Unit 2: Bacterial Replication Flashcards

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1
Q

What is the main difference from prokaryote and eukaryotic replication?

A

how the replication is controlled and linked to the cell cycle. Bacteria divide by binary fission.

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2
Q

What are the steps of bacterial replication?

A

1) A unit cell has a circular chromosome
2) Replication initiates at origin when cell grows to a critical size
3) Replication begins at the origin and produces catenated daughter chromosomes
4) septum divides cell and new chromosomes are partitioned to opposite ends of cell
5) daughter cells separate

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3
Q

How does the cell know when to initiate replication?

A

Possible theories:

1) Initiator protein levels: initiative protein is synthesized constitutively throughout the cell cycle but when it accumulates past a threshold amount it can trigger initiation
2) Inhibitor protein levels: an inhibitor protein could be synthesized or activated at a point when its concentration falls below a critical point by the increasing cell volume

Current model suggest a combo of both

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4
Q

How often does initiation of replication occur in bacterial cell?

A

occurs only once every cycle and it occurs at the same time in every cycle

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5
Q

What is doubling time in bacterial replication?

A

it represents the rate of bacterial growth in case of E coli it can vary anywhere between 80 minutes and 180 minutes.

it takes 40 minutes to replicate the bacterial chromosome which means that the replication fork is moving at a rate of approximately 15 base pairs per minute.

it then 20 minutes from the completion of a round of replication to complete cell division. During these 20 minutes the cell is possibly assembling the components needed for the division into the daughter cells

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6
Q

Fast rates of bacterial cell growth produce what?

A

multiforked chromosomes.

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7
Q

If it takes about 60 minutes for each replication cycle then what happens if the doubling time for the bacteria is less than 60 minutes ?

A

what happens is that the next round of initiation begins even before cell division from the current cycle is completed. For bacteria that has a doubling time of 35 minutes, it shows the status of the bacterial DNA at 5 minute intervals of the cell cycle and the start of the replication cycle which is 0 minutes the cell already has a partially replicated chromosome as a DNA replication would have been initiated 25 minutes before the previous cycle was completed. At about 10 minutes into the cycle even though the replication fork has not reached the terminus the daughter chromosomes have already started the next round of replication producing these multiforked chromosomes. By 15 minutes the replication fork has reached the terminus because DNA replication takes about 40 minutes there. it takes 20 minutes from the completion of DNA replication to cell division so by the end of 35 minutes you have two daughter cells each having an already replicating chromosome and can start the next cycle thus recreating the point at which we started

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8
Q

Poles:

A

the ends of a bacterial cell, usually containing specializes structures for positioning structures

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9
Q

Septum:

A

formation is initiated mid-cell, 50% of the distance from the septum to each end of the bacterium.
this is the site at which the cell divides into the daughter cells

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10
Q

Anucleate cell:

A

bacterial that lack a nucleoid, but are similar shape to wild-type bacteria. Comprise 0.03% of bacterial population.

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11
Q

TRUE OR FALSE: In the case of bacteria, the DNA itself is involved in its segregation unlike eukaryotes where they have an apparatus involved

A

TRUE:

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12
Q

What is a nucleoid?

A

the bacterial chromosome is compacted into an organized structure of protein and DNA called a nucleoid which occupies most of the cell

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13
Q

how do the origin and terminus move around a newly formed cell?

A

in the newly formed cell the origin and terminus of the bacterial chromosome is located in the middle of the cell

as replication is initiated the new origins move apart towards the poles with the terminus remaining in the center

the two origins occupied the 1/4 and 3/4 positions at the time of cell division

following cell division the origin and terminus of each daughter cell comes back to the middle of the cell

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14
Q

Which proteins help maintain the cylindrical shape of E.coli?

A

MreB, PBP2, and RodA

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15
Q

What is the role of MreB

A

MreB is similar to actin protein that is part of the eukaryotic cytoskeleton like actin, MREB forms polymers that moves around the circumference of the cell interacting with PBP2 and other parts of the peptidoglycan synthesis machinery. if MREB is absent cells no longer have a rod shape but become round

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16
Q

What is the family of proteins called SEDS?

