DNA Replication 1

Question 1

why is DNA replication important

Answer 1

it is vital for passing on genetic information from cell to cell as cells divide and from generation to generation as organisms reproduce

Question 2

what is the semi-conservative method of DNA replication proposed by Watson & Crick

Answer 2

Semi-conservative method = double stranded DNA is unwound to produce 2 single strands (parent strands) and each parent strand is used to produce a daughter strand.
Each new molecule has one original parent strand and one new daughter strand

Question 3

describe The Meselson & Stahl experiment proving semi-conservative DNA replication

Answer 3

They relied on gravity and diffusion to make a salt gradient by extracting DNA and centrifuging it to equilibrium in CsCl density gradient

1. initially they grew bacteria in the heavy ^15N DNA - 2 strands of heavy DNA fell to the bottom of the tube
2. then they grew the bacteria in the normal ^14N DNA - first generation daughter molecules produced with hybrid DNA (^15N/^14N) in the middle of the tube
3. continued to grow it in that normal bacteria - second generation daughter molecules produced with light ^14N DNA at the top and Hybrid DNA in the middle of the tube

Question 4

what is a big issue when replicating DNA

Answer 4

the 2 strands are anti-parallel

Question 5

what is meant by the 2 strands of DNA being anti-parallel

Answer 5

each DNA strand has a 5’ phosphate at one end and a 3’ hydroxyl group at the other end, but this orientation is opposite on each strand

Question 6

how can a DNA strand be extended

Answer 6

by adding nucleotides to the 3’ OH end

Question 7

describe the process of DNA polymerase adding nucleotides to the 3’ end of a DNA chain

Answer 7

Needs…
- dNTP’s (triphosphate groups)
- a template (parent strand to copy)
- a primer (another strand paired with the template to give a 3’ end)

DNA Polymerase brings in a nucleotide triphosphate with a base complementary to the template DNA strand and a phosphodiester bond is formed between the incoming nucleotide and the 3’ hydroxyl end of the strand being extended with 2 of the phosphate groups being released.
Repeated for the next base…

Question 8

what is the leading strand

Answer 8

• made continuously
• primer extended by DNA polymerase from left to right (primer orientated 5’ to 3’ and extended from 3’ end)

Question 9

what is the lagging strand

Answer 9

• made discontinuously
• can’t be made in same direction as DNA being unwound
• Need to wait until DNA unwound before adding primer with the lagging strand being made in a series of fragments.

Question 10

what is a double stranded DNA unwound by

Answer 10

DNA helicase

Question 11

what is DNA replication primed by

Answer 11

RNA

Question 12

Why is RNA required to prime DNA replication, and how are Okazaki fragments formed?

Answer 12

• DNA polymerase cannot start DNA synthesis anew
• A short RNA primer is required to provide a free 3′-OH group
• Primase synthesizes the RNA primer
• DNA polymerase III (in E. coli) extends the primer in the 5′ → 3′ direction

Leading vs Lagging Strand:
Leading strand…
- Synthesized continuously from a single RNA primer
Lagging strand…
- Synthesized discontinuously
- Repeated priming produces Okazaki fragments

Okazaki Fragments:
- Length ≈ 1000–2000 base pairs in bacteria
- Each fragment begins with an RNA primer

Key Point:
RNA synthesis does not require a primer, but DNA replication does.

Question 13

how long is an ‘Okazaki’ fragment in length

Answer 13

1000-2000bp

Question 14

In E.coli, what are RNA primers removed by

Answer 14

DNA polymerase I & Rnase H

Question 15

How are RNA primers removed and replaced during DNA replication in E. coli?

Answer 15

RNA primers are removed by:
- DNA polymerase I
- RNase H

• DNA polymerase I has 5′ → 3′ exonuclease activity, which removes RNA primers
• DNA polymerase I simultaneously replaces RNA with DNA
• This leaves nicks (gaps in the sugar-phosphate backbone)
• DNA ligase seals the nicks by forming phosphodiester bonds

Key Point:
RNA primers on Okazaki fragments are removed and replaced with DNA, and the fragments are joined into a continuous strand

Question 16

what does DNA ligase do in DNA replication

Answer 16

it closes the gaps in the DNA backbone between Okazaki fragments between the 5’ and 3’ ends

Question 17

what does the semi-conservative model of replication predicts that replicating DNA would have

Answer 17

a ‘replication fork’: the point at which the double stranded DNA is unwound by the helicase enzyme

Question 18

what is the origin of replication

Answer 18

the point at which replication originates

Question 19

how come DNA replication can be either Bidirectional or Unidirectional

Answer 19

when replication initiates it doesn’t initiate at one end of a linear piece of DNA but initiates somewhere within the double stranded region

Question 20

describe how to detect for movement using radioisotopes

Answer 20

1. grow E.coli in media without radioisotope
2. Grow briefly in low level of radioisotope (3H thymidine labels new DNA)
3. Grow in higher level of radioisotope
4. Isolate DNA on an
   em grid and detect label via
   Photographic emulsion

Question 21

What is meant by bidirectional DNA replication, and how are leading and lagging strands formed?

Answer 21

• DNA replication begins at an origin of replication
• Replication proceeds in both directions, forming two replication forks

Leading strand:
- Synthesized continuously
- Made in the 5′ → 3′ direction, toward the replication fork

Lagging strand:
- Synthesized discontinuously
- Formed as Okazaki fragments
- Each fragment requires a separate RNA primer

Key Concept:
A strand that is the leading strand on one side of the origin becomes the lagging strand on the opposite side
This is due to the antiparallel nature of DNA and the 5′ → 3′ direction of DNA synthesis

Key Point:
Bidirectional replication ensures rapid and efficient duplication of the DNA molecule