Genes in action L3/4 Flashcards
What are main similarities between pro- and eukaryotic replication?
Both use origins of replication, both bidirectional, 5’ to 3’, semi-conservative, semi-discontinous, multiprotein replication complex
What is difference in terms of cell cycle?
Bacteria rapid replication in response to environment/nutrients
Eukaryotes only S phase
Compare size of human chromosome 5 and entire E.Coli chromosome
180Mbp vs 4.6 Mbp
so lots of human origins needed, single human cell has over 10,000
What is the DnaA proetin in eukaryotes, what does it bind and what does it look like?
The Origin Recognition Complex, ORC (a hexamer which binds to AT rich sequences)
What is the DnaC equivalent?
CDC6, recruits the helicase
How are polymerases recruited?
S-phase specific kinases phosphorylate proteins at the origin so polymerases are recruited
DNA pol alpha, delta, eta role?
what enhances processivity?
alpha- Synthesises and elongates RNA primer on leading and lagging strands
delta- Replaces pol alpha to synthesise lagging strand, processivity enhanced by binding of sliding clamp, protein called PCNA
eta- replaces pol alpha to synthesise the leading strand, high level of processivity even without clamp
How is the RNA primer removed in eukaryotes?
No DNA pol 1, instead use Flap Endonuclease 1(FEN1)
Pol delta doesn’t stop when it encounters the last okazaki fragment but continues synthesising new DNA while replacing the primer
pol delta then dettaches and leaves a 5’ structure behind which gets cleaved by FEN1 leaving a nick which is joined by DNA ligase
What are 4 common DNA damage mechanisms and what are each of them caused by?
1) covalent crosslinking of thymidine eg. due to UV light
2) alkylation of nucleotides to form DNA crosslinking eg. due to chemical agents such as EMS
3) Intercalation into DNA eg. ethidium bromide
4) DNA strand breaks by ionising radiation, low levels of x/gamma rays often lead to single-strand breaks and increased exposure leads to double-strand breaks
What is photoreactivation? What enzyme is used and what type of mechanism is used?
UV light makes a cyclobuatane ring between adjacent thymidines, one bond c5-c5, one bond c3-c3
Photolyase works in visible light and uses an electron transfer free-radical mechanism where an FADH- cofactor absorbs a photon to general a free radical that initiates repair
What is nucleotide excision repair?
What causes the damage to be recognised in prokaryotes and what proteins recognise it?
What is different in eukaryotes?
Damage could be any damage to base pairing/structure, recognised due to bulky DNA adducts i.e. DNA bound to a cancer-causing chemical
1) In bacteria distortion of the DNA double helix is recognised by a dimer of protein uvrA with single uvrB
2) UvrB unwinds 5bp resulting in DNA kinking
3) UvrA released, uvrC recruited
4) 12-13 nucleotide fragment removed, replaced by DNA pol 1 and stuck togther by lagase
In eukaryotes much more complex with 15 proteins
What is base excision repair and what enzyme discovers the defect?
When cytosine loses amino group and forms uracil, can’t base pair with G
glycosylase enzyme discovers defect and excises uracil base
more enzymes remove rest of nucleotide
DNA pol fills gap and strand sealed by ligase
What is mismatch repair?
What is it like in prokaryotes and eukaryotes?
This is base-pair specific and highly conserved in pro and eukaryotes
1) 2 enzymes mut S and mutL detect mismatch
2) Another enzyme mut H recognises methyl groups on DNA- only the template strand has methyl groups attached
3) mut H gets activated when bound to mutL and becomes an exonuclease that cuts the mismatch
5) DNA pol fills gap and ligase seals strand
Draw E Coli RNA pol
See notes for diagram
What is found within a bacterial promoter?
A transcriptional start site with purine (A/G) at over 90%
2 6 bp sequences (-35 and -10 sites)
