DNA replication 2

Question 1

why do telomeres cause a problem for replication

Answer 1

DNA polymerase can only synthesize DNA 5′ → 3′ and requires an RNA primer. On the lagging strand, the final RNA primer at the chromosome end is removed, but there is no 3′-OH group available to fill the gap. This causes progressive shortening of chromosome ends, known as the end-replication problem.

Question 2

is the E.coli chromosome circular or linear, and does it have a single or multiple place of origin

Answer 2

• circular
• single origin

Question 3

do most eukaryotic chromosomes have single or multiple origins, and are they circular or linear

Answer 3

• linear
• multiple origins

Question 4

what is the end replication problem

Answer 4

every time DNA is replicated, a little bit of info is lost

Question 5

how does telomerase solve the end replication problem and prevent chromosome shortening during DNA replication?

Answer 5

Telomerase is a ribonucleoprotein enzyme that carries its own RNA template and extends the 3′ end of the telomere by adding repetitive DNA sequences. This provides space for an RNA primer to bind, allowing DNA polymerase to complete replication of the lagging strand without loss of essential genetic information.

Question 6

what are the 5 DNA polymerases that E.coli has

Answer 6

• DNA Pol I: helps remove RNA primer and replaces with DNA in chromosome replication, also has a major role in repair of damaged DNA
• DNA Pol II: restarting replication when blocked by damaged DNA, also a role in DNA repair
• DNA Pol III: Chromosome replication
• DNA Pol IV and V: allow replication to bypass some types of DNA damage, also involved in DNA repair

Question 7

What are the activities of DNA polymerase I and DNA polymerase III in E. coli, and how do their roles differ?

Answer 7

Shared Function (Pol I & Pol III):
- 5′ → 3′ polymerase activity
→ Adds nucleotides to synthesize DNA
- 3′ → 5′ exonuclease activity
→ Proofreading: removes incorrectly added nucleotides to increase accuracy

DNA Polymerase III (Pol III):
- Gene: polC
- Primary replicative enzyme
- Very fast (≈ 250–1000 nt/sec)
- Synthesizes…
>Leading strand continuously
>Lagging strand as Okazaki fragments
- Lacks 5′ → 3′ exonuclease, so it cannot remove RNA primers

DNA Polymerase I (Pol I):
- Gene: polA
- Secondary enzyme, not the main replicator
- Slow (≈ 16–20 nt/sec)
- Has 5′ → 3′ exonuclease activity
→ Removes RNA primers from Okazaki fragments
- Replaces RNA with DNA after primer removal

Key Point:
DNA polymerase III rapidly replicates the chromosome, while DNA polymerase I processes Okazaki fragments by removing RNA primers and filling in DNA.

Question 8

describe the structure of DNA polymerase III in E.coli

Answer 8

• it is called the Clamp Loader complex (composed of 5 subunits)…
• Catalytic core (alpha subunit)
• The Clamp (beta subunit)
• The Tau (holds the 2 halves of the enzyme together)
• E & O subunits (proofreading activity)
• symmetrical (except clamp loader) as it synthesises both strands at the same time

Question 9

what are some other important enzymes apart from DNA Polymerase III involved in DNA replication

Answer 9

• Helicase: unwinds the DNA duplex to produce the replication fork (DnaB gene in E.coli)
• Single stranded DNA binding protein (SSB in E.coli): keeps strands apart and helps prevent stem-loop formation
• Primosome (makes RNA primer): moves with the lagging strand and contains products of DnaC and DnaG genes and others in E.coli

Question 10

describe DNA Polymerase in action

Answer 10

• DnaB (helicase) unwinds the duplex
• alpha subunits (catalytic core) synthesise DNA
• t (Tau) subunits ensures dimerization of polymerase
• beta clamp (not shown) encircles DNA

The leading strand is made continuously by the addition of nucleotides to the 3’ end as it passes through the catalytic core of the enzyme
When sufficient template DNA for lagging strand synthesis has unwound (1-2kb of DNA kept single stranded by SSB), primase synthesises a RNA primer for lagging strand synthesis. The template strand is then pulled through the catalytic core allowing the addition of nucleotides to the 3’ end of the primer

Question 11

How does DNA polymerase III synthesize the lagging strand while moving in the same direction as the leading strand?

Answer 11

• DNA polymerase III is a symmetrical dimer, with two catalytic cores
  -One core synthesizes the leading strand continuously
• The lagging strand template forms a loop so it can be pulled through the replisome in the same direction
• This allows both polymerases to move with the replication fork

Lagging-Strand Synthesis:
- Primase synthesizes an RNA primer
- A β-clamp is loaded onto DNA by the clamp loader
- DNA polymerase III extends the primer to form an Okazaki fragment
- After completion, the loop is released and a new loop forms for the next fragment

Key Point:
The looping of the lagging strand (the trombone model) allows coordinated and efficient synthesis of both DNA strands.

