotide sequences, origins of replication, with short segments of RNA nucleotides called RNA primers.

Rockin’ Reproduction (cont) 3. DNA polymerase attaches to the RNA primers and begins elongation, the adding of DNA nucleotides to the complement strand. DNA polymerase works from 5’ to 3’.

Rockin’ Reproduction (cont) 4. The leading complementary strand is assembled continuously as the double-helix DNA uncoils.

Rockin’ Reproduction (cont) 5. The lagging complementary strand is assembled in short Okazaki fragments, which are subsequently joined by RNA ligase. This is because DNA polymerase can only work from 5’ to 3’. (so it goes backward on this side).

Rockin’ Reproduction (cont) 6. The RNA primers are replaced by DNA nucleotides. (16.13)

IV. Checking It Over A. Proof-reading DNA polymerase double checks each nucleotide as it is adding them. If one is paired wrong, the polymerase will remove the incorrect base pair and add the correct base Like using the delete key

IV. Checking it Over (cont) B. Mismatch Repair Sometime DNA polymerase makes a mistake in proofreading (oops!). Sometimes mutations happen after polymerase is finished. Special enzymes are used to make repairs! Go Enzymes!

Checking it Over (cont) C. Excision Repair More major type of repair Damaged DNA is cut out using a nuclease and the proper bases are replaced using DNA polymerase and ligase.

V. Finishing Replication A. On the lagging strand, it is impossible to finish replication because there is no existing nucleotide sequence to add to B. Bacteria-Don’t have this problem because DNA is circular

V. Finishing Replication C. Eukaryotes Have special sequences called telomeres at the end of their chromosomes These are multiple copies of a short nucleotide sequence (ex. TTAGGG in humans). As the DNA is replicated, it may lose one of these telomeres with each replication Most organisms have 100-1000 copies of this sequence at their end.

AMY’s SECTION AMY’s SECTION

1. Semiconservative Model for replication *Each original strand serves as a template for the replication of a complementary strand

1. Semiconservative Model for replication Original Strand Separates and becomes template A-T A T G-C G C T-A T A C-G C G

2. Conservative Model: Parent double helix remains intact while a second, all new copy is made

3. Dispersive model: Each strand of old and new DNA contains a mixture of old/new DNA

*The semiconservative model was proposed by Watson and Crick *Meselson and Stahl (late ‘50’s) tested the three possible models of replication by labeling the parent DNA with radioactive Nitrogen, allowed replication to occur. Centrifuged and checked for radioactivity.

Found a hybrid of radioactive and regular DNA Found a hybrid of radioactive and regular DNA. When replicated again, they found both regular DNA and hybrid DNA. Conclusion: DNA replicates by semi-conservative replication

C. Methods of Replication Very precise process (one error per billion nucleotides) Occurs in a short period of time. Process involves many enzymes and other proteins

1. Origin of Replication site where DNA begins replicating *bacteria - have only one origin of replication *eukaryotes - hundreds/thousands of origins of replication *Form a bubble and replication occurs in both directions using the parent strands as templates. At end of bubble is a replication fork

2. DNA polymerases enzymes that catalyze the elongation of DNA by adding the correct base to match the template (50-500 per second)

3. Energy substrate is a nucleoside triphosphate with 3, high energy phosphate bonds attached to the nitrogen base *As the base gets added, it loses two phosphate groups that provide the energy needed to form DNA. The other phosphate and the sugar become the backbone of the ladder

4. Strands are Antiparallel run in opposite directions (see picture) *DNA polymerase can only add nucleotides to a 3’ end, not to the 5’ end so DNA can only grow in the 5’----3’ direction. This side of the DNA is called the Leading Strand

*The other strand grow away from the replication fork and is called the lagging strand because it can only add new nucleotides in small segments, as the bubble grows *The lagging strand is created in small fragments called Okazaki Fragments. These must be joined together by an enzyme called DNA Ligase

5. DNA Polymerase can only add to an existing nucleotide (it can’t start synthesis on it’s own). *To solve this problem, a short stretch of RNA is added at the origin of replication called a Primer. An enzyme called Primase joins the RNA bases together.

*For the leading strand, only one primer is needed because the DNA grows continuously *For the lagging strand, each fragment needs its own primer, thus it takes longer to replicate this side of the DNA

6. Helicase and Single Strand binding Protein aid in unwinding the DNA and holding it apart during replication

7. Proof-reading DNA is a very precise process and two main repair mechanisms work to make sure that DNA is replicated accurately

A. Mismatch repair DNA polymerase double checks each nucleotide as it is adding them. If one is paired incorrectly, the polymerase will remove the incorrect base and add the correct one instead (like using the delete key when typing)

B. Excision Repair DNA damage done after replication occurs (due to radioactivity, harmful chemicals, UV light, etc) -damaged DNA is cut out using a nuclease and the proper bases are replaced using DNA polymerase

8. Finishing Replication On the lagging strand, it is impossible to finish replication because there is no existing nucleotide sequence to add to *Bacteria - don’t have this problem because DNA is circular

*Eukaryotes - have special sequences called telomeres at the end of their chromosome. These are multiple copies of a short nucleotide sequence (i.e. TTAGGG in humans) As the DNA is replicated, it may lose one of these telomeres with each replication. Most organisms have 100-1000 copies of this sequence at their end