DNA Replication & Repair

by Stephanie Knol

What is DNA?

Deoxyribonucleic acid is a molecule that carries the genetic instructions used in the growth, development, functioning and reproduction of all known living organisms and many viruses.

Structure:

- Sugar-phosphate back bone
- Hydrogen bonding
- Four nitrogen bases, two pairs
  - - Adenine and Thymine
    - Guanine and Cytosine

The two strands run in opposite directions

DNA Replication

Semi-conserved replication (made of one old, conserved strand, and one new replicated strand)

➾ In the leading strand (5'-3') nucleotides can be added continuously

➾In the lagging strand (3'-5') nucleotides must be added in 𝗢𝗸𝗮𝘇𝗮𝗸𝗶 𝗳𝗿𝗮𝗴𝗺𝗲𝗻𝘁𝘀

Different enzymes come in to help with the process of replication:

1. In order for replication to occur, the two strand must separate

- Helicase: "Unzips" the two strands from each other, making the nitrogenous bases available to bond with other bases
  - - Replication fork creating a template for a new strand of DNA
    - Single stranded binding proteins: attach to the separated strands and prevent them from re-annealing
- DNA gyrase: Untwists strands to relieve tension
- Primase: The initializer/ primer that creates RNA primers to initiate replication in the DNA sequence
- DNA polymerase:
  - - III: Adds nucleotides to the 3' end of the DNA sequence.
    - I: Removes RNA primers, and replaces them with DNA
- Ligase: "Glues" bonds to join all the fragments together

DNA Repair

A collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome

Damage is detected by simply looking for a bulge in the helix of the DNA, detected by the cell itself.

Once damage is localized, specific DNA repair molecules are triggered and they bind at or near the site of damage and repair can start to take place

Single strand repair mechanisms:

Nucleotide excision repair: entire nucleotide is excised (cut out) about 24, and they are replaced
Base excision repair: One enzyme snips out the damages base, and other enzymes come to trim around the site and replace the nucleotide
Mismatch repair: Fix and rematch unmatched nucleotide

Double strand repair mechanisms:

Ionizing Radiation: Gamma rays and x-rays

Non- homologous end joining: ligase connects to split ends of DNA and fuse them back together (isn't as accurate as homologous recombination but is useful when sister DNA isn't present)
Microhomology- mediated end joining: Ligating mismatched hanging strands of DNA, removing overhanging nucleotides and filling extra spaces
Homologous recombination: uses undamaged section of similar DNA as a template. Enzymes inter-lay the damaged and undamaged strands, get them to exchange sequences of nucleotide, and fill them in, ending in two complete double segments

DNA repair process is a constantly active response to damage in the structure of DNA

1 million instances/cell/day!

Proofreading

Sometimes an error occurs (mismatch), about one per million; proofreader enzymes identify and correct most if not all the mistakes (about 3000 per cell division)

Mismatch repair:

- IF a base is mismatched or another error occurs, the DNA replication pauses.
- The mismatched base enters an exonucleus site on the enzyme polymerase.
- Removal occurs in the 3'-5' direction
- After the mismatched base is removed, DNA replication continues

Photo References:

https://5mindna.com/product/info-for-5-min-total-nucleic-acid-extraction-kit/

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