DNA Discovery/Replication + The Central Dogma (not done)

Probably don't need to know all the names but adding them just in case.

What’s an Assay?

A way of measuring something (either a substance or an abstract phenomenon)

What 2 things do you need to purify DNA

1. A method of isolating cell components

2. An assay for genetic materialness

Who (and when) figured out a method for isolating cell components?

What did he call the main constituent of the nucleus?

Fredrich Miescher in 1868

Nuclein (which turned out to be DNA)

Can an extract from dead bacterial cells genetically transform living bacterial cells? Explain the experiment that was done to answer this.

In short, yes it can. Experiment explained in photo

Substance responsible for transformation = the transforming principle

Who figured out what the transforming principle was and how?

Avery, Macleod, and McCarty found that no component of the S bacteria could transform the R bacteria except DNA → therefore DNA = the transforming principle

What is the bacteria lifecycle?

1. Infection (of something)

2. Injection (of DNA)

3. Replication (of DNA)

4. Expulsion (to go infect more things)

Who did the experiment to confirm that DNA was the transforming principle? What was the experiment?

Al Hershey and Martha Chase:

• took a virus made of just DNA and Protein → labled virus with EITHER the

  • DNA with ³²P (radioactive phosphorus)

  • Protein with ³⁵S (radioactive sulfer)

• allowed virus to infect bacteria and then threw the mix in a blender (taking off protein coat) → whatever stayed had to be the genetic material)

• Infected bacteria produced more virus with ³²P but basically no ³⁵S

• SO it had to be the DNA

What did Watson and Crick decide was he key to getting a better understanding of DNA replication? What is the model they eventually put together?

The 3D structure of DNA → made model where the n-bases are in middle and phosphate backbone runs along outside (backbone is negatively charged + exposed to water)

• The hydrogen bonds between A/T and G/C are most stable

• Always pairing a purine and a pyrimidine means that the distance between the 2 strands stays constant

Who’s unpublished data helped figure out the 3D structure of DNA? What did they figure out?

Rosalind Franklin and Maurice Wilkins found that DNA is 4 subunits, the four nucleotides adenosine, guanosine, cytidine and thymidine.

Who showed the thing about ratios of nucleotides in DNA? What did he show?

Erwin Chargaff → amount of A = T and the amount of G = C even though the ratio A+T/G+C can vary widely from one organism to the next

Who showed that DNA is a double helics? How did they show it?

Franklin and Wilkins used the technique of X-ray diffraction (whatever that is…)

Do each individual strand of DNA have a polarity?

Yes

The two strands of DNA are [parallel/antiparallel], and are [complements/reverse complements] of each other.

This structure suggests what 2 things?

Anti-parallel (run in the opposite direction); reverse-complements (one strand complements the other - not mirrored)

1. sequence of nucleotides doesn’t affect overall structure → info can be encoded arbitrarily

2. the 2 strands bind by complementary base pairing so the they both contain identical information → separating the strands and binding them to 2 new strands = DNA replication

3 possible ways that DNA gets replicated + which is the correct way

1. Conservative replication = each strand makes a new strand and the 2 old strands re-bind to each other

2. Semi-conservative replication = each old strand made remains bound to the new strand (THIS IS WHAT ACTUALLY HAPPENS)

3. Dispersive replication = DNA breaks apart and rejoins to produce 4 strands, each with a mix of old and new DNA

How did Meselson and Stahl showed that DNA replication was semiconservative?

• labeled DNA by growing bacteria with a heavier isotope of nitrogen (¹⁵N) → measured weight with centrifuge

• After one duplication in the presence of normal nitrogen the duplicated DNA was ½ as heavy

• After 2 duplications half the DNA was ½ as heavy and ½ the DNA was normal (light) weight.

Only semiconservative replication would produce this result

In a live cell, what (2 things) does DNA need to do to replicate?

Which enzymes within cells help with each problem?

1. Unwind the strands →Helicase

2. Create a ragged end (a stretch of the DNA thats partly double stranded and partly single stranded) → Primase

What is DNA Polymerase?

An enzyme that

1. grabs a nucleotide that is complementary to the next nucleotide in line on the template strand

2. takes nucleotide and breaks the bond between the alpha phosphate (one attached to the sugar) and the beta phosphate

3. Then attaches the alpha phosphate to the 3'hydroxyl group of the last nucleotide on the strand that's being extended (why we say the DNA synthesis always occurs 5' to 3'

What does Helicase do? What forms when they do their job?

Expands energy to unwind DNA → as they do this a bubble forms with “replication forks” on either end of where the old double stranded DNA is being split → act as template for formation of 2 new strands

What does Primase do?

Adds a short stretch of complementary DNA (a primer) which acts as a ragged end.

What are origins of replication?

The specific (non-random) spots where DNA polymerase 3 initiates replication

Leading vs Lagging strand

*Okazaki Fragments

Leading strand → continuous replication

Lagging strand → replicates in short stretches (bc it’s going backwards)

• Continually puts down primers every few hundred bases

• the short stretches of DNA = Okazaki Fragments

DNA Polymerase 3 vs DNA Polymerase 1

P3: Initiates replication

P1: chews up RNA primers + fills in gaps within the lagging strand using newly synthesized DNA as a primer

What does DNA ligase do?

Joins the ends of newly synthesized strands → DNA replication then finished