Replication, Maintenance and Rearrangement of Genomic DNA

Question 1

What is required to copy the genetic material?

Answer 1

It will require a

1. Template: DNA molecule with a 3’OH end for elongation
2. Primer: short RNA molecule
3. one or more enzymes plus replication factors.

(Elongation goes 5’ to 3’)

Question 2

In what phase of mitosis do bacterial DNA replicate?

Answer 2

E. coli replication occurs in the S phase of mitosis.

There is the formation of a replication bubble and two replication forks.

DNA is the circular.

Question 3

What is required for the initiation of replication?

Answer 3

RNA primer

something to melt the double helix

to maintain it single-stranded

and to resolve supercoiling

Question 4

How are the leading and lagging strands synthesized?

Answer 4

1. Both strands are synthesized 5’ to 3’
2. the leading strand undergoes continuous synthesis
3. the lagging strand DNA is added in small unit increments (Okazaki fragments)
   a. The Okazaki fragments are synthesized 5’ to 3’ then the fragment is ligated to the elongating DNA molecule requiring a DNA ligase activity

Question 5

What is the initiation process of Okazaki fragments with RNA primers?

Answer 5

1. DNA melting from double stranded to single stranded DNA
2. An RNA polymerase will add NTPs (4-6) that will be used as primers for the DNA polymerase to elongate

Question 6

How are RNA primers removed and Okazaki fragments joined together?

Answer 6

Once the Okazaki fragments have been synthetized:

1. The DNA polymerase 1 possesses a 5’to 3’ exonuclease activity (nuclease activity that requires a free 5’ end, as oposed to an endonuclease that does not require free 5’ or 3’ ends).
2. The RNA is removed and the DNA polymerase will now add the dNTPs (5’ to 3’) and fill the gap.
3. The DNA ligase will rejoin the two adjacent DNA fragments (ATP is required).

Polymerase I: removes RNA by 5’-3’ exonuclease, the gap is filled with DNA 5’-3’ and DNA ligase joins the DNA fragments (requires ATP)

Question 7

What is the role of DNA Polymerases in E. coli?

Answer 7

Prokaryotes:

Leading strand: DNA polyerase III

Lagging strand: RNA polymerase or primase

DNA polymerase I to remove RNA primer and add new dNTPs

DNA polymerase III is used in the elongation phase

Question 8

What is the role of DNA polymerase in mammalian cells?

Answer 8

Eukaryotes:

Leading strand: DNA polymerase delta and epsilon

Lagging strand: DNA polymerase alpha and primase

The elongation phase uses DNA polymerase delta and epsilon

Question 9

What additional proteins are required for proper replication in Prokaryotes?

Answer 9

In Prokaryotes: Gamma complex interactw with beta protein and promote the recruitment of the DNA polymerase

Question 10

What additional proteins are required for proper replication in Eukaryotes?

Answer 10

Eukaryotes: Replication Factor C (RFC) interacts with proliferating Cell Nuclear Antigen (PNCA) and promote the recruitment of DNA polymerase

Question 11

What are the actions of Hilicases and Single-stranded DNA-binding proteins?

Answer 11

When the replication fork moves along the DNA it needs to unwind the double stranded DNA into single stranded units.

The unwinding is performed by a DNA helicase

The unwound DNA is maintained in the single stranded form by single-stranded binding proteins (SSBP)

Question 12

What is the action of Topoisomerases during DNA replication?

Answer 12

As DNA is unwound topological stress builds up ahead of the replication fork

Prokaryotes: the circular DNA will supercoil

Supercoiling stress is released by a transient break of one DNA strand that will allow free rotation and release of the topological stress

The enzyme that perform the task is the DNA topoisomerase (type I)

Question 13

Label the diagram of the E. coli replication fork

Answer 13

Question 14

How is proofreading performed by DNA polymerase?

Answer 14

When a dNTP is misincorporated DNA polymerase detects the mismatch and cleaves the misincorporated dNTP (3’ to 5’ exonulcease activity) and elongation re-starts with incorporation of the proper dNTP

Question 15

What is special about the bacterial initiation of replication?

