Lecture 3 - DNA Replication Flashcards Preview

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What is the mutation rate during DNA replication? What does this mean for the frequency of protein alteration?

1 per 10^9 (billion) nucleotides

Protein: a 400 AA protein suffers 1 alteration every 200,000 years


How often do cells turnover?

Depends on the cell type:
- Epithelial cells of the gut: every few days
- Most every 7 to 15 years (eg: skeletal muscle)
- Frontal cortex: never


How does the mutation rate of DNA replication limit the size of our genome?

Because we want a genome that is large enough to allow complexity, but not so large that the odds of gaining a mutation is high => limits the genome to 50,000 essential proteins, and our genome is 2/3 of that limit


What is the genetic measurement minute?

1 minute = ~40,000 bp along the DNA molecule


Function of gene mut?



Function of gene dna?

DNA replication


Function of gene pol?

DNA polymerase


Function of gene rpo?

RNA polymerase


Function of gene uvr?

UV resistance


Function of gene rec?



Function of gene dam?

DNA adenine methylation


Function of gene lig?

DNA ligase


Function of gene Ter?

Termination of replication


Function of gene ori?

Origin of replication


What is the Meselson-Stahl experiment?

1. Cells were grown for many generations in a medium containing only heavy nitrogen, 15N, so that all the nitrogen in their DNA was 15N, as shown by a single band when centrifuged in a CsCl density gradient.
2. Once the cells had been transferred to a medium containing only light nitrogen, 14N, cellular DNA isolated after one generation equilibrated at a higher position in the density gradient because it had replicated into hybrid helices with both 15N and 14N
3. A second cycle of replication yielded a hybrid DNA band and another band, containing only [14N]DNA, confirming semiconservative replication


What is a theta, θ, structure?

Structure resulting from the replication of a circular chromosome because both strands are replicated simultaneously and bidirectionally


How many replication forks per origin of replication?



In what direction are nucleic acids synthesized?

5' => 3'


In what direction is the DNA template being read?

3' => 5'


What are the 2 types of eukaryotic replication and which one is more common?

1. Unidirectional
2. Bidirectional (more common)


How to distinguish between unidirectional and bidirectional replication?

Autoradiography: when tritium (3H) is added for a short period just before replication is stopped, the label would be found at one or both replication forks, respectively


How long are Okazaki fragments?

150 to 200 nucleotides long


What enzyme catalyzes DNA elongation during replication?

DNA polymerase


Describe the 2 steps of DNA elongation and write out the equation.

1. Mg2+ on DNA polymerase between 2 Asp residues facilitates the attack of the free 3' (-OH) on primer strand to the alpha phosphate on an dNTP
2. Another Mg2+ on DNA polymerase between 3 Asp residues facilitates the release of PPi

(dNMP)n + dNTP => (dNMP)n+1 + PPi


How do the Mg2+ of DNA polymerase catalyze DNA elongation during replication?

Both ions stabilize the structure of the pentacovalent transition state by interacting with negative charges on Os of the 3 phosphate groups of the incoming dNTP


Describe the mechanism of RNA replication.

RNA polymerases use a similar mechanism than DNA polymerase I


What are the 2 elements that DNA polymerases require?

1. DNA template
2. RNA primer


What are 2 elements that help ensure the high level of accuracy of DNA replication?

1. The standard A=T and G≡C base pairs have very similar geometries, and an active site sized to fit one will accommodate the other, so the active site of DNA polymerase which will be very specific to proper base pair
2. DNA polymerase contains 3' => 5' exonuclease activity to chew back DNA to proofread its own work


How is it possible for an improper base pair to fit in the active site of DNA polymerase?

Chemical or randomly modified base that can fit


Describe the position of the 3' -> 5' exonuclease activity on DNA polymerase and how it works.

The exonuclease activity is located ahead of the polymerase activity as the enzyme is oriented in its movement along the DNA. A mismatched base has different H bonds with its base pair and impedes translocation of DNA polymerase I to the next site. Sliding backward, the enzyme corrects the mistake with its 3′→5′ exonuclease activity, then resumes its polymerase activity in the 5′→3′ direction.