Lecture 13 Flashcards
the double helix
two nucleotide polymers
bound by complementary base pairing
phosphodiester bonds have 5’-3’ polarity
antiparallel strands
helical structure with specific features
Structural challenges of replication
how the new and old stands generate two new double helices
how are the nucleotides exposed and read
how to ensure fidelity
how to organise the enzymatic activities for multiple long DNA molecules
Conservative replication
histone scaffold
breaks hydrogen bonds
new nucleotide pairing without unwinding
need an isomerase activity to turn bases respect deoxyribose
Semi-conservative replication
breaks hydrogen bonds
new double helices are assembled as replication progresses
needs extensive unwinding: potential need for helicases
dispersive replication
no unwinding required
swapping templates strands
but requires multiple single-stranded breaks
assumes polymerisation is in both directions
Taylor, Woods and Hughes
growing root tip of vicia faba
they supplied thymidine labelled with tritium and after labelling the chromosomes incubate in cold medium with colchicine (blocks cytokinesis)
they looked at cells in metaphase and observed distribution of thymidine in chromosomes
Meselson and Stahl
reasoned that DNA with 15N in its nitrogens bases would be denser than with 14N so should lead to different sedimentations behaviour under CsCl gradient centrifugation
conc of Cs ions would be different along the test tube so DNA would sit at different heights based on density
Features of semi-conservative replication
fast
- human genome replicated in around 8 hours
accurate
-error rates of 10-5 in human polymerases but DNA repair mechanisms lower the overall rates to estimate 10-8
not perfect
- some errors , 7-0 new mutations per generation
Highly regulated
- dozens of enzymes participate in the process
Replication initiated at origins
replication proceeds in both ends of the bubble (forks)
however polymerisation is in one direction only
the replication bubble requires unwinding the strands of DNA
a eukaryotic chromomse may have 100s-1000s of origins of replications, this increases speed
replication forks
a very active area where DNA replication takes place
How do replication forks advance
- requires finding the 3-5 DNA polymerase
or - requires finding the small fragments and explaining how they are joined
Okazakis experiments
they used the replication of a phage in E.coli
they used sedimentation to separate DNA in this case of different lengths
they labelled the newly synthesised DNA with thymidine and then measured radioactivity in the distribution of sizes
then they used mutant phages with temperature-sensitive ligase (to join small fragments)
they did many other experiments using 5-3 exonuclease to prove that newly added nucleotides were only incorporated at the 3’ ends
leading strands
can be synthesised in a continuous process
lagging strands
the lagging strand requires additional steps
Essential enzymatic activities
DNA polymerase
can only synthesise in 5-3 orientation
needs a primer that provides 3 end
DNA primases
synthesise 5-10nt RNA primers from scratch using DNA as a template
Helices
unwind the double helix at the replication forks
Topoisomerases
relieve the strain of twisting of the double helix by breaking, swivelling and rejoining DNA strands ahead of the fork
Synthesis of the leading strands
as the fork advances the leading strand keeps elongating
Synthesis of the lagging strand
like with the leading strand first the primase synthesises the RNA primer
DNA polymerase III elongates the primer, until it bumps into the 5’ end of another primer and falls off the DNA
the DNA polymerase I removes the ribonucleotides at the 3’ end of the Okazaki fragment
finally the ligase joins the adjacent nucleotides between two fragments closing the single strand DNA break
The trombone
both strands together
the two polymerases at the fork advance in the same direction
What happens at the end of the DNA
in prokaryotes circular chromosomes, the last active DNA polymerase III will always encounter an RNA primer and DNA polymerase I and ligase will finish the job
but the linear eukaryotic chromosomes have a problem: the last RNA primer of the lagging strand cannot be made into DNA
Telomeres
telomeres are special, repetitive sequences at the end of the eukaryotic chromosomal DNA
Stabilised by proteins
They can be extended after every cell cycle, if the cell has telomerase
They do not prevent but postpone after every cell cycle, if the cell has telomerase
They do not prevent but postpone the erosion of genes near the ends of DNA molecules as they provide a buffer
Effects of telomeres on the germline
loss of telomeres would lead to offspring inheriting chromosomes lacking some genes
Effects of telomeres on somatic cells
progressive loss of terminal genes would impact tissue renewal
Effects of telomeres on medicine
cancer cells need to express telomerase and somatic cells lacking telomerase may be related to aging
Cyclic activity of telomerase
elongation- reverse transcription (RNA to DNA)
translocation- RNA template ends have the same sequence
Replication errors
minimised by proofreading capacity of enzymes but polymerase mistakes and DNA damage due to tension ahead of forks can cause changes in the sequence
If there are errors in the germline:
sequence changes can pass to the next generation
depending on the phenotypic effect the changes may be maintained in the population
this allows evolution
If there’s errors in somatic cells:
cells can become defective and be removed or accumulate as senescent cells decreasing performance of tissue
potential development of cancer (the incidence of tumours is related to the frequency of cell division )
Mutagens
ionising radiation (X and gamma rays)
chemical
Chemical mutagens
Alkylating agents: modify the bases in the DNA making the polymerases pair them with the wrong base during replication
nucleotide analogues: mimic the structure of a nucleotide to fool DNA polymerase to introduce them in DNA
intercalating agents: insert themselves between he nitrobases and can create frame shifts during replication
DNA repair mechanisms
DNA polymerase proofread newly made DNA replacing incorrect nucleotides
in mismatch repair of DNA repair enzymes correct error in base pairing
in nucleotide excision repair, DNA is scanned for structural deformations, a nuclease cuts out and replaces damaged stretched of DNA and the gap is closed by polymerase/ligase activities