A

SEDS (Shape, elongation, division, and sporulation)

found in all bacteria that have a peptidoglycan cell wall, of which RodA is a member

Each member of the SED family is linked to a specific transpeptidase for its function. a transpeptidase catalyzes the formation of the crosslinks in the peptidoglycan layer

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17
Q

What is the role of RodA?

A

Rod a functions along with its specific transpeptidase PBP2

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18
Q

how do we know that these proteins (MreB, PBP2, RodA) are necessary for the elongated structure of the bacterial cell wall?

A

mutations and any one of the genes that encode for these proteins and or depletion of these proteins results in the cells no longer retaining their cylindrical shape and instead look more rounded

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19
Q

What is FtsZ and what is it required for?

A

FtsZ is a GTPase (hydrolyzes GTP and uses energy to form oligomeric ring) that forms a ring (the Z-ring or septal ring) on the inside of the bacterial envelope.

The product of ftsZ is required for septum formation at pre-existing sites. FtsZ recruits other proteins needed for the synthesis of the septum

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20
Q

Where is ftsZ found before the cell divides?

A

FtsZ is found throughout the cytoplasm but before the cell divides it localizes along the circumference at the midpoint of the cell forming a ring shaped structure called the Z ring.

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21
Q

What determines position of the septum?

A

Position of the Z ring.

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22
Q

FtsZ is analogous to what in eukaryotes?

A

FTSZ resembles tubular and the formation of the ring could be analogous to the formation of the microtubules in eukaryotes

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23
Q

How do ZipA and FtsA interact with FtsZ?

A

ZipA is links FtsZ ring to the membrane.

Once the FtsA has been incorporated other Fts proteins joined the Z ring to form the septal ring.

This multi protein structure is thought to have the ability to constrict the membrane

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24
Q

TRUE OR FALSE: The Z ring will form if both ZipA and FtsA are absent

A

FALSE:

The z ring will form if either ZipA or FtsA are absent but does not form if both are absent suggesting that they may play a role in stabilizing the Z ring and possibly linking it to the membrane

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25
Q

How do we know that ftsZ is necessary for septum formation?

A

because in Fts mutants the septum does not form but instead long filaments are formed.

Surprisingly chromosome replication and segregation are not affected.

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26
Q

What happens if FtsZ is over-expressed in a cell?

A

if FTSZ is overexpressed then too many septum are produced resulting in the formation of minicells.

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27
Q

What is a minicell and how does it differ from anucleate cells?

A

the mini cell is small and lacks DNA

Minicell: small in size and lacks DNA
Anucleate: retains normal size and lacks DNA

Minicells: too many septum affect it
Anucleate: generated b/c of the failure of chromosomes to segregate properly

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28
Q

Genes that regulate the location of the septum were determined by studying what?

A

minicell mutants;

The minC, D, and E are part of the minB locus. so in cells that were lacking the minB locus the septum formed at the poles instead of the middle. this means that MinB was needed to prevent formation of the septum at the poles.

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29
Q

What is the role of MinC and MinD?

A

proteins minC and MinD together form what is called a division inhibitor.
MinD activates minC with
MinC preventing FtsZ from forming the Z ring.

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30
Q

What determines the number and location of the septa?

A

The ratio of the levels of MinCD to minE

MinCD/MinE

31
Q

What occurs if MinE levels are similar to MinCD? What if levels of MinCD are higher than MinE? Or if MinE is absent?

A

if minE levels are similar to minCD then the inhibition is confined to the poles as you would want in normal growth. however if the levels of CD are higher or if minE is absent then division is inhibited throughout the cell.

32
Q

What type of protein is MinD?

A

MinD is a ATPase and oscillates from one pole to another taking minC with it, as minC cannot move on its own.

33
Q

what is the role of MinE

A

minE forms a ring at the edge of the minCD front. minE is required for the hydrolysis of ATP and then the release of MinD from the membrane. the minE ring then disassembles and reforms at the opposite pole where the minCD zone is now formed. due to this oscillation between the poles the concentration of minC is minimal at the center while highest at the poles. Thus the Z ring whose formation is inhibited by minC will now form in the mid cell region and not at the poles.

