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1: GENET 270 > Bacterial Chr. Pt 2 > Flashcards

Bacterial Chr. Pt 2 Flashcards

1
Q

4 characteristics of bacterial chr.

A

-often (but not always) circular
-less structured than eukaryotic chromosome
-may have multiple copies of genes present when
new rounds of replication initiate before others
finish
-no use of telomeres to replicate ends

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2
Q

What is the general overview of replication of bacterial chr.

A

1) replication initiates at specific site, oriC
2) proceeds bidirectionally around chromosome
3) terminates at unique site (Ter) OR when replication forks run into each other

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3
Q

How many bps is oriC in DNA? What does the oric site contain?

A

<260 bp
-binding sites for proteins needed to start replication, including many DnaA, IHF, Fis sites

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4
Q

What are the name of the high affinity sites that are in oriC & the proteins that bind to them?

A

-DUE, t, I sites: DnaA-P
-R1-5 sites: DnaA (uncharged) - bound all the time

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5
Q

How does DnaA open up DNA for chromosome replication?

A

-ATP-bound DnaA binds I, t DUE sites to form large multimer
- Then binds to IHF & Fis bound to bend DNA to open up double helix

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6
Q

What does the DnaC protein do in chr replication?

A

loads DnaB helicase onto oriC and then falls off

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7
Q

What is the DnaB protein and function in chr replication?

A

helicase, interacts with DnaA to further unwinds DNA,

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8
Q

What is the DnaG protein? What kind of complex does it form and what is its function? primase: travels across DNA laying down RNA primers

A

-primase
- DnaG and DnaB form primosome complex which travels across DNA laying RNA primers

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9
Q

What is RNAP job(s) in chr replication?

A

can eaither lay down first primer or transcribe through oriC to open up DNA for DnaA protein binding

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10
Q

What are the two possibilities for termination of replication?

A

1) Replication forks run into ter sites
2) replication forks run into each other, meeting one another cause termination of chr. replication.

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11
Q

What are ter sites? How does it cause replication termination?

A

-22bp long site that traps replication fork by allowing it can only pass through in one direction to ensure that forks meet and terminate at a discrete location

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12
Q

What are the two types of ter sites in E.coli?

A

ter A,D,I,H = replisome can pass through clockwise but not counter-clockwise
ter B,C,F,G,J = replisome can pass through counter-clockwise but not clockwise

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13
Q

what is chromosome segregation?

A

the separation of replicated chromosomes prior to partitioning into daughter cells

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14
Q

chromosome segregation is complicated by a couple of things? what are they? (3)

A
  • large size of chromosomes
  • possible joining of replicated chromosomes by recombination
  • tangling
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15
Q

chromosome segregation studied by which technique?

A

labelling chr with GFP (DnaA) & imaging dividing cells using fluorescence microscopy

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16
Q

what are chromosome dimers?

A

-double length circular chromosomes joined by recombination that are common in dividing cells (15%); they prevent segregation of chromosomes

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17
Q

chr. dimers are resolved by what kind of protein? at which sites?

A
  • Xer recombinase at dif sites (different from area of crossover)
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18
Q

How does Xer recombinase resolve chromosome dimers?

A

XerC/D binds at dif sites and cuts dsDNA & ligates at dif site

19
Q

How do chromosome dimers form?

A

-accidental homologous recombination by RecA protein (no DNA added/lost)

20
Q

How does Xer Site-Specific Recombinase System work?

A

-XerCD binds to dif sites near ter when 2 copies (of genes) exist on 1 DNA & interacts with FtsK motor at septum. FtsK motor moves replicated chr into daughter cells and recombinase system resolves chromosomes

21
Q

What is FtsK protein and where does it act? What does it do?

A
  • it is cell division protein that is localized to division septum
  • it acts as DNA translocase that moves dif site to septum using the KOPS sequence to know which way to pump chromosome
22
Q

What is the KOPS sequence?

A

-FtsK-orienting polar sequence
-5’ GGGNAGGG 3’

23
Q

What are catenanes?

A

daughter DNAs that are linked, like links on a chain, formed during DNA replication (two circles wrapped up)

24
Q

How are Catenanes formed?

