B3- Replication Of Kinetoplast Dna Flashcards
How many copies of kinetoplast are there
1 on the base of flagella
Why are they on flagella base
For the energy and motility
How is ktdna constructed
5000 minicircles with valency of 3 (link to 3) and also 40 maxi circles in the network forming long strands
(Kinetoplast disc )
What do maxicircles encode like mt dna
Rrnase
Energy transduction genes like nadh dehydrogenase, cox subunits, atp synthase
What do these transcripts undergo for maturation
Rna editing where u added or deleted
How do mini circles allow this
Encode grnas for this
Which conserved 100-200 bp sequence in all mini circles is for leading strand synthesis initiation
Universal minicirlce sequences (UMS)
Which site is for Okazaki fragment stsrt site/ lagging strand
Hexamer invariant site (6 residuesh
What are the dna structures in minicircles also conserved in very heterogenous minicircles for bending dna
A-tracts of 5+ residues for bending dna for organisation
When are the dna networks replicated which differs to mt dna
S phase
Explain to hat happens first in replication of minicircles
The central zone (closed circles) released from network by topoisomerase II into the kinetoflagellar zone (kfz)
THEN REPLICATED LIKE PLASMIDS (theta structure replication)
Why do nicks form in replication
To make sure only replicated once
Where do they go after kfz for further rep help and ligation
2 antipodal sites on each end of dna network
Where do they reattach after antipodal site by topoisomerase II
Periphery of the network (ends)
Since it was found they reattach at two poles, what are the 2 suggestive ways they then become evenly distributed across network
The disc rotates so each point makes contact with the 2 replication proteins on the periphery
Or they attach at the poles then redistribute
What does this explain
How at the start there is poles of higher activity during replication (until rotates completely )
- then even just before division of meshwork/network
How does maxi circle replication occur according to crithidia model
Theta structure/rolling circle model like plasmids
Whilst still attached to the network
Where does it originate
Conserved sequences like in minicircles ums
What is segregation of kdna done by
Tripartite attachment complex (tac)
Meaning it is dependant on flagellar-basal body to segregate it
Before division what holds the 2 networks and what cuts them
Maxi circle threads
Topo isomersse II
What types of filaments in kinetoplast are between the mt membrane down to surface of the network (pass the kfz) (part of tac) - what do they do to help division
Unilateral filaments
Rotation of the networks before division occurs
What are the filaments attaching basal body to outside of mt membranes (part of tac)
Exclusion zone filaments
Which cdc2 - yeast related gene in crithidia found in the kt and what does cdc2 do - potential role??
CRK gene in crithidia
Mitotic regulation/cell cycle regulation
What ss dna binding protein found in crithidia whcih stops winding back to ds dna - potential role in ktdna??
Rep protein A
Of 5 purified cross linked proteins from crithidia kt, how many found to have a 9aa import sequences suggesting nuclear encoded AND ALSO how many of the proteins have lysine rich regions perfect for dna binding
2 lysine rich
3 have import sequences
Why was crithidia shown to hve something that resembles dna pol B - potential role in gapnfilling ?
Because was same size , had no proofreading/exonucelase activity , and was able to gap fill Okazaki fragment
Why would error rate or 1/1200 for pol B explain minicircle sequences
Very heterogenous apart from the 3 conserved motifs
Why are they so heterogenous
Encode many grnas for editing of maxi circle
Which 2 topoisomerase a found in crithidia
A tetramer localised within kt
Also a hkmodimer mroe local to the antipodal sites (probably for reattachment)
Which protein binds ums and also the hexamer invariant site
Umsbp
Where is it located
2 sites of the kfz
What does it binding to okazaki site suggest
Okazaki gap last repaired. Potentially stop premature repair
What is p38 suggested to do in antipodal sites
Speculate it allows helicase together with topoI to unwind dna helix
Because p38 binds replication origin
Which polymerases in the kfz
Pol 1b and 1c
Why is pol 1b likely a hlmodimer
Synthesises both leading and lagging strand
Where is the pol B identified in crithdiia located for gap filling and what does it pair with
Antipodal sites and pairs with ligase kappabeta kb to anneal
Which helicase needed for primer removal in brucei and is located in antipodal sites
Pif5 helicase
Which pol and ligase complex in the network for final reannealinf
Pol b-pak and ligase ka
What 2 primates are in antipodal site and why - suggested by rnai experimentation
Primase 1 is for maxi circle rep
Primase 2 for minicircle (rnai causes increase in covalently closed)
Which pif helicase necessary for maxi circle formation
Pif2
Which protease targets pif2 to regulate replication
Hslvu
What does oxidation of umsbp do to make sure replication only happens in s phase
Inactivates it to control reg
By blocking it’s interaction with zinc (it’s a zfb protein)
Only reduced form is in s phase
What is the 3rd component of tac
The mt membrane
Which 2 proteins seem to have effect on correct segregation (based on rnai experiments) of kdna networks and where are they located
P166 and aep-1 likely in the unilateral filaments close to the network/kdna
When interfered with it stops correct segregation
Are their roles known specifically yet
No
What do the kfz zones contain vs antipodal
Umsbp,Pol 1b and 1c
Antipodal has topo 2, primase 1 and 2, pol beta, ligase kbeta, sse-1, p38, p93
Is the role of pol 1C and D known?
No but likely involved in maxicircle since pol 1b produces both minicircle strands
