Lecture 5 - DNA Replication

Question 1

Explain Meselson and Stahl’s experiment

Answer 1

1. They used density labelling to distinguish parent DNA strands from new DNA strands
2. DNA was labelled with 15N, making it more dense
3. Bacteria was grown in this ‘heavy’ medium then transferred to a normal ‘light’ medium (14N)
4. At t=0 all DNA was heavy
5. After two generations DNA was half ‘light’ and half ‘intermediate’ proving the semiconservative replication

Question 2

Primase

Answer 2

Synthesizes short RNA sequences called primers that serve as a starting point for DNA synthesis.
In eukaryotes they are about 10 nucleotides long and made at intervals of 100-200 nucleotides on the lagging strand.

Question 3

DNA polymerase I

Answer 3

1. Removes RNA primers in lagging strand and fills in the nucleotides which are necessary for the formation of DNA in the direction 5’ to 3’.
2. Helps in proof reading to see if there are any mistakes done while replicating and matching base pairs
3. ONLY ADDS NUCLEOTIDES, DOESN’T JOIN THEM

Question 4

DNA Polymerase III

Answer 4

Essential for the replication of the leading and lagging strands

• Alpha subunit: responsible for the DNA polymerase activity
• Epsilon subunit: proof reading exonuclease activity
• Theta subunit: smallest of all and helps in enhancing proof reading properties of epsilon

Question 5

Ligase

Answer 5

Facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond.
On the lagging strand it joins the 3’ end of the new DNA to the 5’ end of the previous one.

Question 6

Helicase

Answer 6

Uses hydrolysis of ATP to “unzip” or unwind the DNA helix at the replication fork to allow the resulting single strands to be copied.
1000 nucleotide pairs/second
Has 6 subunits

Question 7

Telomerase

Answer 7

Made of protein and RNA subunits that elongates chromosomes by adding TTAGGG sequences to the end of existing chromosomes at the 3’ end.

Question 8

Nuclease

Answer 8

Catalyses the cleavage of phosphodiester bonds

Question 9

Topoisomerase

Answer 9

Regulates the over winding or under winding of DNA. Separates two chromosomes in prokaryotic DNA

Question 10

Single-strand binding proteins

Answer 10

-Helix-destabilizing protein
Binds tightly and cooperatively to exposed single-strand DNA without covering bases, which therefore remain available as templates

Question 11

Sliding clamp

Answer 11

Keeps the polymerase firmly on the DNA when it is moving, but releases it as soon as the polymerase runs into a double-strand region of DNA

• First discovered in dividing cells therefore called proliferating cell nuclear antigen (PCNA)
• Helps to orient the polymerase for substrate binding
• Removes the pre-replication complex from ori

Question 12

Direction of synthesis for DNA replication

Answer 12

Synthesis of DNA replication is done from the 3’ to 5’ so that the new strand is in a 5’ to 3’ direction

Question 13

Synthesis of leading strand

Answer 13

Made by continuous synthesis from a single primer

Question 14

Synthesis of lagging strand

Answer 14

Made by synthesis of short Okazaki fragments from multiple primers.
Fragments nearest to the fork are the most recent.
Direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth

Question 15

Pre-replication complex

Answer 15

Large protein complex that interacts with the template strands. Bind to a DNA sequence in the ori

Question 16

Origin of replication (ori)

Answer 16

• Ori near genes that are actively transcribed then to fire earlier because the chromatin is looser and accessible
• When DNA is tightly wound, hetero chromatin, they fire later
• Some may never fire at all; they are there as spare in case another ori might stall for example

Question 17

Replication forks

Answer 17

They move bi-directionally as the template DNA is copied. Eukaryotes have many ori, this ensures that replication can be finished in a reasonable time

Question 18

DNA Polymerase

Answer 18

1. Shaped like an open right hand:
   - ‘Palm’ brings the active site and the substrates into contact
   - ‘fingers’ recognize the nucleotide bases
2. Cells usually have several DNA polymerases
3. They are processive; they catalyze many polymerization each time they bind to DNA

Question 19

Why is RNA degraded faster?

Answer 19

Because it is less stable than DNA

Question 20

What would happen if there was no sliding DNA clamp?

Answer 20

The polymerase would fall off the template after 20-100 cycles of polymerisation.
With it the polymerase can carry out >50,000 cycles