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L4 Molecular Microbiology > Week 1: Prokaryotic Cell Cycle : Replication > Flashcards

Week 1: Prokaryotic Cell Cycle : Replication Flashcards

1
Q

What is the key enzyme for firing replication at the origin?

A

Dna A

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2
Q

What is the role of DnaA?

A
  • Central to control of initiation of DNA replication
  • Not the whole story
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3
Q

What does Dna bind beofre DNA?

A

ATP

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4
Q

Why does DnaA bind ATP?

A

Has higher affinity for
binding sites
* Link to metabolic state of
the cell
* Model that ratio of DNA- ATP to DNA-ADP peaks for initiation

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5
Q

Where does DnaA-ATP bind on DNA?

A

Dna box
* Binding sites throughout chromosome
* multiple low affinity sites at origin
* Vary in affinity for binding

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6
Q

What is downstream of the ATP box?

A

AT rich region (AT Rich DNA Unwinding Element “DUE)
Weaker bonds, susceptible to meting

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7
Q

What is the AT rich region?

A

Origin of replication
245 base pairs long

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8
Q

What is replication dependent on?

A

linked to metabolic state of the cell.
Has an element of control
ATP>ADP = Dna-ATP

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9
Q

What happens when Dna-ATP binds to the Dna box?

A

Conformational change, allows the binding of the replisome components (DNA replication machinery)

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10
Q

How many replisomes of each DNA strand?

A

2 on each strand!!
bi-orientataion

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11
Q

How many replisomes of each DNA strand?

A

2 on each strand!!
bi-orientation

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12
Q

What eventually happens to the Dna-ATP?

A

hydrolyses and decreases affinity for the Dna box
half-life

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13
Q

When replication occurs, is the OriC available for new repliaction?

A

NO, delayed reassembly of the replisome

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14
Q

What is the period called when newly replicated DNA is not available for replication?

A

eclipse period- cant fire another round of replication
(14-18 mins) e.coli

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15
Q

How is the origin inactivated/negatively regulated after replication?

A
  1. SeqA
  2. Dam methylase
  3. DnaA-ATP
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16
Q

How does SeqA delay replication?

A
  • Prefers newly replicated hemi- methylated sites
  • Overlap some of the DnaATP binding sites, make them inaccessible physically.
  • targets GATC sites in origin region & behind advancing replication site
  • Delays DnaA-ATP re-binding
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17
Q

How does Dam methylase delay replication?

A
  • GATC binding sites
  • Methyl group
  • Delay in fully methylating new strand
    When it is activated it can displace SeqA and construct proper site
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18
Q

How does DnaA-ATP delay replication?

A
  • General DnaA-ATP levels lowered after firing of the origin
  • Bound DnaA-ATP hydrolyses to DnaA-ADP increase
  • Hda Protein, datA locus increase hydrolysis following initiation
19
Q

Why is there a delay for Ori accessibility?

A

dont put too much stress on material.
Allow the newly replicated strand to have correct nucleotide formation …
selected for in microbes

20
Q

What happens to the location of the origin during replication?

A

move apart

21
Q

What protein separate the strand ahead of the replication fork and subsequently bind DNA back together after?

A

topoisomerases
helicases and gyrases

22
Q

What force is creates as the DNA is replicated?

A

torsion

23
Q

What enzymes use the energy created by torsion?

A

E.g. MukBEF and ParCE

24
Q

What is MukB similar to (EUK)?

A

smc V-shaped dimers

25
Q

What is MukBEF analagous to?

A

condensins

26
Q

What is the role of ParC and ParE topoisomerases?

A

localisation of MukB

27
Q

What is the process of OriC movement during replication?

A

Ori moves closer to the terminator

Replicated origins stay localised. Ordered movement of chromosomes driven by replication forks, supercoiling enzymes, & dedicated SMC proteins.

As the origin moves closer to the ter, the proteins associated with MukB separate them…

28
Q

What approach was used to understand the movement of the origin?

A

labelled DNA binding protein e.g. lac repressor
Fuse WITH GFP
bind in clumps and GFP would get brighter as the origin and ter moved closer.

29
Q

What is a problem during DNA replication?

A

concatemers
Recombination events can lead to concatamers being formed (inter- linked circular chromosomes e.g. dimers)

30
Q

Does DNA replication occur alongside other DNA activities?

A

DNA synthesis continues in a bidirectional manner at the same time
chromosome is being transcribed, methylated, moved & repackaged.

31
Q

What is the sparation of linked chromosmes called?

A

Resolution of chromosome dimers (CDR) into monomers at a specific site dif

32
Q

What is the site called where linked chromosomes are resolved?

A

dif, enhanced at the termination site

33
Q

How are linked chromosomes resolved, what process?

A

homologous recombination.
XerCD proteins bind to the dif sites.
Dedicated proteins XerCD help resolve interlinked chromosomes to
enable segregation.

34
Q

What proteins help to resolve interlinked chromosomes (bind dif)?

A

XerC and XerD

35
Q

What is dif?

A
  • Two 11bp flanking sites
  • 6bp region where cleavage, strand exchange & ligation occurs
36
Q

What is XerC and XerD?

A
  • Chromosomally encoded tyrosine recombinase
  • Tyrosine refers to AA involved in link between protein & phosphate at cleavage site
  • Site specific
  • 28bp dif site located in terminus region
37
Q

How do you study the function of specific proteins?

A

mutate

38
Q

What is FtsK?

A

1329 AA membrane protein
DNA translocase

39
Q

Where does FtsK bind?

A

KOPS
FtsK orientating polar sites
close to the terminus region

40
Q

What is the N-terminus of FtsK involved in?

A

N-terminus membrane spanning involved in cytokinesis &
hexamer formation

41
Q

What is the role of the FtsK’s role in

A
  • FtsK orientating polar sequences direct the FtsZ ring towards terminus wherever it assembles on chromosome as a ring structure.
  • assists the XerCD/dif resolution and then translocates terminus region into cell cytoplasm away from the septum/division site.
  • Slides recombinases towards dif sites
  • Once they have finished their work, rapidly allow complete segregation of chromosomes (generated movement)
  • Frees up space for septum to come through
42
Q

Where is DNA recombination located?

A

terminus

43
Q

If there is a mutation in Ftsk, what can occur?

A

gilitine
cell hasnt completely separated because chromosome come through and entrap DNA so its stuck in the middle

L4 Molecular Microbiology (53 decks)

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