34
Q

Which Min protein inhibits the formation of the z ring?

A

minC

35
Q

How does the cell prevent the septum being formed over the bacterial chromosome?

A

proteins slmA or Noc prevent that from happening. slmA binds to specific sites on the DNA and this binding activates slmA which like minC inhibits the polymerization of FtsZ and hence the formation of the Z ring. Noc plays a similar role in another bacterial species bacillus subtilis.

36
Q

Describe the oscillation of Min proteins in E.coli

A

1) Min D binds to membrane at one pole
2) Min C binds to MinD
3) A minE ring forms at the edge of MinD
4) MinE ring promotes dissociation of MinD from the membranes
5) MinD diffuses and binds to opposite pole
6) MinE ring dissociates and reforms at the edge of new MinD zone

37
Q

The location of the septum is controlled by what proteins?

A

minC, D, E and by noc/slmA

38
Q

Dynamic movement of the Min proteins in the cell set up a pattern that causes what?

A

a pattern in which inhibition of the z ring assembly is highest at the poles and lowest at mid-cell

39
Q

TRUE OR FALSE: Chromosomal segregation may require site-specific recombination

A

TRUE

40
Q

Following replication, what kind of recombination can occur in newly formed copies?

A

Intermolecular recombination; 2 monomers coming together to form a dimeric circle we call this a generalized recombination as it is not site specific. another recombination event an intramolecular recombination has to occur to break up the oligomers into monomers this second recombination is usually site specific

41
Q

Regardless of whether there was general recombination or not, eventually each daughter cell will have what kind of chromosome?

A

monomeric circular chromosome

42
Q

When general recombination occurs (movement of chromo is constrained), the bacteria needs a system in place to generate the monomeric circles. What system is this?

A

Most bacteria have the Xer site specific recombination system that consists of the XerC and XerD proteins which are recombinases. these proteins target the site in the terminus region called the diff site.

43
Q

What is the role of XerC

A

XerC forms the Holliday junction by binding to a pair of the dif sequences.

44
Q

WHat is the role of FtsK in the Xer site specific recombination system

A

FtsK is localized to the septum and is required for the actual separation of the copies into two single circles

45
Q

How does the bacteria determine that it needs to use a site-specific recombination system?

A

The system needs to come into play only if there has been a general recombination event to form the dimeric circle, otherwise the site specific recombination itself can generate a dimer.

it is thought that as of chromosome start segregating following replication if there is no recombination, the two copies should be able to separate easily, however if there has been a general recombination event, then there will be some constraint felt, forcing the terminus region to linger at the septal region exposing the diff side to Xer system

46
Q

What is partitioning?

A

Partition is the physical separation of the daughter chromosomes on either side of the septal region

47
Q

What must happen before partitioning occurs?

A

DNA needs to be disentangled a role carried out by topoisomerase.

48
Q

For the replication apparatus to work, what must happen to the parental chromosome?

A

parental chromosome has to be centrally located and has to decondense for the replication apparatus to work and after the newly formed daughter chromosomes are distentangled they have to be re-condensed

49
Q

What is the role of MukBEF protein complex?

A

it re-condenses the daughter chromosomes after they have been decondensed from the parental chromosomes.

MukBEF is considered a condenser

50
Q

replicon

A

a unit of the genome in which DNA is replicated. Each contains an origin for initiation of replication

51
Q

origin

A

a sequence of DNA at which replication is initiated

52
Q

terminus

A

a segment of DNA at which replication ends

53
Q

single-copy replication control

A

a control system in which there is only one copy of a a replicon per unit bacterium. The bacterial chromosome and some plasmids have this type of regulation

54
Q

multi-copy replication control

A

occurs when the control systems allows the plasmid to exist in more than one copy per individual bacterial cell

55
Q

semiconservative replication

A

replication accomplished by separation of the strands of a parental duplex, w/each strand then acting asa template for synthesis of a complementary strand

One parent strand is retained in the daughter duplex DNA

56
Q

TRUE or FALSE: A replicated region appears as a bubble w/in non-replicated DNA

A

TRUE

57
Q

A replication fork is initiated where?

