Lecture B3 - Replication of Kinetoplast DNA Flashcards
Where does kinetoplast DNA lie?
Within the single mt of the organism and found at the base of the flagellum.
What is the kinetoplast composed of?
5000 minicircles
40 maxicircles
Describe the kDNA network?
Interconnected meshwork of DNA molecules composed of mini and maxi circles.
Each mini circle can link to 3 other mini circles.
What does DNA Pol I do?
Repairs tears in the DNA. Active base goes in and if there are tears in the DNA then it will go a dark colour.
A halo of dark staining suggests that all of the DNA around the kinetoplast has tears in it.
Describe maxi circles.
Resemble mitochondrial DNAs.
Transcripts are cystic and undergo RNA editing to produce functional mRNA - uridine residues added or deleted to create ORFs.
Describe mini circles.
Encode small guide RNAs to control editing specificity.
Heterogenous sequence but contain conserved regions, universal mini circle sequences, leading strand synthesis.
Hexamer invariant site.
Bent DNA structures.
Why is replication of the DNA networks hard in trypanosomes?
40 maxi circles and 5000 mini circles must be accurately doubled and then segregated into daughter cells.
What is a good model organism for trypanosomes and why?
Crithidia fasiculata - a parasite of insects not humans, can grow to large quantities in cheap media allowing access to good amount of DNA and RNA.
When is there a slow release of minicircles?
Throughout the S phase there is a slow release of minicircles by topoisomerase II in to the Kinetoglagellar zone.
Why are holes left in the DNA of the mini circles?
Machinery leaves holes in the DNA to tell the cell machinery that the mini circle has been copied already and does not need to be copied again.
How does the daughter cell repair the holes in the DNA?
Progeny minicircles reattached by topoisomerase back to the periphery of the network.
Peripheral zone only (contains nicked minicircles that have been replicated once).
Two minicircles reattach for each one removed.
When replication complete all nicks are repaired by being reattached to the network periphery.
Why does the kDNA need to be packaged?
The isolated network is larger than the parasite cell.
What does the model for kDNA replication predict?
Covalently closed minicircles are released from central region of the network.
Migrate to one of two replication protein complexes
The replicated nicked progeny are then attached to the network periphery.
What did using greater resolving power of em autoradiography show about minicricle replication?
That replicated minicircles are in distributed uniformly around the network periphery not on the end.
This led to the hypothesis that the two replication complexes are fixed and the kt disc rotates.
How do maxi circles appear to replicate in C fasiculata?
Like rolling circles whilst attached to the network.
After replication the rolling circle tail is removed and the linearised molecule recircularises and reattaches to the network.
