GENET 270: Lec 3 - Bacterial chromosomes pt. 2

Is the bacterial chromosome more or less structured than eukaryotic chromosome?

Less structured

True or false: Many rounds of replication can occur at the same time?

True

• multiple copies of genes present when new rounds of replication initiate before other rounds finish

****What does the simultaneous rounds replication result in?

POLYPLOIDY until cell division catches up to DNA replication

Do bacterial chromosomes use telomeres?

No 

• not linear therefore don’t need buffer of degradation

***Where does replication initiate for bacteria?

Specific site = OriC

How many replication forks are there for bacterial replication?

2

• bidirectional

***What is the typical size range of oriC?

Less than 260 bp of DNA

*****What does oriC contain?

Binding sites for proteins required to initiate replication

• Many DnaA Binding sites

• IHF binding sites

• Fis binding sites

****What does DnaA do?

DNA unwinding element/ opens Helix

***What do IHF + Fis each do?

Regulate DNA replication

Integration host factor = BENDS dna

Fis= prevent premature DnaA binding to for multimer

*****Where are the 2 places DnaA can bind on the oriC?

1. High affinity sites

2. I, sigma + DUE sites

****When is Dna A bound to each site?

1. DnaA = ALWAYS bound to High affinity sites

2. Only ATP-bound DnaA can bind to I, sigma + DUE sites

*****Why does Replication only occur when the Energy stores of the bacteria are favorable

Only ATP bound DnaA can bind to the I, sigma and DUE sites

• When these sites are bound it forms a large MULTIMER

*****List the process by which DnaA opens the helix

• DnaA-ATP binds oriC

  • DnaA forms a multimeric filament

• Fis vs. IHF regulation

  • Fis bound at oriC prevents too-early DnaA assembly → acts like a checkpoint.

  • IHF sharply bends DNA (~160°), bringing distant DnaA boxes into proximity.

• DNA bending + DnaA multimerization

  • DnaA-ATP filament wraps the oriC DNA, causing torsional strain.

• Opening of AT-rich region

  • The strain unwinds the AT-rich DNA next to the DnaA boxes.

  • This creates the open complex.

***After DnaA opens up the helix @ oriC, what 2 enzymes come to further open the helix?

1. DnaC

2. DnaB

***What does DNA C + B each do during initiation?

C = Loads DNA B onto OriC

B = Helicase, interacts with DNA A + further unwinds the DNA

***What is the Next Enzyme that comes into play with initiation after A B+ C?

DNAG

****What is DNA G + What does it do?

DNA G = PRIMASE

• Lays down RNA primers

***What is the PRIMOSOME made of + what does it do?

helicase–primase complex in bacteria (DNA G + B)

• Job is to unwind DNA and then synthesize RNA primers so replication can begin

****What is an alternative to DNA G/ Primase when it is not available?

RNAP/ RNA poly

• it can lay down the first primer instead of DNA G

****What is the other function of RNAP?

can transcribe through oriC to help open up DNA for DnaA binding

****What all the enzymes involved with Initiation?

DnaA = binds to high affinity sites + I, sigma + DUE sites = forms multimer that creates tension + pulls helix apart (with the help of IHF + Fis)

DnaC = Loads DnaB

Dna B = Helicase

Dna G = Primase (makes RNA primers on DNA)

Primosome = Dna G + B

What are the 2 ways that DNA rep of bacterial chromosomes terminate?

1. At Ter sites

2. When replication forks run into each other

***How long are Ter sites?

22 bp long

*****How do Ter sites work?

Act as traps

• Replication fork can only pass through in ONE DIRECTION

Like the spikey tire trap speed bumps

******Which Ter sites block what direction of travel? (there are 9 ter sites)

A, D, H + I = Allow Clockwise movement but not counterclockwise (on the right side of the circle pointing clock wise)

B, C, F, G + J = Allow counter clockwise not clockwise (on left side of circle pointing counter clockwise)

*****What is the purpose of ter sites?

Ensure replication forks meet + terminate at discrete locations

• in case of stalled DNA poly, don’t want the the other DNA poly to go into area of replication where head on collision with RNA poly can occur

******In what scenarios are Ter sites used and in what scenarios do the forks run into each other for termination?

Ter sites = Stalling of replication for or the 2 DNA poly not in sync

• can cause head on collisions with RNA poly destroying DNA

Run into each other = Replication forks = at same speed

****What must occur before the septum formation + partitioning into daughter cells?

Chromosome segregation

****What 3 things Complicate Chromosome Segregation?

2. Large size of chromosomes

3. Possible joining of replicated chromosomes by recombination

3. Tangling

How was chromosome segregation studied?

