DNA replication, the cell cycle and mitosis

Question 1

Mechanism of DNA replication: explain the reaction catalysed by DNA polymerases and the functions of the components of the replication complex, explain semi-conservative replication

Answer 1

DNA Replication: Semi-conservative model

• When a DNA strand replicates, the 2 strands in the PARENT DNA strand separate
• Each parent DNA strand becomes one-half of the two DAUGHTER DNA strands
• The Parent strand acts as the template for attaching the correct complimentary bases to form the other half of the Daughter strand
• Each Daughter strand contains
  
  • 1 old strand (from Parent DNA)

DNA replication is performed by a group proteins that work together with DNA polymerase and primase, forming a multi-enzyme protein machine (image)

• • 1 new strand

DNA Replication: Separation

• Hydrogen Bonds broken via DNA HELICASETemplate strand bases now exposed
• DNA PRIMASE (a type of RNA polymerase) then synthesises a short complimentary RNA primer sequence to the primer region in the DNA
  Note: the RNA primer-making enzyme is called DNA primase, because it “primes” the DNA for replication
• By this point, the cell has ensured there is a healthy supply of dNTPs (deoxynucleotide triphosphates)
• dNTPs line up with their complimentary bases on the template strand

1 DNA Replication: Initiation

• DNA POLYMERASE attaches to the RNA primer
• This is necessary because DNA POLYMERASE cannot start a new strand from scratch, it needs:

1. a template strand
2. an oligonucleotide primer
3. a supply of deoxynucleotide triphosphates (dNTPs)

• DNA POLYMERASE then runs down the template strand, attaching the dNTPs onto the 3’ position of the pentose sugar (-OH group) and forming phosphodiester bonds
  IMPORTANT: replication must occur in the 5’ to 3’ direction
• Energy is released by hydrolysis of the triphosphate. This drives the reaction.

2 DNA Replication: Replication Fork

• Replication begins at discrete points on the DNA molecule called origin of replication
• The site of DNA synthesis is called a replication fork: the fork moves along during the process
• The replication fork will move along the DNA, synthesising the daughter strands in its wake
• Replication must occur in the 5’ to 3’ direction, as the dNTPs are being added to the 3’ hydroxyl group
• The oposite strand is synthesised in the opposite direction to the movement of the replication fork in fragments of DNA called OKAZAKI FRAGMENTS

The Replication Fork is asymmetric:

1. LEADING STRAND synthesised continuously, in direction of replication fork
2. LAGGING STRAND synthesised discontinuously, in opposite direction of replication fork

3 DNA Replication: Synthesis of lagging strand

• DNA polymerase adds to RNA primer, starting new Okazaki fragment
• DNA polymerase finishes DNA fragment
• Old RNA primer erased and replaced by DNA
• DNA ligase joins new Okazaki fragment to growing chain
• A special ribonuclease removes RNA primer using a 5’ to 3’ exonuclease activity
• A repair DNA polymerase then replaces RNA with DNA.
• DNA ligase joins the two fragments together using ATP. This makes the DNA strand continuous.

DNA Replication: Inhibition

4 Drugs to learn: Classified as NUCLEOSIDE ANALOGS

MOA: Replace dNTPs, and terminate the chain

They do not have hydroxyl group at 3’ position, so DNA polymerase cannot add any more dNTPs to the strand

1. Dideoxycytidine (ddC) / Zalcitabine: Has 3’ (-H) group, used to treat HIV
2. Azidothymidine (AZT) / Zidovudine: Has 3’ (-N3) group, used to treat HIV
3. Acyclovir: Does not have a ring structure to its sugar group (so not a sugar at all), used to treat Herpes
4. Cytosine Arabinose: Has a tetrose sugar instead of pentose, used in Chemotherapy​

Question 2

DNA replication fidelity: explain how accuracy is maintained by proof-reading, DNA repair and the use of RNA primers

Answer 2

Proofreading mechanism:

The high fidelity of DNA replication requires a proof-reading mechanism to ensure no mistakes are made. Mutations (changes in DNA sequence) are very dangerous to the organism. Any errors in replication cannot be repaired.

DNA replication has an error frequency of about 1 change per 109 base pairs.

Before a new nucleotide is added, the previous nucleotide is checked for correct base-pairing.

Any incorrect bases are removed by 3’ to 5’ exonuclease activity of DNA polymerase. A new, correct nucleotide is then added.

Question 3

Chromosome replication: summarise the replication of mammalian chromosomes, explain replication of the E. coli chromosome by drawing a diagram

Answer 3

Prokaryote:

• In E.coli, replication starts at a unique origin, OriC.
• Two replication forks proceed simultaneously in opposite directions.
• The two forks meet on the other side of the circular chromosome.
• Bi-directional replication

Eukaryote:

• Chromosomes are linear and very long
• Multiple replication origins are distributed at intervals of about 100 kilobase pairs
• Each replication origin gives bi-directional replication forks.
• Replication is finished when all the forks have met.

Draw a diagram (image)

Question 4

The cell cycle: define the different phases of the cell cycle, explain how the chromosomes segregate at metaphase by drawing a diagram

Answer 4

The cell cycle

G1, G0, S, and G2 are Interphase

• M phase: Mitosis; cell division; 1hr
• G1 phase: Gap phase 1 (prior to DNA syntesis); 10 hrs
• S phase: period of DNA synthesis (replication); 9hrs
• G2 phase: Gap phase 2 (between DNA synthesis and mitosis); 4 hrs
• G0: cells which have stopped dividing

Chromosomes during cell cycle

• G1: DNA of each chromosome present as a single linear double helix of DNA
• S phase: the DNA is replicated
• G2: each chromosome has two identical sister chromatids
• Mitosis: the two chromatids separate to the daughter cells

Stages of mitosis I

1. Interphase (G2)
   Chromosomes not visible
2. Prophase

Condensed chromosomes, each contains two sister chromatids

3. Metaphase
   Condensed chromosomes, aligned on central plane of spindle
4. Anaphase
   Sister chromatids separate & are pulled to spindle poles
5. Telophase
   Sister chromatids move to opposite poles of spindle
6. Cytokinesis
   Division of cytoplasm
   Formation of two daughter cells

Interphase (G1): Condensation process reversed

• During Early Prophase: Chromosomes pair up + Crossing Over occurs
• During Late Prophase: Chromosomes condense and become visible + Spindle fibres move to opposite ends of cell

Draw a diagram: image