BIOL253 - Lecture 5

  • Created by: Katherine
  • Created on: 07-04-17 11:43
Where does termination of replication occur?
At the ter sites, opposite side of the chromosome from the ori.
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What is ter bound by?
A replication terminator protein called Tus.
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What does Tus do?
When replication forks hit Tus they stop and replication complex disassembles.
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Remaining unreplicated DNA is rilled in by
DNA pol I.
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What is decatenation?
When the daughter molecules are separated.
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When the daughther molecules are linked what are the called?
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How are eukaryotic chromosomes replicated?
There are multiple origins of replication. Termination can occur anywhere on the chromosome where two replication forks meet. Forks and replication machinery disassemble and remaining DNA filled in. DNA are entwined and separated by topoisomerases.
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What is the problem of replicating a completely linear chromosome in eukaryotes?
Lagging strand synthesis cannot replicate the very end of a linear chromosome. This is the end replication problem.
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What is the end replication problem?
RNA prier removal leaves a gap that cannot be filled in. Dissolution of the replication fork, when the lagging strand is finished, before lagging strand synthesis is complete - could lead to the loss of the terminal Okazaki fragment.
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What is the issue associated with the end replication problem?
Leads to loss of chromosome ends over several rounds of replication.
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End replication is not a problem for organisms that have...
circular chromosomes
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What solves the end replication problem?
Telomeres and telomerase
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Eukaryotic chromosome ends have simple sequence repeats;
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Telomeres consist of a ...
long, double stranded repeat region with a single strand tail at the end.
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The sindle strand DNA tail...
goes towards the chromosome end.
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Which nucleotide is Telomere rich?
It is G rich
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Why do telomers form an unusual DNA structure?
To mark the end of the DNA as the end - it makes them distinguished for DNA breaks which is essential for chromosome stability.
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What is the name of the unusual structure?
T loop - formed by base pairing of the overhang with the double stranded region.
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What is a four stranded structure called?
G quadruplex
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How are telomeres maintained?
Using telomerase ( a specialised reverse transcriptase).
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What are reverse transcriptases?
DNA pols that copy RNA into DNA.
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How do reverse transcriptases work?
They use single stranded RNA as a template, but are structurally and catalytically similar to DNA pols
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What is the role of RNA
To act as a template for synthesis of repeats and has a role in enzyme activity
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What is TERT?
Protein telomerase reverse transcriptase.
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What does telomerase do?
It binds to ** telomere DNA, elongating telomere
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How does telomerase b
it base pairs the DNA to the RNA
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How often can E.coli divide?
As often as once every 20 mins.
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How long does it take to replicate E.coli bacterial genome/
50 mins
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How many mechanisms are there that regulate initiation of regulation in E.coli?
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How is regulatory inactivation of DNA done?
DnaA binds and multimerizes at oriC when bound to ATP, and unwinds adjacent to DNA. Helicase initiates synthesis. After initiation, sliding clamp stimulates ATP hydrolysis. DnaA-ADP dissociated and cannot reinitiate origin firing, preventing further.
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What does RIDA stand for?
Regulatory inactivation of DnaA - it blocks replication initiation.
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How does E.coli protect itself from pathogens?
It modifies it DNA at specific sites (GATC)
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How does E.coli modify it's DNA?
It methylates the A residue in GATC sites
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What methylates GATC sequences?
Dam methylase
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What are the steps of regulation of initiation In E.coli?
A residue in GATC methylated by Dam methylase. DnaA binds Dna A boxes at oriC when GATC fully methylated. After replication DnaA boxes are transiently hemi-methylated. SeqA binds to hemi-methylated GATC sites.
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What does the binding of SeqA to hemi-methylated GATC sites result in?
Prevents DnaA rebinding DnaA box. Prevents Dam methylase methylating at GATC.
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How does Seq A work?
Seq A binding causes a temporary block. Seq A dissocites - may be replaced by another Seq A, or dam methylse may bind and methylate at GATC (blocking further Seq A binding).
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Origin is usually fully methylated after ...
10 mins after initiation.
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A fully methylated chromosome is ready for...
DnaA binding. This is initiates a new round of replication.
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What is the 3rd mechanism to prevent initiation of replication?
DnaA sequestration
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How does DnaA sequestration occur?
Just after replication, the datA region is duplicated, sequestering more DnaA, making it unable to bind to oriC.
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What happens after segregation of chromosomes?
There is one copy of datA so there is again enough DnaA available to bind to ori C and promote initiation
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What is the 4th mechanism of regulation of initiation of replication in E.coli?
Recycling of DnaA-ADP to DnaA-ATP
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What is the process of recycling of DnaA-ADP to DnaA-ATP?
DnaA-ATP must be provided for the next round of initiation. Dna A ATP is newly synthesized, and DnaA-ADP is recycled to DnaATP. DnaA binds to DARS regions of DNA. DARS serves as cofactors to stimulate exchange of ATP for ADP.
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What does DARS stand for?
DnaA Reactivating Sequences
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In Eukaryotes, when does chromosomal DNA replication occur?
During the S phase
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What happens in the S phase?
All the DNA in the cell must be duplicated once.
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Incomplete replication in Eukaryotes leads to
Inappropriate links between daughter chromosomes.
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Segrgation of linked chromosomes leads to...
Breakage or loss and re-replication can have severe consequences.
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How is chromosome replication controlled?
A subset of origins are activated to initiate replication. Origins are selected in G1. Origins are activated in S, and cannot be re-used until re-prepared in another G1 phase. Origins are inactivated when replicated.
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What does the mechanism of replication control prevent in Eukaryotes?
It prevents any part of a chromosome being replicated more than once in any cell cycle.
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How is the pre-replication complex made?
Origins are selected in late M/G1. ORC binds to origin, recruits cdc6 and cdt1. ORC, Cdc6 and cdt1 co-operate (as helicase loaders) to load double hexmer of the MCM helicase. Each MCM, loaded head to head, encircles both strands of DNA - inactive.
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What is each MCM helicase?
It is a heteromeric hexamer
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The pre-replicative complex can only be assembled in
G1 (low Cdk)
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Card 2


What is ter bound by?


A replication terminator protein called Tus.

Card 3


What does Tus do?


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Card 4


Remaining unreplicated DNA is rilled in by


Preview of the front of card 4

Card 5


What is decatenation?


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