DNA Replication Flashcards
Which direction do polymerases work?
5’-3’
What kind of DNA does polymerase 1 bind too?
Binds to nicked or gapped DNA and works on a the 3’ end, can’t bind ssDNA or dsDNA. No primer or 3’ end
What is nick translation?
When a phosphate bond is broken (open 3’ end) so start building that while degrading next bonds to rebuild them.
If treated by trypsin what happens to DNA polymerase?
Forms the Klenow fragment
What can the Klemperer fragment do?
It’s has a a 5’ exonuclease activity
What is the mechanism of the Klemperer fragment?
It’s got 2 magnesium on aspartate residues. Then positions itself at 3, end of primer and incoming dNTPs to enhance catalytic reactions.
How do polymerases know the right nucleotide to use?
- formation of correct H-bonds
- shape of pair as wrong pairs cause changes in helix structure
- cannot incorporate ribonucleotide triphosphates due to steric clashes
What is steric clash?
Steric clash is when there is an unnatural overlap of two no binding atoms in protein structures
How does polymerase decide whether to use polymerase or exonuclease site?
Polymerase is fast but is retarded by addittion of the incorrect base allowing time for the strand to come into contact with the exonuclease site.
How many types of polymerases are there?
5
What each polymerase used for?
Pol 1: repair+replication Pol 2:repair Pol 3: replication (main polymerase) Pol 4:repair Pol 5: repair
How many DNA polymerase 3 per a cell?
10
How fast is DNA synthesis with polymerase 3?
1600 nucleotides/second
What can Polymerase 3 do with what subunit?
Alpha subunit is used for polymerisation and the epsilon subunits performs exonuclease proofreading.
Why so many enzymes?
- antiparallel strands so different enzymes
- primers need enzymes to be made
- DNA plectonemically bound so enzymes need to separate strands
How is the lagging strand synthesised?
3’-5’ strand know as lagging strand and is formed in chunks known as Okazaki fragments and they are joined using DNA ligase.
Describe Okazakis’ experiment:
He used ecoli with radioactive thymine, and took regular samples of DNA he used alkali to make ssDNA. Performed sucrose density centrifuge and found you have groups of small fragment and big fragments.
Why can you find uracil in DNA?
-due to similar shape Pol 3 could of used it in replication
-could occur because of spontaneous deamination of Cytosine Due to water.
Binds Adenine instead of guanine now
How is uracil removed from DNA?
Uracil-N-glycosylase cuts out the base. Pol 1 fills gap via nick translation. Only U opposite G is removed, producing pseudo-Okazaki fragments
What’s a pseudo Okazaki fragment?
When uracil removed from the DNA produce pseudo Okazaki fragments.
Which subunit of DNA polymerase performs exonuclease activity?
DNA Q subunit
What enzyme synthesises primers?
Primases
What are Okazaki fragments primers made of?
RNA
What happens to Okazaki fragments when new strand meets it?
Polymerase 1 removes the primer via nick translation -use exonuclease function. The gap is sealed by DNA Ligase.
What is the mechanism of fork opening on DNA helix?
DNA helicases are used to unwind the helix. It’s a DNA dependent ATPase acting from 5’-3’ on the lagging strand
How is ssDNA stabilised once unwound?
Single strand binding proteins bind cooperatively on the lagging strand
What is the use of the beta clamp?
It forms a ring around the DNA holding the complex to it.
What are topoisomerases used for?
Enzymes the relieve superhelical stress this is produced in front and behind the replication fork. Also seperate daughter DNA molecules after replication.
How long is 1 helical repeat?
10.5 base pairs
What is the linking number?
Number of helical repeats
What is the Equation for linking number?
Lk=N/h
N-numbers of base pairs
h-helical repeats size
How many types of topoisomerases are there?
2
What is type 1 topoisomerase?
Catalyses relaxation of supercoils
What is type 2 isomerase
Utilise free energy from ATP hydrolysis to add negative supercooled DNA.
Mechanism of topoisomerase?
Break one or two strands, pass a segment through the gap and reseal the DNA.
What does DNA gyrase Do?
Introduces negative supercoils and decrease Lk by 2
Where does DNA replication initiation take place?
It starts at Ori-C and goes on till it reaches Ter-C
What does Ori-C contain?
4x9bp repeats
3x13bp repeats
All are AT-rich
What is DNAa role in initiation?
It binds to 9bp of Ori-C interacting with the 13bp sequence melting the strands.
What is the function of DNAb?
Moves along the lagging strand opening up the replication fork.
What’s the function of DNAq?
DNAg associates to DNAb and acts as a DNA primer producing an RNA primer at each replication fork.
What is the control of DNA synthesis done by DAM?
New DNA stands don’t replicate until they are methylated. New DNA is only hemimethylated so DAM (DNA adenosine methylase) methylates N6 adenine. Activating DNA initiation as fully methylated.
What is DAM in respect to DNA replication?
DAM (DNA adenosine methylase) methylates N6 adenine, however is a very slow enzyme, but it allows for DNA to be replicated only once.
How is DNA replication terminated?
It reaches the Ter region which is six homologous 23 basepair sequences that bind protein Tus. Tus prevents movement of the fork in one direction only.
What does protein Tus do?
Tus binds to the Ter region preventing fork movement in one direction.
How are the two circular daughter chromosomes separated?
Seperated by topoisomerase 4
What is the main differences between eukaryotic and prokaryotic replication?
Eukaryotes don’t replicate from a single point
Eukaryotic polymerases are slower
Okazaki fragments are smaller
What are 5 main eukaryotic DNA polymerases?
Alpha Beta Gamma Sigma Epsilon
How long are eukaryotic RNA A primers?
About 10base pairs
What is another name for the beta clamp?
Proliferating cell nuclear antigen (PCNA)
What is polymerase alphas functions?
It has a primase function but no beta clamp so doesn’t remain attached for long
Main function of polymerase sigma?
Associates with beta clamp and performs progressive synthesis
What is polymerase epsilon’s functions?
It repairs DNA (analogous with polymerase 1)
How are eukaryotic primers removed ?
Removed by FEN 1 ( flap exonuclease) cut out primer for DNA ligase to join back bone
What is the hayflick limit?
It’s a limit on DNA replication (40) as the end primer cannot be replicated so reduced in size till zero which is senescence.
What is senescence?
Senescence is the loss of a cells power to replicate
How is hayflick limit problem solved?
By adding repeated units of simple sequences to chromosomal ends. Done by an enzyme called a telomerase.
What is a telomerase?
Protein that adds simple sequences to the telomere of DNA
How is telomere length regulated?
By binding proteins such as TRF1/2
What can high levels of telomerases be linked to?
Tumours
What’s replication Like for RNA genomes?
RNA genomes are copied by an RNA polymerase plus strand directly copied from minus. Self priming replication with low to no proof reading
What is reverse transcriptase?
An enzyme that copies RNA into DNA.
How does reverse transcriptase work?
First synthesises a copy strand cDNA, tRNA LYS is used as a primer. RNA strand is then degraded by RNAse H.
What is reverse transcriptases primer?
tRNA Lysine
