B3- Replication Of Kinetoplast Dna

Question 1

How many copies of kinetoplast are there

Answer 1

1 on the base of flagella

Question 2

Why are they on flagella base

Answer 2

For the energy and motility

Question 3

How is ktdna constructed

Answer 3

5000 minicircles with valency of 3 (link to 3) and also 40 maxi circles in the network forming long strands

(Kinetoplast disc )

Question 4

What do maxicircles encode like mt dna

Answer 4

Rrnase
Energy transduction genes like nadh dehydrogenase, cox subunits, atp synthase

Question 5

What do these transcripts undergo for maturation

Answer 5

Rna editing where u added or deleted

Question 6

How do mini circles allow this

Answer 6

Encode grnas for this

Question 7

Which conserved 100-200 bp sequence in all mini circles is for leading strand synthesis initiation

Answer 7

Universal minicirlce sequences (UMS)

Question 8

Which site is for Okazaki fragment stsrt site/ lagging strand

Answer 8

Hexamer invariant site (6 residuesh

Question 9

What are the dna structures in minicircles also conserved in very heterogenous minicircles for bending dna

Answer 9

A-tracts of 5+ residues for bending dna for organisation

Question 10

When are the dna networks replicated which differs to mt dna

Answer 10

S phase

Question 11

Explain to hat happens first in replication of minicircles

Answer 11

The central zone (closed circles) released from network by topoisomerase II into the kinetoflagellar zone (kfz)

THEN REPLICATED LIKE PLASMIDS (theta structure replication)

Question 12

Why do nicks form in replication

Answer 12

To make sure only replicated once

Question 13

Where do they go after kfz for further rep help and ligation

Answer 13

2 antipodal sites on each end of dna network

Question 14

Where do they reattach after antipodal site by topoisomerase II

Answer 14

Periphery of the network (ends)

Question 15

Since it was found they reattach at two poles, what are the 2 suggestive ways they then become evenly distributed across network

Answer 15

The disc rotates so each point makes contact with the 2 replication proteins on the periphery

Or they attach at the poles then redistribute

Question 16

What does this explain

Answer 16

How at the start there is poles of higher activity during replication (until rotates completely )

• then even just before division of meshwork/network

Question 17

How does maxi circle replication occur according to crithidia model

Answer 17

Theta structure/rolling circle model like plasmids
Whilst still attached to the network

Question 18

Where does it originate

Answer 18

Conserved sequences like in minicircles ums

Question 19

What is segregation of kdna done by

Answer 19

Tripartite attachment complex (tac)
Meaning it is dependant on flagellar-basal body to segregate it

Question 20

Before division what holds the 2 networks and what cuts them

Answer 20

Maxi circle threads
Topo isomersse II

Question 21

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

Answer 21

Unilateral filaments

Rotation of the networks before division occurs

Question 22

What are the filaments attaching basal body to outside of mt membranes (part of tac)

Answer 22

Exclusion zone filaments

Question 23

Which cdc2 - yeast related gene in crithidia found in the kt and what does cdc2 do - potential role??

Answer 23

CRK gene in crithidia
Mitotic regulation/cell cycle regulation

Question 24

What ss dna binding protein found in crithidia whcih stops winding back to ds dna - potential role in ktdna??

Answer 24

Rep protein A

Question 25

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

Answer 25

2 lysine rich 3 have import sequences

Question 26

Why was crithidia shown to hve something that resembles dna pol B - potential role in gapnfilling ?

Answer 26

Because was same size , had no proofreading/exonucelase activity , and was able to gap fill Okazaki fragment

Question 27

Why would error rate or 1/1200 for pol B explain minicircle sequences

Answer 27

Very heterogenous apart from the 3 conserved motifs

Question 28

Why are they so heterogenous

Answer 28

Encode many grnas for editing of maxi circle

Question 29

Which 2 topoisomerase a found in crithidia

Answer 29

A tetramer localised within kt Also a hkmodimer mroe local to the antipodal sites (probably for reattachment)

Question 30

Which protein binds ums and also the hexamer invariant site

Answer 30

Umsbp

Question 31

Where is it located

Answer 31

2 sites of the kfz

Question 32

What does it binding to okazaki site suggest

Answer 32

Okazaki gap last repaired. Potentially stop premature repair

Question 33

What is p38 suggested to do in antipodal sites

Answer 33

Speculate it allows helicase together with topoI to unwind dna helix Because p38 binds replication origin

Question 34

Which polymerases in the kfz

Answer 34

Pol 1b and 1c

Question 35

Why is pol 1b likely a hlmodimer

Answer 35

Synthesises both leading and lagging strand

Question 36

Where is the pol B identified in crithdiia located for gap filling and what does it pair with

Answer 36

Antipodal sites and pairs with ligase kappabeta kb to anneal

Question 37

Which helicase needed for primer removal in brucei and is located in antipodal sites

Answer 37

Pif5 helicase

Question 38

Which pol and ligase complex in the network for final reannealinf

Answer 38

Pol b-pak and ligase ka

Question 39

What 2 primates are in antipodal site and why - suggested by rnai experimentation

Answer 39

Primase 1 is for maxi circle rep Primase 2 for minicircle (rnai causes increase in covalently closed)

Question 40

Which pif helicase necessary for maxi circle formation

Answer 40

Pif2

Question 41

Which protease targets pif2 to regulate replication

Answer 41

Hslvu

Question 42

What does oxidation of umsbp do to make sure replication only happens in s phase

Answer 42

Inactivates it to control reg By blocking it’s interaction with zinc (it’s a zfb protein) Only reduced form is in s phase

Question 43

What is the 3rd component of tac

Answer 43

The mt membrane

Question 44

Which 2 proteins seem to have effect on correct segregation (based on rnai experiments) of kdna networks and where are they located

Answer 44

P166 and aep-1 likely in the unilateral filaments close to the network/kdna When interfered with it stops correct segregation

Question 45

Are their roles known specifically yet

Answer 45

No

Question 46

What do the kfz zones contain vs antipodal

Answer 46

Umsbp,Pol 1b and 1c Antipodal has topo 2, primase 1 and 2, pol beta, ligase kbeta, sse-1, p38, p93

Question 47

Is the role of pol 1C and D known?

Answer 47

No but likely involved in maxicircle since pol 1b produces both minicircle strands