Answer 15

Bacteria possess only one origin of replication where replication will be initiated

1. binding of initiator protein
2. Synthesis of RNA primers

unwinding of DNA by helicase and single-stranded DNA-binding proteins

3. formation of two replication forks

Question 16

Why is replication in Eukaryotic chromosomes different the Prokaryotes?

Answer 16

Eukaryotes hare a larger genome to be replicated but have only a small amount of time to achieve replication

Theycompensate the size difference by making use of multiple origins of replication which involves strand displacement

Question 17

How are origins of replication in yeast identified?

Answer 17

Two plasmids, plasmid I and II contain a selectable marker (LEU2) that allows transformed cells to grow on medium lacking leucine

Only plasmid II contains an ARS (Autonomously Replicating Sequence)

Yeast transformed with plasmid I will only replicate if the plasmid gets integrated in the chromosomal DNA (under the control of endogenous ARS)

Plasmid II containing its own ARS is able to replicate autonomously therfor the number of transformants is much higher

Question 18

Describe the origin of replication in Yeast

Answer 18

Autonomous Replicating Sequence or ARS:

1. 11 bp sequence is a key element in ARS (also called ACS)
2. Sequences B1 to B3 are also important for efficient initiation of replication
3. Composition of replication in Yeast is more complex than its bacterial counter-part: Origin of Replication Complex or ORC binds to ARS and promote recruitment of additional factors (licensing factors CDC-6, CDT-1 and MCM).
4. As the replication complex moves it will displace the MCM proteins

Question 19

What is the action of Telomerase?

Answer 19

1. Telomerase carries its own RNA complimentary to telomeric DNA
2. Telomeric DNA with overhanging 3’ end:
   a. Telomerase carries its own RNA that is complementary to sequences present at the end of chromosomes and hybridizes to the protruding ssDNA (3’ overhang)
   b. reverse transcription occurs (elongation)
   c. Extension follows by standard DNA polymerase activity
   d. RNA primer is removed and telomere has benn elongated by one repeat unit
   e. the enzyme translocates and the process can repeat
3. 5’ end has the newly synthesized lagging strand
4. Binding to telomerase RNA
5. Telomerase RNA
6. 3’ end of leading strand elongated by one repeat unit
7. Extension of lagging strand by primase and polymerase
8. Telomeric DNA extended by one repeat unit

Question 20

What are the characteristics of chromosomes?

Answer 20

1. Chromosomes are GT rich
2. the RNA in telomerase is AC rich
3. Shortening of telomeres is catastrophic for cells and leads to chromosome breaks and rearrangements

Question 21

What are the properties of telomeres?

Answer 21

1. Telomeres are built by telomerase
2. it adds copies of the telomeric repeat sequence (TTGGGG for Tetrahymena) to the end of the chromosome
3. to do this it has an internal RNA template with the sequence complementary to the repeat CAACCCCAA
4. Each species telomerase carries an RNA template with the sequence that the mechanism of telomer synthesis
5. The loop structure at the end is stabilized by base pairing of a non-standard type between the G bases
6. this has to unfold to allow telomerase to add new repeats, after which refolds
7. DNA polymerase fills the gapped region and there is incomplete ligation leaving a few nicks
8. The nicks allow DNA to unfold at the nest round of replication

Question 22

Know the schematic representation of the composition of telomeric complexes, telomerase and proteins implicated in cellular signaling from the telomere (telomer repair)

Answer 22

Telomerase is composes of #1 which includes the template for telomere DNA synthesis, and the protein catalytic component #2

1. hTR: RNA component
2. hTERT: catalytic component

The proteins in #3 and TRF2, tankyrase, RAP1 are all invoved in interacting with the telomere and may regulate the opening/closing of free telomere end and access to the telomere by other protein complexes such as telomerase

3. TRF-1, TIN2, POT1: maintenance of telomere structure