Lecture 3 - DNA Replication Flashcards Preview

SMP - Genetics > Lecture 3 - DNA Replication > Flashcards

Flashcards in Lecture 3 - DNA Replication Deck (78)
Loading flashcards...
1

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

2

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

3

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

4

What is the genetic measurement minute?

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

5

Function of gene mut?

Mutagenesis

6

Function of gene dna?

DNA replication

7

Function of gene pol?

DNA polymerase

8

Function of gene rpo?

RNA polymerase

9

Function of gene uvr?

UV resistance

10

Function of gene rec?

Recombination

11

Function of gene dam?

DNA adenine methylation

12

Function of gene lig?

DNA ligase

13

Function of gene Ter?

Termination of replication

14

Function of gene ori?

Origin of replication

15

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

16

What is a theta, θ, structure?

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

17

How many replication forks per origin of replication?

2

18

In what direction are nucleic acids synthesized?

5' => 3'

19

In what direction is the DNA template being read?

3' => 5'

20

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

1. Unidirectional
2. Bidirectional (more common)

21

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

22

How long are Okazaki fragments?

150 to 200 nucleotides long

23

What enzyme catalyzes DNA elongation during replication?

DNA polymerase

24

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

25

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

26

Describe the mechanism of RNA replication.

RNA polymerases use a similar mechanism than DNA polymerase I

27

What are the 2 elements that DNA polymerases require?

1. DNA template
2. RNA primer

28

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

29

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

30

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.