Using GFP

****What are Chromosome Dimers?

Double length circular chromosome joined by recombination = PREVENT SEGREGATION

• Complicate segregation #2: Possible joining of replicated chromosomes by recombination

***WHAT resolves the Chromosome dimers caused by recombination?

Xer Recombinase at dif sites

****What occurs during a recombination event? what is it? and WHAT MAKES THE RECOMBINATION?

DNA = broken = double strand break

DNA crosses over and the 2 are ligated together

• Rec A makes the cross over

****Is any DNA added or lost during this process?

No

****What is the way Xer can resolve dimers?

• Resolution by Xer CD

• Xer Site-specific recombinase system

*****Explain how Xer CD works

Xer C + D cut dsDNA at specific dif sites —> REVERSE CROSSOVER = Ligates at dif sites

= 2 separate chromosomes instead of 1 dimer

****explain how Xer Site-specific Recombinase system works

Xer CD binds to Dif sites near ter

• occurs when there are 2 copies of dif sites present on DNA

Interact with FtsK motor that channels dimer back + forth until all KOPS sequences = aligned in the right direction in each DNA + Dif site = in middle/ DNA evenly split

Xer CD = cut + ligate at dif site into 2 separate strands

****What is the Kops sequence?

Goes in opposite direction in each DNA

• 1 side of KOPS = 1 diff site + the other side of KOPS = other diff site

Helps the Ftsk recognize which way to pump/channel the DNA + when they have evenly split the DNA between the 2 cells.

****Does the resolution of Dimers always occur where the recombination occurred?

NO

• recombination occurs anywhere BUT Resolve = ALWAYS AT dif Sites

****What is FtsK? What role does it play in chromosome segregation? How does it do its role?

a CELL division protein

• In chromosome segregation it acts as a DNA TRANSLOCASE that moves dif to septum

Uses KOPS (FtsK-oreinting polar sequence) to know which way to move the chromosome

*****Where is FtsK located?

At the DIVISION SEPTUM

• anchored to CELL WALL AT SEPTUM

***What is a Catenanes?

Tangling of 2 separate circles of daughter DNA + formed during DNA replication

• Complication #3 of chromosome segregation

*****How are Catenanes resolved?

Topoisomerase IV

****Of the 2 types of Topoisomerase, what type is IV?

TYPE 2

= Cuts BOTH STRANDS of one DNA + passes the other DNA through

*****What is Topoisomerase also in association with?

FtsK

*****How are Catenanes FORMED during DNA rep?

Positive supercoiling ahead of the fork

****What is the process of resolving catenanes called?

decatenation

****How does FtsK + Topoissomerase IV work to resolve catenanes?

Similar mechanism to Xer

• THREAD DNA back + Forth until TANGLE = ALIGNED in the Middle/SEPTUM

• Topoisomerase = Cut 1 strand + thread other through

Does the decatenation process use KOPS?

NO

• instead aligns the tangle with the middle

*****Finish the Sentence: Topoisomerase IV is active based on _____?

Based on DNA strain

****What is Condensin? What does it Do?

Protein that works with TOPO IV to condense DNA after decatenation.

• Condense newly replicated chromosomes' so there is less change of tangling

*****What is the condensin protein in E.Coli?

MukB

*****What does MukB look like + how does it work?

Dumbbell shaped proteins

• Bind DNA + allows binding of Muk EF to hold it apart in large loops

*****What are the 2 types of Chromosome Partitioning?

1. Pulling mechanism

2. Pushing Mechanism

Fill in the blank: Partitioning systems resemble those of ______ and vary between bacteria.

Resemble plasmids

*****What PROTEINS are involved in the process of Partitioning for the pushing + pulling mechanisms? What general way do they achieve this?

Par proteins (actin like)

• Push by polymerization

• Pull by Depolymerization

*****What are the 2 MECHANISMS that work together + are responsible for Coordinating cell division + chromosome partitioning How do Cells divide in the middle + ensure each daughter cell gets 1 chromosome?

