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DNA metabolism lecture notes
The Human Body (PY4010)
Kingston University
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DNA metabolism (N)
Characteristics of DNA the antiparallel strands are templates for each other specific base pair hydrogen bonding allows for accurate copying tightly wound helical structure is stable and needs to have the hydrogen bonds broken for strands to unwind torsional stress in winding the extremely long molecule need to be overcome DNA replication process DNA Helicase breaks hydrogen bonds between bases, unwinds double helix into two strands which both act as templates Free floating DNA nucleotides attracted to exposed bases via specific complementary base pairing, hydrogen bonds form (adenine-thymine and cytosine-guanine) DNA polymerase joins adjacent nucleotides on new strand by condensation, forming phosphodiester bonds (= sugar phosphate backbone) Replication is semi-conservative – each new strand formed contains one original / template strand and one new strand Ensures genetic continuity between generations of cells Incorporation of new nucleotides Nucleotide triphosphate is attacked by the OH group of the ribose. It forms a phosphodiester bond with the release of pyrophosphate.
Semi-conservative replication
Two copies of the original DNA molecule are produced, each copy conserving the information from one half of the original DNA molecule.
Sample 3 in Meselson Stahl experiment
50% DNA molecules contain 1 original ‘heavy’ and 1 new ‘light’ strand, 50% contain both ‘light’ strands.
DNA replication in bacteria (in depth)
Replication starts from one point of origin. Specific proteins recognise DNA sequences at the origin and bind to the DNA and begin to open out the helix and unwind it. This forms a replication bubble, which allows access to proteins that synthesise the new strands. They move away from the origin as they make new DNA. A replication fork is produced. Typically happens in both directions (bidirectional replication). The replication bubbles merge and coalesce.
Semi-discontinuous replication
One strand is copied continuously by adding to the 3’ end (leading strand). The other strand is replicated in small sections, as a discontinuous process (lagging
DNA polymerase = an enzyme responsible for elongation of the DNA. The strands must have a template and extend on the 3’ end. They also require a primer, nucleotide triphosphate and magnesium ions. DNA primase = an enzyme that synthesizes short RNA sequences called primers. These primers serve as a starting point for DNA synthesis. Ligase = joins the Okazaki fragments together to make one strand. DNA helicase = separate double-stranded DNA into single strands in order to open out the DNA helix. Single-stranded binding protein = protects the single stranded DNA from being attacked by nucleases or becoming tangled. Topoisomerase = prevents the DNA from getting too tightly coiled and untangles DNA. Clamp proteins = usually multimeric proteins with a ring shape, and are capable of encircling duplex DNA. The DNA polymerase is physically linked to clamp which increases its processivity.
Extra info
The fidelity of a DNA polymerase refers to its ability to accurately replicate a template. Correct base pairing is favourable for the enzyme. The DNA polymerase itself has a proofreading activity. The enzyme has a 3’ to 5’ exonuclease activity. This means it checks the base pairing as it goes. An incorrect base pair activates a second catalytic site, which removes the incorrect base pair.
DNA damage
Endogenous sources of DNA damage ⇒ replicative errors, oxidative damage by free radicals, spontaneous alteration in DNA and alkylating agents. Exogenous sources ⇒ UV, pollution, carcinogens, radiotherapy and chemotherapy Damage to DNA includes ⇒ bases become oxidised, damaged, alkylated, deaminated or lost. Bases can become dimerised (covalently bonded). DNA backbone can break. Strands can break or become crosslinked.
DNA repair
Direct reversals ⇒ i. when the bases become covalently bonded or methylated, an enzyme is used to directly repair the bases
Nucleotide excision repair ⇒ a nucleotide containing the error is removed and DNA ligase is used to fill in the space
Base excision repair (BER) ⇒ only the affected base is removed ⇒ the damaged base is removed leaving a AP site ⇒ DNA polymerase and ligase add new DNA and seal the gap (AP site)
Mismatch repair ⇒ recognition of the deformity caused by the mismatch, determining the template and non-template strand, and excising the wrongly incorporated base and replacing it with the correct nucleotide
Recombinational repair ⇒ DNA is repaired using sequences from a homologous piece of DNA
Non homologous end joining ⇒ binding together of 2 broken DNA ends. May be different or original strands. Very toxic form of DNA damage and very prone to error.
DNA metabolism lecture notes
Module: The Human Body (PY4010)
University: Kingston University
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