Lecture 6 - Chromosomal Segregation Flashcards
Describe the Replication Factory model in terms of:
(i) Structure
(ii) Chromosomal Segregation
(1 Point, 2 Points)
(i) Stationary structure at cell centre, which consists of two replisomes back-to-back that pull DNA in opposite directions
(ii) Replisomes = powerful molecular motors, which push daughter chromosomes in opposite directions
* Not strong enough to push DNA (0.5-1µm) to poles, hence other factors co-operate
Define:
(i) Linking Number
(ii) Twist
(iii) Writhe
(i) Linking Number (L) = Writhe (W) + Twist (T)
(ii) Twist - number of times single strands are twisted round one another
(iii) Writhe - number of times double-stranded helix crosses over itself (supercoils)
Explain:
(i) The Supercoiling Problem of Circular Chromosome Replication
(ii) Relevance of Linking Number (L), Writhe (W) and Twist (T)
(i) Unwinding of Parental DNA Duplex generates torsion ahead of the replication fork, which would resist further unwinding if too much tension accumulated
(ii) Linking Number (L) cannot change in a closed topological system, therefore if a twist is removed (i.e unwinding), the writhe must increase (supercoiling)
How can torsion be removed to allow continued unwinding of DNA double helix?
(3 Points)
- Can be removed in several different ways:
1. DNA Gyrase/Topo IV - remove positive supercoils ahead of replication fork by creating double strand break, then passing single strand through break
- Topo I/III - work by generating a single strand break through which the other strand of duplex is passed
Describe SMC Protein in terms of:
(i) Monomeric Structure
(ii) Dimeric Structure
(i) SMC Monomer - consists of N and C-terminal domains which are separated by a hinge region, and interact to form a Head domain with ATPase activity
(ii) SMC Monomers dimerise via their hinge domain, producing a “pincer-like” structure that opens/closes dependent on the nucleotide present in head domains (ATP vs ADP)
What is the function of SMC?
(2 Points)
- Not involved in the segregation of replicated DNA, but is instead involved in trapping segregated daughter chromosomes at poles
- This occurs via the formation of Foci (Flower-like), which occurs due to interaction between head domains of adjacent SMC dimers
How are SMC Proteins regulated?
- ScpA/ScpB - bind to SMC head domains and reduce SMC ATPase activity, thereby organising SMC-DNA complexes
- (i.e. determine whether clamp is open or closed)
(i) Give an Example of the role of Actin-like proteins in Chromosomal Segregation
(2 Points)
(i) MreB - actin-like filament, produces ATP-dependent filaments that organise into helical elements near inner membrane
* MreB Filaments perfom dynamic movement via polymerisation/depolymerisation events, moving newly formed genome towards cell poles
How does Bacterial Gene Transcription play a role in Chromosomal Segregation?
(2 Points)
- RNAP (RNA Polymerase) has strong motor activity, with its moving away from the origin causig movement of transcribed regions towards poles
- On Most bacterial Chromosomes the orientation of transcription is away from the origin, with highly transcribed genes being found closer to the origin
How does the MreB-RNAP interaction Facilitate chromosomal segregation?
- RNAP molecules may be fixed directionally in such a way as to effect movement of duplicated DNA towards poles (pulling effect)
Define SpoIIIE in terms of:
(i) Structure
(ii) Function
(i) Hexameric Ring protein
(ii) Recruited to division septum, where it acts as a rescue system to move entrapped chromosomes into daughter cells by pulling DNA in a single direction
Recombination is frequent event during Chromosomal segregation. How can it go wrong?
A Chromosomal Dimer can be formed when an uneven number of crossover events occur, making segregation into daughter cells impossible
Describe the Mechanism by which Chromosomal Dimers are resolved?
(3 Points)
- Chromosomal dimers can be resolved by final round of recombination between sites close to terminus known as Dif sites
- These Dif sites are recognised and used for dimer resolution be two site-specific Tyr Recombinases (e.g., XerC/XerD in E. coli)
- The Dif sites are aligned at the division septum by the action of DNA Pumps (SpoIIIE), where Recombinases catalyse final recombination, producing two separate chromosomes
(i) Describe the Formation of the Axial Filament during sporulation
(ii) Why is the Axial Filament produced?
(4 Points)
(i):
* RacA, a sporulation induced protein, binds to several sites along chromosome, but predominantly at oriC
* RacA interacts with DivIVa, a membrane protein present exclusively at cell poles, attaching origins to cell poles
* This Chromosomal Structure linking the two poles is known as the Axial Filament
(ii) Axial Filament Organisation of Chromosomes allows incorporation of one chromosome into the forespore during polar cell division
How is the Daughter Chromosome incorporated into the budding spore?
(2 Points)
- As Division Septum at pole of the cell begins to form it starts crushing the chromosome, recruiting SpoIIIE that pumps entire chromosome into the forespore
- To Ensure that Chromosome is only pumped into forespore, only SpoIIIE in the mother cell assembles correctly
