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Microbial Genomics > chromosomal replication > Flashcards

chromosomal replication Flashcards

1
Q

define a replicon.

A

A DNA unit capable of Replication and Inheritance

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2
Q

who discovered replicons?

A

Jacob, Brenner & Cuzin, 1963

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3
Q

give 2 examples of replicons

A

chromosomes and plasmids

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4
Q

4 facts of chromosomes

A

– Essential for viability
– Generally 1; sometimes >1 (Vibrio = 2)
– Sizes 0.5 - 5Mb (500-5000 genes); some ~30MB
– Mostly ccc DNA ; some linear DNA (Borrelia)

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5
Q

3 characteristics of plasmids

A

– Super-numerary, non-essential for viability
– May confer new phenotype (eg. resistance, catabolism)
– Smaller than chromosomes: 1 – 200kb

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6
Q

how is replication controlled

A

ori usage and copy number

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7
Q

who discovered Isolated Nucleoids?

A

Pettijohn (1971)

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8
Q

what do nucleoids contain?

A

DNA, RNA, Proteins

– Major proteins; RNA Polymerase DNA Gyrase

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9
Q

what was pettijohns experiment?

A
  • Treatment with detergent & protease
  • Releases anchored loops of DNA
  • DNA in loops is supercoiled
  • loops isolated & anchored at base
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10
Q

who discovered the Folded bacterial Chromosome?

A

Worcel & Burgi, 1972

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11
Q

what are the 4 Chromosomal Macrodomains?

A

Ori, Left, Right, Ter

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12
Q

name 3 Domain-specific binding proteins?

A

– SeqAp
– SlmAp
– MatPp

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13
Q

what does SeqAp do?

A
  • binds Ori, R, L localises oriC

- Negatively regulates timing of replication + initiation.

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14
Q

SlmAp do?

A
  • binds Ori, R, L

- chromosome positioning

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15
Q

what does MatPp do?

A
  • binds Ter and localises/anchors terC

- Stops premature chromosome segregation

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16
Q

types of DNA compaction in Nucleoid

A
  • Long range loop domain folded chromosome x25 compaction

* Short range supercoiling condensed nucleoid x860 compaction.

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17
Q

DNA concentration in nucleiod?

A
  • 2-20mg/ml DNA

- 80% mass nucleoid, only 5% vol

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18
Q

what are the Structural proteins of the Nucleoid?

A
  • Nucleoid Associated Proteins (NAPs)

- SMC (chromosome organising) proteins

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19
Q

name 3 NAPS

A

HU, IHF, H-NS

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20
Q

whats HU?

A
  • 9.5kDa NAP
  • binds structural distortions/stabilises bends
  • binds & constrains supercoils
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21
Q

whats IHF?

A
  • sequence-specific

- bends DNA

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22
Q

what does H-NS do?

A
  • binds AT rich bent DNA
  • forms bridge between two DNAs
  • DNA compaction.
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23
Q

what are NAPS?

A
  1. small DNA binding proteins
  2. abundant
  3. basic
  4. dimeric
  5. highly conserved
  6. nonessential
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24
Q

what is the structure of SMC proteins?

A

– Large V-shaped.

– Two DNA-binding domains connected by long coiled region with flexible hinge

25
Q

how does MukBEF act as a Condensin?

A
  • Nucleoid compaction & organisation

- hinge and arms and ATP.

26
Q

how do NAPs affect gene expression?

A
  • Interactions with DNA affecting shape
  • bind AT-rich promoters
  • Sets expression state of genes
27
Q

whats the most stable form of bacterial DNA?

A

360bp covalently closed circle of B-Form DNA

28
Q

what is b-form DNA?

A
  • Right-handed helix
  • 10.5bp/helix turn
  • Bases lie flat & perpendicular to helix axis
29
Q

how does b form supercoil?

A
  • breaks 1 strand + Unwinds 4 helix turns and reseals break

- DNA changes shape to its most stable state lowest free energy

30
Q

what are the Effects of EthBr intercalation into DNA?

A

-unwinds negative supercoil to introduce a positive sc

31
Q

whats the Action of E.coli Topoisomerase 1 on DNA?

A
  • type-I topoisomerase Breaks 1 DNA strand, holds ends, passes 1 (other) strand through, reseals nick.
  • introduces one positive SC
  • releases one negative SC
32
Q

Action of E.coli DNA Gyrase?

A
  • A type-II topoisomerase
  • Breaks 2 strands of DNA, passes duplex through, reseals break
  • introduces two negative SCs
33
Q

How are the linked circles decatenated?

A

Type II Topoisomerase

In E.coli, TopoIV

34
Q

how do fast growing cells differ from slow?

A

larger with more DNA than slow cells

35
Q

how does Origin/Terminus ratio change with growth rate?

A
  • Fewer Ter/Ori at faster growth rates

- dosage of genes near Ter falls at fast growth rates

36
Q

describe Replication of the E.coli Chromosome?

A
  1. 2 Rep Forks each traversing half chromosome
  2. Cell division occurs constant time after Termination
  3. 1 replication fork polymerises 2.3Mb in 40 minutes
37
Q

how does Rate of Cell Growth regulate frequency of initiation?

