DNA Replication and Recombination II

Question 1

What have we established about replication

Answer 1

1. It is under the direction of DNA polymerase III
2. semiconservative in nature
3. and proceeds in two directions
   (physically in two directions, i.e. bidirectional
   – BUT proceeds in one biochemical direction only: 5’ to 3’)
   2 replication forks move away from each other–> away from the origin of replication

Question 2

A model for DNA synthesis

Answer 2

1. Must be a mechanism by which the double helix can unwind locally where the replication is taking place.
   = Replication bubble
   > must be kept in an open state for the enzyme to proceed with
   synthesis
   DNA is double-stranded, but the enzyme must work on a single-stranded region of the DNA so that the existing nucleotides can attract their complementary partners.

• As DNA unwinds- will create tension further down the strand
  2. Must be a mechanism in place to reduce this tension - So that the DNA does not snap

3. A primer…
   must be synthesized for DNA polymerase III to be able to commence with the polymerization process
   – polymerase cannot initiate de novo synthesis
   > Needs a free 3’- OH end onto which the next nucleotide can
   be added.
   THEREFORE: needs to be a short double-stranded region (primer) to continue with synthesis
   This primer is synthesized by RNA nucleotides - Not DNA
   (provides free 3’OH)

Question 3

A model for DNA synthesis continued

Answer 3

4. Because DNA replication is always performed in the 5’→3’
   direction, and the strands of the parental molecule are orientated
   antiparallel to each other, continuous synthesis of complementary DNA strands by DNA polymerase III is possible on one strand (leading strand) only.
   Synthesis on the other strand (lagging strand) is discontinuous.
5. Primers (consisting of RNA nucleotides)
   must be removed (can’t have RNA nucleotides), and the gaps left after their removal filled up (with deoxyribonucleotides), prior to completion of replication.
6. The DNA synthesized to fill the gaps caused by primer removal, must be ligated to (joined with) their adjacent DNA strands.
7. Even though DNA polymerases are very accurate, occasional errors are made - a proofreading mechanism must exist to correct such errors and to make sure the DNA is not mutated.

Question 4

Unwinding the Helix

Answer 4

Most prokaryotes and viruses have a single origin of replication on their chromosome.
The origin of replication in E.coli has been well studied
> Called oriC- consists of 245 base pairs (bp)
- It is characterized by repetitive sequences of 9 (X5) and 13 (X3)
bases
- (9mers & 13mers) and are AT-rich
- A protein, DnaA, initiates the unwinding of the helix by first
binding 9mer ( then changes in conformation slightly) and then
binds to 13mer regions
- This puts tension on the DNA strands and causes them to
separate
- DNA helicase (DNA B) then binds the ssDNA region and recruits
the DNA pol III holoenzymes to bind to the newly formed
replication fork
- Once the helix has been opened, single-stranded binding
proteins (SSBPs) bind ssDNA and ensure that re-annealing
(base-pairing) does not occur
- As unwinding proceeds, tension is created ahead of the replication fork – the result is supercoiling of the helix.
- Tension is relieved by the action of gyrase, an enzyme that is part of a bigger family of proteins called topoisomerase.
> It cuts one of the strands of DNA and removes a loop before
resealing the DNA strand- Prevents supercoiling

Question 5

Initiation of DNA synthesis

Answer 5

1. Initiation occurs as soon as a small portion of the helix is unwound.
2. DNA polymerase III requires the free 3‘ end of a primer (3’-OH) in order to start elongation of a polynucleotide chain.
3. RNA primers (5-15 bases) are synthesized on the DNA template (parental strand) by an RNA polymerase called primase
   
   • doesn’t need free 3’-OH (de novo; but is template-dependent).
     > Provides a 3’end for DNA polymerase III to add nucleotides
4. Elongation is performed by DNA polymerase III.
5. Synthesis continues until another RNA primer is encountered. 6. DNA polymerase I (5’-3’ exonuclease activity) removes RNA primers and fills the gap with dNTPs - complementary to the parental strand. 6. Ligase seals the strands.

Question 6

Continuous and Discontinuous synthesis

Answer 6

• DNA strands are unwound and the replication fork progresses
  down the helix.
• Since strands are anti-parallel, one strand will run 3’-5’ (direction in
  which fork is moving)
• While the other runs 5’-3’
  HOWEVER:
  DNA polymerase III can only synthesize DNA in 5’-3’ direction
  = only on one strand is the direction of synthesis the same as the direction of movement of the replication fork
• Only one strand (leading strand- 3’-5’ direction) can serve as a template for continuous strand synthesis.

Various initiation points are needed on the other strand
(lagging strand- 5’-3’)
– DNA synthesis is, therefore, discontinuous on this strand.
- Discontinuous DNA fragments (1000-2000 nucleotides) are called:
Okazaki fragments
> Each begins with an RNA primer
> Later on, DNA polymerase I, will remove the RNA primer, and replace them with deoxyribonucleotides
- Okazaki fragments are joined together at a later stage by an
enzyme called DNA ligase.
> creates a new phosphodiester bond between the two
strands

Question 7

Concurrent synthesis on the leading and lagging strands
How does DNA polymerase III manage to concurrently synthesize DNA on both the leading and the lagging strand? …or do two enzymes synthesize DNA in opposite directions?

Answer 7

The DNA polymerase III holoenzyme is a dimer.
- It contains 2 core enzymes
(one for leading and one for lagging strand)
= One of the enzymes sits on each of the parental strands

In order to deal with the problem of the replication fork moving away from the lagging strand:
[Concurrent synthesis at the replication fork is achieved by forming a loop in the lagging strand.]
- The lagging strand is looped out and inverted for a short region
=The core enzyme can synthesize along the lagging strand in the same direction as the replication fork is moving

HOWEVER:
This lagging strand template will eventually need to be repositioned
=Start of new Okazaki fragment

The orientation of the lagging strand is thus inverted - the biochemical direction is unaffected, however.
Each parental strand can now be replicated by one of the monomers of the DNA polymerase III holoenzyme.
> β-subunit clamp keeps the core enzyme in position.
[To maintain processtivity]

Remember, in each replication bubble, there are 2 replication forks that move in opposite directions - away from origin of replication.

This process happens in both replication forks at the same time

Question 8

Proofreading
Even though DNA synthesis is normally very accurate, it is not perfect: Non-complementary nucleotides are sometimes incorporated.

Answer 8

1. To compensate for this, both DNA polymerase I and III have 3’-5’
   exonuclease activity.
   - This provides them with the ability to detect and correct
   mismatched nucleotides.
2. In the case of the DNA polymerase III holoenzyme, the
   proofreading function is attributed to the epsilon (ε) subunit.

Question 9

Answer 9