Question 12

what are the 2 key polymerases in mammalian cells

Answer 12

delta (operates on lagging strand) and epsilon (operates on the leading strand) polymerase

Question 13

what are the main polymerases operating in mammalian cells (replication), what are their functions and what are their locations

Answer 13

Alpha:
- function = initial synthesis (the 1st 20-30 nucleotides) at RNA primer (iDNA)
- location = nucleus

Delta:
- function = DNA replication (lagging strand), 3’-5’ exonuclease
- location = nucleus

Epsilon:
- function = DNA replication (leading strand),3’-5’ exonuclease
- location = nucleus

Beta:
- function = repair
- location = mitochondrion

Question 14

describe the differences in the way RNA primer is removed in mammals and E.coli

Answer 14

• In E. coli RNA primers are removed by DNA POL I which degrades from the 5’ end and is replaced with DNA
• In mammals the RNA primers are removed by an endonuclease FEN1
• When an Okazaki fragment is synthesised and reaches the 5’ end of the next fragment, it displaces the RNA primer used to initiate that fragment and ‘flaps’ around
• FEN1 is known as a ‘flap endonuclease’. It cuts off the RNA primer and degrades internally rather than at the end as in DNA POL1.
• DNA ligase then fills in the gap

Question 15

what are the advantages and disadvantages of DNA being supercoiled

Answer 15

benefit = more compact

disadvantage = difficulties in replication as its more difficult for polymerase to access

Question 16

when does supercoiling occur

Answer 16

when the ends of liner/circular DNA is constrained

Question 17

what are the 2 types of supercoiling

Answer 17

Positive supercoiling = when the right-handed double helical conformation of DNA is twisted in a right handed fashion (overwound) (in front of the replication fork

Negative supercoiling = twisted in a left-handed fashion (unwound) (behind the replication fork)

Question 18

what is the shared function of Topoisomerase and Gyrase

Answer 18

they restore the balance in super coiled regions during DNA replication

Question 19

what is the function of Gyrase

Answer 19

removes positive supercoils (relaxes overwound molecule)

Question 20

what is the function of Topoisomerase I

Answer 20

can remove both positive and negative supercoils, main function in DNA replication is to remove negative supercoils (tightens underwound molecule)

Question 21

where do bacterial replication forks initiate and terminate

Answer 21

they initiate at the origin

they terminate at the terminus

Question 22

what do the terminators do

Answer 22

they keep the whole process in check by causing the replication fork to terminate if either the replication fork goes beyond the half way point (e.g. if one fork slows due to DNA damage

Question 23

describe the events occurring at the bacterial origin

Answer 23

• DnaA (replication initiation protein) binds to the 9 bp repeats
• DNA melts at the 13bp repeats (full of AT) i.e. denatures - 2 strands come apart
• this allows Helicase, primers and DNA polymerase to start replication
• Bidirectional replication forks begin from origin

Key point:
DnaA wraps the DNA around itself leading to the unwiding of the 13bp (AT rich) repeats, allowing DNA replication machinery to then access the DNA > replication can then commence

Question 24

how long does it take E.coli. to replicate

Answer 24

• E. coli has 4.6 x 106 bp of DNA in its single circular chromosome.
• DNA Pol III synthesises DNA at 900 nt per second.
• Divide the total length of the genome by 900 = gives 5111 seconds (÷60) is 85 minutes. However, there are 2 replication forks so the answer is:
  42 minutes

Experiments have then shown that after the 42 minutes of replication, E.coli also requires 20 mins to form a septum (divides cell), to finally complete division

BUT…
E.coli can divide at much faster rates (20-35 mins) in good conditions

Question 25

How does E. coli manage to divide every 20–35 minutes when a single round of chromosome replication typically takes ~40 minutes

Answer 25

Key Mechanism: - Overlapping Cycles: A new round of replication begins at the origin (ori) before the previous round reaches the termination site (ter). - Inheritance: Daughter cells inherit chromosomes that are already partially replicated. Cycle: - A cell division the chromosome is already partly replicated and the newly formed cell inherits a chromosome like this - After 10 minutes the next round of replication begins before the previous round is complete - After 15 minutes the 1st round of replication is complete, the second round continues (in the last 20 mins, there is the assembly of cell division apparatus, septum formation and cell division)