DNA Replication and Recombination I

Question 1

All organisms have evolved a __________

Answer 1

highly accurate system of DNA replication

Question 2

DNA replication is an essential function of genetic material
Accuracy:
Error rate of 10-6
Still implies

Answer 2

~3000 mistakes per replication cycle in the human genome

Question 3

Watson & Crick (1953) model

Answer 3

• Semi-conservative replication

- Based on complementarity of nucleotide bases

Question 4

semiconservative replication (model)

Answer 4

Watson and Crick:
1. Each DNA strand is a potential template
2. Complementary bases attract relevant partner in new
developing strand:
>Adenylic acids attract thymidylic acids; cytidylic acids
attract guanidylic acids.
3. Each replicated DNA molecule consists of one “old” and one
“new” string

Question 5

Besides Watsons and Cricks semi-conservative model, there are two other possible mechanisms:

Answer 5

1. Conservative
2. Dispersive

• Also rely on parental strands as templates

Question 6

In conservative replication:

Answer 6

Complementary polynucleotide chains would be synthesized as previously described.
- BUT after the synthesis of the new strand, the parent strands
would reassociate (join) and the two new strands would anneal
= one original molecule of DNA and one new one
= The parental molecule is CONSERVED

Question 7

In dispersive replication:

Answer 7

Parts of the parental strand are dispersed into new strands.
= Each new double stranded DNA molecule contains parts of the original DNA and parts of the new DNA

Question 8

why is dispersive replication ‘rare’

Answer 8

This involves the cleavage of parental strands during replication
=Most complex mechanism out of the three possible mechanisms of replication

Question 9

The origin of replication:
Is replication initiated at a specific position on the chromosome, or are the chances equal all along the length of it?
In what direction does replication proceed: In one direction only (unidirectional), or in two directions away from the origin (bidirectional)?

Answer 9

For unidirectional replication to occur, the two DNA strings have to first unwind to form a replication fork. The fork then moves along the DNA strand as replication proceeds.
If replication is bidirectional, two forks will be present, migrating in opposite directions from the origin of replication.

Question 10

replicon

Answer 10

The length of DNA that is replicated following one initiation event at a single origin

Question 11

Origin of replication in prokaryotes

Answer 11

For most prokaryotes
(where only a single circular chromosome is present)
> only one origin of replication is found
(in E. coli this specific region is called oriC)
…and therefore also only one replicon.

1. Replication starts at oriC and proceeds bidirectionally with two replication forks.
2. It terminates at a termination region (called ter in E. coli) after replicating the entire chromosome.

In eukaryotes replication also proceeds bidirectionally, but from multiple origins and with various replication forks migrating along the chromosome. In the end, all forks merge, resulting in a completed round of replication for the chromosome.

Question 12

Kornberg et al. isolated an enzyme from E. coli that had the ability to synthesize DNA in ‘n cell-free (in vitro) system:

Answer 12

DNA polymerase I

Question 13

Three major requirements for in vitro DNA synthesis to be successful:

Answer 13

1. All four deoxyribonuleoside triphosphates must be present
   (dATP, dGTP, dCTP, dTTP) = (dNTP)
   Precursors Had to be dNTP, otherwise no synthesis
   (Mono and diphosphates didn’t work)
2. Template DNA
   Synthesis needed a DNA template (3) primed with a double-stranded region with a free 3’-OH for the next dNTP to attach to.
3. primer

Question 14

chain elongation direction

Answer 14

5’ to 3’ direction

Question 15

Explain chain elongation:

Answer 15

[5’ to 3’ direction]

1. two terminal phosphates from the 5’-C of each dNTP are removed by DNA polymerase I.
2. The remaining phosphate then binds to the 3’-OH group of the last dNMP in the chain to which it is added.
3. By the addition of a nucleotide at each step to the growing 3’ end, providing a newly exposed 3’ OH to participate in the addition of the next nucleotide

Question 16

Can DNA polymerase I perform DNA synthesis in cells (in vivo)?

Answer 16

Based on:
Rate of synthesis
Nuclease activities

DNA polymerase I adds +/- 15-20 nucleotides per second to growing DNA chain.
However, DNA replication in vivo was known to occur at a rate of 1000 nucleotides/second

BUT:
Kornberg et al. proved that DNA polymerase I is capable of synthesizing the complete DNA of a small phage in vitro, and that this DNA could be used to successfully transfect E. coli protoplasts.

Question 17

DNA polymerase II, III, IV and V

Answer 17

New questions arise after the discovery of Pol A1.
At least one more enzyme that is capable of DNA synthesis in vivo must be present in E. coli
DNA polymerase I may serve only a secondary function (fidelity of replication) in DNA synthesis.
To date, four other unique enzymes, DNA polymerase II, III, IV and V have been discovered.

Question 18

Can any of the five polymerases initiate double-stranded synthesis from a single-stranded template?

Answer 18

No, none of them can.

- All of them need a double-stranded region with a free 3’-OH to which they can add the next dNTP in sequence

Question 19

5 polymerases properties

Answer 19

• All 5 have 5’-3’ polymerize activity

- As well as 3’-5’ exonuclease activity = allows them to proofread their activities

Question 20

DNA polymerase III

Answer 20

Responsible for DNA synthesis during replication.

3’-5’ exonuclease activity serves as a proofreading mechanism.

Question 21

DNA polymerase I

Answer 21

• Removes RNA primers and fills the gaps left after their removal.
• Has a unique 5’-3’ forward exonuclease activity which allows it
  to remove the RNA primers ahead of it
• Exonuclease activity serves as a proofreading mechanism and
  also allows it to participate in DNA repair.

Question 22

DNA polymerase II, IV and V

Answer 22

Probably involved in repair of DNA that has been damaged by external forces (e.g. UV light).

Question 23

The DNA Pol III

[holoenzyme]

Answer 23

> DNA Pol III is a very large (900kDa) and complex enzyme
The active form (called a holoenzyme)
–is a dimer consistingof 10 polypeptide subunits, each with a
specific function during DNA synthesis
Comprises of 2 core enzymes; sliding clamp loader, and sliding
clamp
The holoenzyme + other proteins at replication fork is known as
replisome
(analogous with ribosome–Replisome doesn’t produce protein,
instead, it extends the DNA to replicate it )

By clamping the hollow enzyme to the DNA strand, keeps the enzyme in contact with the substrate for longer, increasing processivity.

Question 24