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Lecture notes Year 1 MBChB
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Concise lecture notes from the nucleic acids strand of the IMS module taught in the first year of the MBChB course at the University of Leeds!
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NUCLEIC ACIDSDNA STRUCTURE AND REPLICATION
Evidence for DNA’s biological role
Frederick Griffith
Streptococcus pneumoniae- cause of fatal pneumonia in humans
Two strains- one grew as ‘rough’ colonies & non virulent, the other
as ‘smooth’ colonies and was virulent
Oswald Avery Live smooth + rough
From purified DNA, RNA, protein, lipid and carbohydrate only DNA strains isolated
from heat killed virulent strain could induce virulence in the non- from dead mouse
virulent strain
Hershey and Chase
The protein and DNA components of bacteriophage were labelled
with different radioactive molecules
Erwin Chargaff – studied base composition of DNA in different species and the proportion of bases
DNA structure
Sugar phosphate backbone, adjacent deoxyribose sugars linked by phosphodiester bonds
5’ and 3’ gives DNA strand directionality, A=T C≡G
5’ 3’ (coding strand in protein synthesis) and 3’ 5’ (template strand in protein synthesis)
DNA + histone protein = chromatin
DNA is tightly coiled with help of proteins to fit its considerable length into the nucleus
Active genes more loosely coiled then silent ones
Cell cycle
Interphase (90% of the time) – chromatin is disordered and uncondensed, it condenses in the cell cycle
Evidence for semi-conservative replication
Meselson and Stahl grew E.coli for 14 generations in a medium containing 15N (heavy). After 14 generations, most of
the DNA in the bacteria would be heavy due to 15N
They then transferred these bacteria into a medium containing 14N, and left them to replicate once
After 1st division: hybrid DNA, so it doesn’t replicate conservatively
After 2nd division: after being centrifuged, two bands of DNA were produced, one hybrid and one light, showing
replication is semi-conservative
Light DNA increases and heavy DNA decreased after subsequent divisions
DNA replication
DNA synthesis occurs at the origin of replication and uses a replication fork
o At the fork:
o Helicases breaks H bonds and unwind double stranded DNA
o Single strand binding protein stabilise denatured DNA, they ensure chains stay
separated
o RNA primase makes short RNA primers to let replication happen
o The RNA primer allows DNA polymerase to start the replication as the primer
provides an OH group on carbon 3 to allow phosphodiester bonds to form with
free nucleotides – forms sugar phosphate backbone
o DNA polymerase III brings in dNTPs which bind together, the third phosphate
group breaks off which releases energy for the phosphodiester bond to form
o DNA polymerase carries out the elongation of the new strand of DNA, which
forms by complementary base pairing to the template strand
Okazaki fragments - relatively short fragment of DNA synthesized on the lagging strand during DNA replication
DNA polymerase III carries out DNA replication in prokaryotes
DNA synthesis
, Occurs in 5’ to 3’
DNA polymerase catalyses the formation of a new phosphodiester bonds
Leading and lagging strands
DNA made in short strands on the lagging strand, each one primed with a new DNA primer
DNA polymerase I replaces the RNA primers with DNA and DNA ligase seals the gaps between fragments
DNA polymerase can only function in 5’ to 3’ direction, with nucleotides being added from the 3’ (on the original
strand)
DNA polymerase travels along the leading strand in the 3’ to 5’ direction, adding new nucleotides to the 3’ end of the
new strand
Mutations
DNA polymerase has a 3’ to 5’ editing function, to remove incorrectly inserted bases, reduces error to 1 in 10 7
Enzymes present to also check for mismatches, reduces error to 1 in 10 9
Occurs due to errors in replication, induced by DNA damage, radiation, mutagens (such as carcinogens)
Base substitution, deletions, insertions, rearrangements – can be silent mutations
Deletions and insertions causes frameshift
o Consequences:
Germ cells – can be inherited, could result in new genetic disease
Somatic cells – not inherited but can lead to cancer
Mutagens
Example is ethyl methane sulphonate (EMS) which alkylates DNA to form the O6-ethylguanine adduct
This adduct mis-pairs with thymine during replication producing GC ® AT mutation
EMS, N-Ethylnitrosourea (ENU) and other alkylating agents used to generate mutations in organisms
This can help to find novel genes involved in disease processes
DNA repair 1
Endogenous and exogenous chemicals damage the DNA
DNA repair proteins remove the damage before replication occurs
Base excision repair proteins cut out damaged bases- they are specific to specific types of damage
Nucleotide excision repair proteins are less specific and cut out sections of the damaged DNA strand
DNA repair 2
DNA polymerase I replaces the DNA by copying the intact strand, and DNA ligase seals the gap
Goes wrong or too much to repair - mutations may occur, increasing the chance of cancer
Cytosine deamination
Occurs slowly in aqueous solution and changes the sequence of the DNA strand
Uracil N glycosylase
Recognises uracil in DNA and cuts it out - base excision repair enzyme
specific for its substrate
THE HUMAN GENOME
The human genome consists of about 3 x 109 nucleotides distributed between 22 autosomes, 2 sex chromosomes (X and
Y) and a small amount of DNA that is present in mitochondria
RBC’s lose their nucleus to squeeze through capillaries but other cells (e.g. hepatocytes and megakaryocytes) copy their
DNA several times without dividing and become 4n, 8n, 16n etc.
Women have one inactive copy of their X chromosome in each cell of their body and push it to the edge of the nucleus
(Barr body)
Synteny - where long DNA sequences (e.g. genes) are present in the same order across species
Translocation
Chromosome breakage and reforming, can cause disease in patients
Translocations can cause cancer and cause developmental abnormalities and can be inherited
Transcription of genes
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