A

at the origin and then moves sequentially along DNA

58
Q

What is bidirectional replication?

A

this involves the formation of two replication forks that progress in opposite directions away from the origin. Replication continues until it encompasses the entire replicon.

59
Q

What is unidirectional replicaiton?

A

this occurs when a single replication fork is formed at the origin

60
Q

What happens once DNA has been completely replicated?

A

the two daughter chromosomes are catenated, that is they are linked together by one pass so enzymes are needed to separate the two chromosomes

61
Q

What is OriC?

A

the site where replication is initiated and the two replication forks proceed at the same rate to the termination point.

Oric is 245 base pairs in length and contains binding sites for DnaA: dnaA-boxes

oriC also contains 11 GATC/CTAG repeats that are methylated on adenine on both strands

OriC has four 9-mer base pairs (on right side of oric) and three 13-mer base pairs repeats (left side of oric)

62
Q

What is DnaA?

A

an ATP binding protein and serves to initiate replication. It is a licensing factor and binds to short repeated sequences and forms an oligomeric complex that melts DNA

63
Q

What is one method of ensuring the cell replication is only initiated once at the origin?

A

one method of control is through methylation. An enzyme the dam methyltransferase adds a methyl group to the adenine on GATC or CTAG. There are eleven such repeats at the oriC region. In addition to the DNA binding site typically the genome is fully methylated at the GATC sequences. however this changes when replication is initiated

64
Q

What happens to methylation when replication is initiated?

A

the newly formed daughter strands will contain unmethylated adenine residues. Thus, the parent strand in the duplex is methylated, while the daughter strand is not. This is known as hemimethylated DNA and there’s a 30 minute delay before the dam methylase comes in and methylates the unmethylated adenines on the daughter strands.

65
Q

TRUE OR FALSE: methylated DNA cannot undergo replication

A

FALSE: hemimethylated DNA in the OriC cannot undergo initiation. The initiation process requires a large protein complex to for at the OriC.

66
Q

How does the initiation process (for replicatoin) start?

A

It starts out by DnaA and ATP binding to the fully methylted OriC region. DnaA is only ative when bound to ATP, and will become inactive when it hydrolyzes ATP to ADP when initiation ends.

Associatoin of DnaA to the oriC prevents the newly synthesized strand from getting methylated unit the DNA gets degraded.

67
Q

The four 9-mer base pair sequence at the right side of the ori is the binding site for what? What kind of binding is this?

A

binding site for DnaA ATP and this binding is cooperative

68
Q

What role does DnaA play on the three 13 base repeats?

A

DnaA is in the extended form initially and changes to the more compact form when activated resulting in the opening up of the DNA molecule. All 13-mer repeats need to be in the open state before the next step.

69
Q

What forms the replication fork at oriC?

A

DnaA-ATP binds and DnaC bind to of DnaB helicase forming the prepriming complex and is recruiting to the oriC and forms at each replication fork

DnaC represses role of DnaB (helicase activity until needed, which displaces DnAATP) DnaB then recruits DnaG which binds to the complex and creates the replication forks.

Gyrase and SSB are also needed

70
Q

what mechanisms exist to prevent premature reinitiation of replication?

A

1) SeqA
2) dat locus
4) Hda protein

71
Q

What is the role of seqA?

A

prevents premature reinitiation of replication. SeqA binds to hemimethylated DNA and is required for delaying re-replication

SeqA may interact with DnaA

As they origins are hemimethylated they bind to the cell membrane and may be unavalable to methylases

72
Q

What is the role of dat locus?

A

dat locus contains DnaA-binding sites that titrate availability of DnaA protein (preventing rereplicaiton)

73
Q

What is the role of Hda protein?

A

Hda protein is recruited to the replication origin to convert DnaA-ATP to DnaA-ADP (preventing premature reinitiation of replicaiton)