DNA Replication

and Repair

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DNA Replication

• genetic information is passed on to the next generation

• semi-conservative

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Parent molecule with two

complementary molecules

Parental strands

separate

Each parental strand is a template

Each daughter DNA molecule consists of one

parental and one new strand

Overview of replication

• DNA is unwound and stabilized

• Origins of replication: Replication bubble and replication fork

Initiation

• RNA primers bind to sections of the DNA and initiate synthesis

Priming

• Leading strand (5’ 3’) synthesized continuously

• Lagging strand synthesized discontinuously then fragments are joined

• RNA primer replaced by DNA

Elongation

• Mismatch repair by DNA polymerase

• Excision repair by nucleases

Proofreading

Review of DNA structure

• double helix

• each strand has a 5’ phosphate end and a 3’ hydroxyl end

• strands run antiparallel to each other

• A-T pairs (2 H-bonds), G-C pairs (3 H-bonds)

STEP 1 Initiation at origins of replication separation sites on DNA strands

• Depend on a specific AT-rich DNA sequence – Prokaryotes – one site – Eukaryotes – multiple sites

• Replication bubble • Replication fork • Proceeds in two directions from point of

origin

The proteins of initiation 1. Helicase –

unwinds double helix

2. Single-strand binding proteins – holds DNA apart

3. Topoisomerase – relieves strain by breaking, swiveling, rejoining strands

STEP 2 Priming initiation of DNA synthesis by RNA

RNA primers bind to unwound sections through the action of primase

– leading strand – only 1 primer

– lagging strand – multiple primers

– replaced by DNA later

STEP 3 Elongation of a new DNA strand lengthening in the 5’ 3’ direction

DNA polymerase III can only add nucleotides to the 3’ hydroxyl end

Leading strand - DNA pol III – adds nucleotides

towards the replication fork; - DNA pol I - replaces RNA with

DNA Lagging strand - DNA pol III - adds Okazaki

fragments to free 3’ end away from replication fork

- DNA pol I - replaces RNA with DNA

- DNA ligase – joins Okazaki fragments to create a continuous strand

STEP 4 Proofreading correcting errors in replication

Mismatch repair • DNA pol III – proofreads

nucleotides against the template strand

Excision repair • nuclease – cuts damaged

segment • DNA pol III and ligase – fill the

gap left Telomeres at 5’ ends of lagging

strands • no genes, only 100 – 1000

TTAGGG sequences to protect genes

• telomerase catalyzes lengthening of telomeres

DNA Replication and Repair 1. Summarize the central dogma in a diagram. 2. Define antiparallel and semiconservative in terms of

the structure of DNA. 3. Use the following terms associated with replication

and create a flowchart showing the different stages: replication bubble and replication fork, helicase, single-strand binding proteins, RNA primer, primase, leading strand, lagging strand, DNA polymerase III, DNA polymerase I, DNA ligase, Okazaki fragments, and 5’à 3’.

4. Differentiate between mismatch and excision repair. 5. What are telomeres and what role do they play in

protecting the integrity of the lagging strand of the DNA?

Modelling

1. By team, create a DNA strand that is at least 20 nucleotide pairs long with at least one stretch that has the sequence ATATAA

2. One member should be sketching the DNA strand on the sheet provided

3. Indicate the 5’ end and the 3’ end for each strand

Modelling

4. You are modeling eukaryotic DNA. How would prokaryotic DNA be different?

5. Use the clay to create helicase, topoisomerase, and single-strand binding proteins.

6. Show how these act in unwinding, stabilizing and holding the strands apart.

Modelling

7. In real life, RNA primers are 7-10 nucleotides long. Create two 3-nucleotide long RNA primers that would correspond to the sequence complementary strands closest to the two replication forks.

8. Create primase using clay and use it to attach the RNA primers to the correct sequences on the complementary DNA strand.