1. Min proteins

2. Nucleoid Occlusion

*****1. How do Min proteins work to coordinate cell division?

Ensures septum formation only occurs in the MIDDLE

• Septum cannot form where there is min

works either by oscillation mechanism or by polar localization

******2. How does Nucleoid occlusion work to coordinate cell division?

Nucleoid occlusion proteins bind DNA + prevent FtsZ division ring/septum

• Prevents cell division from cutting chromosome in half before DNA is finished replicating

*****What are the 2 nucleoid occlusion proteins talked about in class?

1. Noc

2. SImA

****In which ORGANISMS do theses 2 proteins each occur?

Noc = B. Subtilis (GRAM +)

SImA = E.coli = (GRAM —)

*****What are the 3 MECHANISMS responsible for controlling the timing of chromosome replication? How does it a cell know when to replicate?

1. Cell Mass

2. DnaA binding sites

3. Hemi-methylation + sequestration

******Which scientists ran an experiment that determined CELL MASS was a mechanism of control for replication?

Helmstetter + cooper

• baby machine experiment

*****How did the baby machine experiment work?

Measure DNA conenta @ different stages of cell cycle

1. Attachment of “mother” cells

   • sticking them onto a membrane filter

2. SYNCHRONIZE BACTERIA

   • Newly divided cell = not attached + WASHED into the flowing medium.

   • These newly released cells were newborns (age = 0 minutes).

3. Collecting synchronized cells

   • The flow system continuously washed away “babies,” giving a pure, synchronized population of cells all starting at the same cell cycle point.

4. Measure radioactive DNA content to determine how much chromosome replicated at time X

********** What did Helmstetter + cooper DIVSOVER? What 2 conclusions did they make?

1. Time BETWEEN initiation of replication differed

2. Time taken for replication events + cell divisions events to be completed = CONSTANT

******What affected the time BETWEEN initiation of replication?

Nutrient availability + RATE OF GROWTH

• high availability = faster growth = faster imitation of replication = decrease time between

******What does the 2 conclusion from the baby machine experiment mean/ tell us?

More than one replication round may be initiated in the same cell when growth rates are fast

******What are the 2 WAYS DnaA binding sites affect Timing of replication?

1. Cellular Concentration of Dna A

2. ATP vs. ADP bound state of Dna A

*****how does CELLULAR CONCENTRATION of DnaA influence rate of replication?

Replication = Number of oriC sites DOUBLED

• DnaA binding = slower

Other DnaA binding sites also compete with oriC for binding DnaA

= Less DnaA available to bind to Duplicated OriC site to initiate replication

*****how does ATP vs. ADP bound state of DnaA of DnaA influence rate of replication?

Only ATP-Bound DnaA can bind oriC DnaA boxes (I, sigma + DUE) to initiate replication

• DnaN = causes DnaA to HYDROLYZE bound ATP = no ATP-bound not available = Inactivate initiation until there is more ATP

inactivate DnaA-ATP so oriC doesn’t fire again.

*****What is DnaN + what does it do?

AKA = Beta sliding-clamp = ATPase

• Hydrolyses ATP bound to DnaA to prevent immediate replication

***What are the 3 things Replication depends on?

1. DnaA

2. # of DnaA binding sites

3. [DnaA-ATP]

How can replication be initiated faster?

High concentration of ATP = can replenish ATP faster of DnaA

***********how does Hemi-methylation + sequestration influence rate of replication?

DNA = methylated

1. After replication DNA = HEMIMETHYLATED (only one strand methylated) 

2. SeqA binds to oriC in un-methylated areas = blocks binding of DnaA-ATP

3. Bound SeqA = drawn to cell membrane + hidden from methylation enzyme

4. Once found + methylated, SeqA falls off of oriC = DnaA-ATP can bind

*****What ENZYME methylated DNA in E.Coli?

Dam methylase

****Where is E.coli methylated? What specific sequence?

5‘- GATC -3’

****How many GATC sites are there in oriC in which SeqA can bind to the unmethylated version?

11

*****What is the purpose of all the replication timing mechanisms?

Not start replication too quickly

• ensure there is enough E

• Prevent DNA rep from going out of hand