A
  1. Initiator Accumulation- Initiator made proportional to growth rate accumulate to critical level for initiation Initiator consumed at initiation
  2. Initiator Dilution- Inhibitor produced at initiation Diluted by cell growth (volume increase) Initiation when concentration falls below threshold
38
Q

where is E.coli’s Initiation?

A

oriC

39
Q

describe oriC?

A
  1. Unique origin of bidirectional replication
  2. Isolated DNA replicates freely as minichromosome
  3. Minimal sequence 245bp
    – 5 x DnaA-ATP binding sites
    – 3 x AT-rich 13mer sequences
    – binding sites for IHF, FIS, HNS
40
Q

what is DnaA?

A
  1. 52kDa protein monomer
  2. binds ATP hydrolysed to ADP during replica’s
  3. DnaA-ATP binds dnaA boxes in oriC, PdnaA, datA
  4. DnaA-ATP opens oriC at AT rich repeats
41
Q

how is DnaA expression controlled?

A

auto-regulation via dnaA box in promoter limits amount of protein made

42
Q

describe Initiation at oriC.

A
  1. DNA in correct topology negatively sc
  2. binds DnaA protein which Binds ATP
  3. DnaA-ATP binds to dnaA boxes in oriC Wraps oriC DNA around multimer of DnaA-ATP
  4. Opens oriC at A T rich region
43
Q

how does replication proceed at initiation at oriC?

A
  1. DnaB/C helices complex loads, unwinds DNA
  2. DnaG primas enters, synthesises primer
  3. Pol III complex with DnaN sliding clamp loaded
  4. DnaA-ATP hydrolysed to ADP
44
Q

what are the negative controllers of OriC?

A
  1. SeqA - Sequestering protein
  2. idaB- Replication Inhibition of DnaAp
  3. datA- DnaA titration locus
45
Q

how does SeqA work?

A

sequesters hemi-methylated oriC and PdnaA to membrane

46
Q

how does idaB work?

A

– hydrolyses ATP to ADP inactivates DnaAp

47
Q

how does datA work?

A

– depletes cell of free DnaA-ATP protein

48
Q

describe Initiation control by sequestration of oriC by seqA

A
  1. oriC becomes hemi-methylated by initiation
  2. seqA protein binds to hemi- methylated DNA
  3. sequesters to membrane
  4. methylation of other strand by dam blocks seqA binding
49
Q

how is dnaA activity controlled by ATPase action of idaB of the Sliding Clamp?

A
  1. idaB hydrolyses ATP on dnaA to ADP

2. dnaA-ADP unable to initiate replication

50
Q

whats the net result of OriC control cycle?

A

All active DnaA removed oriC unaccessible

51
Q

how is the oriC cycle switched on?

A
  1. oriC free, fully methylated, supercoiled
  2. DnaA-ATP accumulates to critical concentration autoregulation of dnaA gene.
  3. DnaA-ATP binds to oriC initiation triggered
  4. oriC replicated converted to hemi-methylated state
52
Q

how is OriC switched off?

A
  1. Hemi-methyl’d oriC sequestered to membrane by SeqA
  2. oriC unavailable
  3. beta-clamp hydrolyses ATP to ADP; DnaAp bound at oriC inactivated
  4. dnaA gene replicated GATCs hemi-methylated & sequestered,DnaA synthesis stops released & activated after delay
  5. datA locus replicated binds any free DnaA-ATP
53
Q

how is danA switched off at Initiation at oriC?

A
  1. idaB hydrolyses ATP dnaA-ADP inactive for initiation
  2. hemi-methylated oriC sequestered by seA
  3. excess dnaA removed by datA-binding
54
Q

whats The Chromosomal End Game?

A
  1. Termination of replication
  2. Resolution of multimers to monomers
  3. Decatenation of linked products
  4. Segregation to daughter cells at division
55
Q

describe Cell Division of E.coli?

A
  1. FtsZ polymerises into Z-ring at mid-cell needs GTP hydrolysis
  2. MinCDE proteins control position of Z-ring
    – MinCD oscillates from pole to pole every ~30sec establishes conc gradient; low at middle high at poles
  3. Other proteins attach to Z-ring Septal Ring
  4. Z-ring septal ring contracts inwards pulling in membrane and wall
  5. Nucleoid occlusion stops Z-ring complete contraction and closure
  6. FtsZ controls TopIV decatenation and Xer/difresolution of multimer products of replication.
56
Q

whats the Movement of Nucleoid at chromosome segregation?

A
  1. oriC foci at 1⁄4 and 3⁄4 positions SeqA moves oriC
  2. Pol III replic’n complex at mid cell – Spools DNA through
  3. terC at mid cell
  4. MukFEB complex (cohesin/condensin) moves replicated chromosomes apart
57
Q

what do MinCD proteins do?

A

determine site of cell division septum by oscillating every 30 sec inhibiting FtsZ polymerisation

58
Q

example of an SMC protein

A

MukBEF

59
Q

what do SMC proteins do?

A
  • compaction
  • cohesion
  • loop domain- organisation,
  • movement/segregation of nucleoid

Microbial Genomics (7 decks)

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