Week 5- DNA Replication

Question 1

What direction do DNA polymerases synthesize DNA?

Answer 1

5’ to 3’

DNA polymerases can only add nucleotides to the free 3’ end of a growing strand.

Question 2

What are the two types of activity that DNA polymerase possesses?

Answer 2

3’ to 5’ exonuclease activity

This activity allows for proofreading during DNA replication.

Question 3

What is the size of DNA molecules typically measured in?

Answer 3

Base pairs (bp)

1 bp corresponds to approximately 0.34 nm of length along the strand.

Question 4

What is the range of DNA sizes mentioned?

Answer 4

5 kb to over 250,000 kb

kb = kilo base pairs, Mb = mega base pairs, Gb = giga base pairs.

Question 5

What temperature can E. coli undertake DNA replication down to?

Answer 5

70°C

Some extremophiles, like P. halocryophilus, can replicate DNA at -150°C.

Question 6

Who won the Nobel Prize in 1959 for the discovery of the mechanisms of DNA synthesis?

Answer 6

Arthur Kornberg

He shared the prize with Ochoa.

Question 7

What is the primary role of DNA polymerase I?

Answer 7

DNA repair

It is involved in removing RNA primers during DNA replication.

Question 8

Which DNA polymerase is the major replicative enzyme in E. coli?

Answer 8

DNA polymerase III

Temperature-sensitive mutants of Pol III cannot replicate DNA at restrictive temperatures.

Question 9

What are the nucleotides used in DNA synthesis called?

Answer 9

Nucleoside triphosphates (dNTPs)

Examples include dATP, dTTP, dGTP, and dCTP.

Question 10

What is the rate of elongation for DNA replication in bacteria?

Answer 10

500 nucleotides per second

In human cells, the rate is approximately 50 nucleotides per second.

Question 11

What initiates the synthesis of RNA primers during DNA replication?

Answer 11

RNA primase

DNA polymerase III cannot initiate synthesis de novo.

Question 12

What are Okazaki fragments?

Answer 12

Short segments synthesized on the lagging strand

They are later joined together by DNA ligase.

Question 13

How many origins of replication do prokaryotes have?

Answer 13

One origin of replication

This leads to a single replication bubble.

Question 14

What is the typical replication time for prokaryotes?

Answer 14

<1 hour

Eukaryotes, with multiple chromosomes, take approximately 2-3 hours.

Question 15

What is the role of helicase in DNA replication?

Answer 15

Unwinds the double helix

ATP is required for this process.

Question 16

What is the purpose of single-strand binding proteins during DNA replication?

Answer 16

Prevent the re-annealing of unwound DNA strands

They stabilize the unwound template strands.

Question 17

What is the mechanism of proofreading during DNA replication?

Answer 17

3’ → 5’ exonuclease activity

This activity clips back unpaired or misinserted nucleotides.

Question 18

What must occur before DNA polymerases can add nucleotides?

Answer 18

A primer must be synthesized

The initial nucleotide chain produced is actually a short stretch of RNA.

Question 19

Fill in the blank: The lagging strand is synthesized as a series of _______.

Answer 19

Okazaki fragments

Question 20

True or False: DNA polymerases can initiate the synthesis of a polynucleotide.

Answer 20

False

They can only add nucleotides to an existing chain.

Question 21

What are the four main proteins involved in the initiation of DNA replication?

Answer 21

• Topoisomerase
• Helicase
• Primase
• Single-strand binding proteins

Question 22

What is the role of DNA ligase during DNA replication?

Answer 22

Joins Okazaki fragments

It seals the gaps between newly synthesized DNA segments.

Question 23

What is the outcome of the proofreading mechanism in DNA replication?

Answer 23

Reduces error rate to ~1 in 10^9

This results in approximately 0.04 potential mutations per genome replication.

Question 24

What is the primary function of DNA ligase?

Answer 24

Joins two Okazaki fragments

DNA ligase is essential in DNA replication to connect segments of DNA.

Question 25

What is the role of DNA polymerase I?

Answer 25

Removes RNA primer from 5’ end of 2nd fragment and replaces with DNA ## Footnote DNA polymerase I is crucial for DNA replication, particularly in the removal of RNA primers.

Question 26

How does eukaryotic DNA replication differ from that in E. coli?

Answer 26

Has significant similarities and differences ## Footnote Key differences include initiation mechanisms and genome size.

Question 27

What are the key characteristics of bi-directional DNA replication?

Answer 27

Leading and lagging strands, primers, and proofreading ## Footnote This is a fundamental process in DNA replication ensuring accuracy and efficiency.

Question 28

List the major differences in eukaryotic DNA replication compared to prokaryotic.

Answer 28

* Different DNA replication initiation * Larger genomes * Multiple replication sites * Linear chromosomes * Lower rates of DNA synthesis * More complex enzymology ## Footnote These differences reflect the complexity of eukaryotic organisms.

Question 29

What is the significance of the Origin Replication Complex in humans?

Answer 29

Loads onto DNA first for fork initiation ## Footnote The Origin Replication Complex is crucial for starting DNA replication in eukaryotic cells.

Question 30

What is the primary function of DNA polymerase α?

Answer 30

Chromosomal replication 5’ to 3’ but lacks exonuclease activity ## Footnote DNA polymerase α is always associated with a primase for initiation.

Question 31

What is the role of DNA polymerase δ?

Answer 31

5’ to 3’ polymerase activity and a 3’ → 5’ exonuclease activity ## Footnote DNA polymerase δ is involved in the elongation phase of DNA replication.

Question 32

What is the mitochondrial genome structure?

Answer 32

Circular, 16,569 base pairs encoding 37 genes ## Footnote Mitochondrial DNA is inherited maternally in most species.

Question 33

Which DNA polymerase is specialized for mitochondrial DNA replication?

Answer 33

Pol γ/PolG ## Footnote This polymerase is essential for the replication of mitochondrial DNA.

Question 34

What is the composition of telomerase?

Answer 34

Catalytic subunit (hTERT) and template RNA (hTR) ## Footnote Telomerase plays a critical role in maintaining telomere length during cell division.

Question 35

Fill in the blank: Telomeres in humans consist of repeated sequences of _______.

Answer 35

(TTAGGG)n ## Footnote These repeated sequences help protect chromosome ends.

Question 36

True or False: Telomerase is active in most somatic cells.

Answer 36

False ## Footnote Telomerase is repressed in most somatic cells, leading to telomere shortening.

Question 37

What happens when D loop integrity is compromised in telomeres?

Answer 37

Triggers irreversible exit from the cell cycle producing replicative senescence ## Footnote This process is crucial for cellular aging and the prevention of uncontrolled cell division.

Question 38

What is the primary challenge posed by linear chromosomes during replication?

Answer 38

Okazaki fragment placement cannot be replicated by conventional machinery ## Footnote This challenge leads to the need for specialized mechanisms like telomerase.

Question 39

List the methods that can be used to assay DNA replication.

Answer 39

* 32P incorporation * Fluorescence ## Footnote These methods allow researchers to track and analyze DNA replication processes.

Question 40

What is a characteristic of the chloroplast genome?

Answer 40

Circular and typically 12-17 Kbp, encodes ~100 genes ## Footnote Chloroplasts, like mitochondria, are derived from ancient prokaryotic organisms.