A

-DNA is opened by helicase and causes positive supercoiling of DNA
-supercoiling of DNA form precatananes
-catenanes formed (during DNA replication)

25
Q

How are Catananes resolved?

A

-topoisomerase IV cuts both strands one DNA & passes other DNA through to remove catenanes; uses FtsK to center DNA in middle

26
Q

what is the job of condensins?

A

-condense newly replicated chromosomes so less chance of entanglement and work with topoisomerases to condense DNA after decatenation

27
Q

What is MukB protein & what does it look like?

A

-condensin in E.coli
-dumbbell shaped proteins that binds DNA and holds it in large loops

28
Q

What is chromosome partitioning? We are still ensure of how it occurs but the systems are similar to that of plasmids.

A

apportionment of one segregated, replicated chromosome to each daughter cell

29
Q

what proteins are involved in chr partitioning and what are their function?

A

DNA binding, actin-like Par proteins (SegA & Seg B)
they pull chromosomes apart by cycling through periods of polymerization & depolymerization

30
Q

There are two partitioning models with Par proteins, SegA & SegB. What are they?

A

-Pulling: depolymerizing SegA proteins pull DNA apart
-Pushing: polymerizing SegA proteins push DNA apart

31
Q

Cell Division & Chromosome Partitioning are coordinated by which two mechanisms?

A

1- Min Proteins
2- Nuceoid Occlusion

32
Q

How do Min proteins coordinate cell division and chromosome partitioning?

A

MinCD proteins prevent the formation of septum where the chromosome is either by localization at the ends of cells or through a oscillation mechanism

33
Q

How does Nucleoid Occlusion coordinate cell division and chromosome partitioning?

A

DNA binding proteins prevent formation of FtsZ division ring/septum to stop cell division from cutting chr in half until replication is done. Then nuceloid occlusion zone is lost and the septum forms

34
Q

What is the Nucleoid occlusion DNA binding protein in B.subtilis? E.coli?

A

-Noc
-SlmA

35
Q

What are the 3 mechanisms used to control the timing of chromosome replication?

A

i) Cell mass
ii) DnaA: Cellular concentration of DnaA and ATP vs. ADP bound state of DnaA
iii) Hemi-methylation & Sequestration

36
Q

What is the Helmstetter & Cooper 1968 “Baby Machine Experiment” generally?

A

-they measure DNA content at different stages in cell cycle to determine how chromosome replication & cell division coordinated

37
Q

How did they do the baby machine experiment?

A

-they labelled DNA of growing bacteria with radioactivity
-Synchronize bacteria: attach to solid surface; collect released daughter cells
-All daughter cells collected at given time = same age
-Measure radioactive DNA content to determine how much of parent chromosome replicated at time x

38
Q

What were the conclusions of the Baby machine experiment?

A

-Time between initiation of chromosomal replication events correlated with growth rate
-Time for chromosome replication & time between completion of replication and cell division constant
*which means that more than one replication round may be initiated in same cell when growth rate fast

39
Q

How does the cellular concentration of DnaA control the timing of chromosome replication?

A

-After replication, number of oriC sites doubled so DnaA binding to oriC is slower because of the competition so replisome initiation is inactive

40
Q

How does the ATP vs ADP bound state of DnaA control the timing of chromosome replication?

A
  • since only the ATP bound form of DnaA can bind the oriC & initiate replication, less ATP-DnaA = no replisome initiation after DNA replication starts
    -Loading of sliding-clamp DnaN causes DnaA to hydrolyze bound ATP so no DnaA-ATP is immediately available
41
Q

How does Hemi-methylation & Sequestration control the timing of chromosome replication?

A

-DNA in E. coli methylated at 5’-GATC-3’ sites by Dam methylase
-After replication, DNA is transiently hemi-methylated
-SeqA protein binds to 11 hemi-methylated GATC sites in oriC right after replication and delays DnaA-ATP binding and initiation of DNA replication

42
Q

After Dam methylase fully methylates DNA, what happens to SeqA?

A

SeqA falls off oriC, now DnaA-ATP sites exposed again, and DnaA-ATP can bind again and start replication

43
Q

When does Dam methylase methylates DNA?

A

-minutes after replication because it does not travel with the DNAP replisome

1: GENET 270 (15 decks)

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