DNA Replication I + II

Question 1

What were the three original theories of DNA replication?

Answer 1

-Semi-conservative theory
-Conservative theory
-Dispersive theory

Question 2

What theory did the Messelson-Stahl experiment support?

Answer 2

This experiment supported the theory that DNA replication is semi-conservative

Question 3

What are the 3 basic steps of the Messelson-Stahl experiment?

Answer 3

1. Grow bacteria in solution of heavy nitrogen-15 isotopes, where both DNA strands will be tagged with H(eavy) isotope
2. Transfer ‘heavy’ bacterial solution to a solution of light nitrogen-14, so that as replication progresses the new DNA strands will be identifiable by the L(ight) nitrogen
3. Run the bacterial solution thorugh a density-gradient centrifuge for 48 hours; observe that the DNA has become separated based upon density

Question 4

What are the 4 steps of density gradient centrifugation?

Answer 4

1. Mix CaCl solution and cell extract, place in centrifuge
   
   2. Centrifuge at high speed for ~48 hours
   3. Molecules move to positions in the tubes where their density equals that of the CaCl solution
   4. Proteins and nucleic acids absorb UV light, their positions can be seen using UV optics

Question 5

What are the relative densities of RNA, DNA and proteins?

Answer 5

Protein densest, DNA middle, RNA least dense

Question 6

True or false: DNA replication can begin at any sequence as long as it is complimentary to a polymerase enzyme.

Answer 6

False; DNA replication is initiated at substrate-specific sequences

Question 7

What technique proved the bidirectional nature of DNA replication?

Answer 7

Fibre autoradiography, whereby following replication initiation low specific (radio)activity thymidine was mixed with the replicating DNA, then later high specific (radio)activity thymidine was added to the DNA mixture

Question 8

What were the results of fibre autoradiography and what did they indicate about DNA replication?

Answer 8

The results of fibre autoradiography showed the radioactivity of the thymidine tags on the replicating DNA to increase on both sides of the origin of replication as distance increased

Question 9

In which direction does DNA polymerase synthesize new nucleotides?

Answer 9

This enzyme synthesizes in the 5’ –> 3’ direction

Question 10

What does semi-discontinuous replication refer to?

Answer 10

This phrase refers to the consequence of the singular orientation by which DNA polymerase can synthesize new nucleotides, which is 3’ –> 5’; consequently, new nucleotides are discontinuously synthesized to the ‘lagging’ strand, in the 5’ to 3’ direction away from the replication fork

Question 11

How were Okazaki fragments discovered?

Answer 11

1. Pulse cells with 3H- thymidine to label DNA fragments
2. Centrifuge DNA to determine relative sizes of labelled DNA
3. Short labelling periods allows labelling of small DNA fragements (1000-2000 nt)

Question 12

Which enzyme joins together Okazaki fragments?

Answer 12

DNA ligase

Question 13

True or false: The average okazaki fragment length is longer in eukaryotes and archaea, at 1000-2000nt, compared to prokaryotes, at 100-150nt.

Answer 13

False; Okazaki fragments are longer on average in bacteria then in eukaryotes and archaea

Question 14

What must happen before DNA polymerase can bind to an unwound DNA strand to begin synthesis?

Answer 14

RNA primers, synthesized by primase enzymes, must be applied to a DNA strand before DNA polymerase is able to bind and synthesize new nucleotides.

Question 15

Describe the loop or trombone model of DNA replication.

Answer 15

In this model of DNA replication, the lagging strand of DNA ‘loops’ so that the DNA polymerase III complex on each strand can synthesize nucleotides on both strands at the same time in the same direction.

Question 16

Name and briefly describe 4 key proteins that can be found at the bacterial DNA replication fork.

Answer 16

1. Helicase
   -involved in a protein complex (UMG) which unwinds the DNA double helix
2. DNA Polymerase III
   -synthesizes new nucleotides along unwound DNA strand
3. Primase
   -synthesizes RNA primer sequences
4. DNA Polymerase I
   -joins at the junction of the okazaki fragments

Question 17

How does proofreading occur during DNA replication?

Answer 17

Exonucleases are attached to the DNA polymerase so that nucleotidal errors are immediately detected and removed during replication.

Question 18

What is the en vivo error rate of DNA replication?

Answer 18

This error rate is 1 x 10^-11

Question 19

Which experimental method was used to map replication origins across the yeast genome?

Answer 19

The Meselson-Stahl method was used for this purpose.

Question 20

Describe steps of the Meselson-Stahl method toward observing the replication origin sequences across a yeast genome.

Answer 20

1. Grow yeast culture for many generations in “heavy” medium to generate HH DNA
2. Arrest yeast cells in G1 phase of cell cycle then release into ‘synchronous’ s-phase in ‘light’ medium to produce HL DNA (semi-conservative replication)
3. Label DNA fractions and hybridize to genomic DNA microarrays
4. Compare the hybridization signal obtained with HH and HL microarray probes

Question 21

Describe the mechanism of DNA microarray probing.

Answer 21

Labelled (HH and HL) DNA sequences are added to a microarray slide which contains thousands of microscopic DNA sequences; the DNA sequences which are complementary to the sample DNA sequences will glow

Question 22

What exact feature of the microarray display indicates the replication origin of the sample DNA sequence?

Answer 22

Upon displaying the complementary DNA sequences during replication, the middle sequence of a microarray slide may, on its own, glow. This is the sequence of the replication origin.