genet 270 lec 3- bac chromo
What is cell division?
Process by which one cell splits into two daughter cells.
What is the typical shape of the bacterial chromosome?
Often circular (but not always).
How is it different from eukaryotic chromosomes?
Less structured; no telomeres (to replicate ends)
Can multiple copies of genes exist during replication?
Yes, if new replication rounds start before previous ones finish.
Where does replication initiate in bacteria? How does replication proceed? Where does replication terminate?
At a specific site called oriC.
Bidirectionally around the chromosome.
At a unique site or when replication forks meet.
What is oriC?
~260 bp region with binding sites for replication proteins. (intiates replication)
Which protein binds oriC to initiate replication?
DnaA (ATP-bound form- only type that can bind t and I)
How does DnaA promote unwinding?
Binds high-affinity sites, recruits IHF & Fis, bends DNA, and opens the helix at the DUE (DNA unwinding element).
what happens after DnaA binds at due site
large multimer is formed (roll up the DNA), when paired with IHF and FIs it bends DNA (strains h bonds) to open up the double helix
What does DnaC do?
Loads DnaB helicase onto oriC.
What does DnaB do?
Functions as helicase; interacts with DnaA to further unwind DNA.
What happens to DnaC afterward?
It leaves the complex once DnaB is loaded.
What does DnaG do?
Acts as primase; with DnaB forms the primosome → lays down RNA primers.
What role can RNA polymerase (RNAP) play?
May lay down the first primer or transcribe through oriC to help DnaA bind.
What are the two ways replication terminates?
(1) Forks reach ter sites, or (2) forks run into each other.
How do ter sites control replication fork movement?
they are 22 bp long sites that act as one-way “traps” → forks can pass in one direction but are blocked in the other.
what are the two groups of ter sites?
terA, D, I, H: replisome can pass through clockwise but not counter clockwise
terC, B, F, G, J: replisome can pass through counter clockwise but not clockwise
Why are ter sites important?
Ensure forks meet and terminate at discrete locations.
What must happen before daughter cell partitioning?
Replicated chromosomes must be separated.
What complicates chromosome segregation?
Large chromosome size, recombination joining replicated chromosomes, and tangling.
How can chromosome segregation be studied?
By labeling chromosomes with GFP and using fluorescence microscopy.
What are chromosome dimers? Why are dimers a problem? How are they resolved?
Double-length circular chromosomes joined by recombination (~15% of cells). They prevent chromosome segregation. By Xer recombinase acting at dif sites near the terminus.
accidental homologous recomb: recomb can happen anywhere on chromosome circle, does this process add or loose dna?
no, the dna is broken between bases then backbone is ligated back together
Which proteins are involved in resolution?
XerC and XerD recombinases.
what does xercd do?
XerCD binds dif sites near ter, cuts dsDNA, exchanges strands, and ligates at dif to convert dimers into two monomers.
What other protein is associated with this process? (xer site specific recombinase system)
FtsK (motor), which helps position chromosomes for resolution. accuratley moves replicated chromos into daughter cells
what is the FtsK? how does it work?
FtsK is a cell division protein at the septum. It acts as a DNA translocase, moving dif sites to the septum and activating XerCD to resolve chromosome dimers. It uses KOPS sequences to know which way to pump the chromosome- locates midpoint of dimer.
What are catenanes?
Daughter DNA molecules linked like rings in a chain- formed during dna replication.
How are they resolved? (catenanes). What other protein is associated with this process?
By Topoisomerase IV (Type II topo), which cuts both strands of one DNA, passes the other through, then reseals. FtsK, which helps position chromosomes for resolution.
What do condensins do? work with who?
Condense newly replicated chromosomes to prevent entanglement. work with topoisomerases to condense DNA after decatenation.
Which condensin is in E. coli?
MukB.
What is the structure of condensins?
Dumbbell-shaped proteins that bind DNA and hold it in large loops.
What is chromosome partitioning? Is the mechanism fully known? Which proteins are involved?
Apportioning of replicated, segregated chromosomes into daughter cells.
No, it varies between bacteria, but many systems resemble plasmid partitioning.
Actin-like Par proteins, which push/pull chromosomes via polymerization and depolymerization cycles.
Why must chromosome segregation be coordinated with cell division?
To ensure each daughter cell gets one complete chromosome.
Which two systems regulate division site placement?
Min proteins and nucleoid occlusion (NO).
How do Min proteins ensure proper division site placement?
By preventing septum formation at the poles, either through oscillation (E. coli) or polar localization (other bacteria), ensuring the FtsZ division ring forms only at midcell.
what is the min protein oscillation mechanism vs polar localization?
In E. coli, Min proteins oscillate between poles, keeping MinC levels lowest at midcell, allowing FtsZ to form the division ring at the center.
In some bacteria, Min proteins stay at the poles, permanently blocking septum formation there, ensuring FtsZ assembles at midcell.
What does nucleoid occlusion do?
DNA-binding proteins (Noc in B. subtilis, SlmA in E. coli) prevent FtsZ ring/septum from forming over the nucleoid. this stops the cell division from cutting the chromo in half.
How is replication timing studied?
Using the Helmstetter & Cooper “Baby Machine” experiment (1968).
what is the baby machine experiement
objective: dna content measured at diff stages in cell cycle to see how rep and division coordinated
1. label dna of bacteria with radioactivity
2. synchronizse bac: attach to solid surface and collect released daughter cells
3. all daughter cells collected at given time = same age
4. measurace radioactive dna to determine how much of parent chromo replicated at time x
what was the conclusions of the baby machine experiment?
Time between initiation of chromo replication events correlates with growth rate.
Replication time and time between replication completion & division are constant.
At fast growth, multiple replication rounds may initiate before earlier ones finish.
How do extra oriCs affect DnaA activity after replication?
DnaA must now bind more oriCs, slowing initiation.
How do DnaA binding sites around the chromosome regulate replication timing?
They compete with duplicated oriC sites for DnaA binding, reducing available DnaA and preventing premature re-initiation of replication.
What regulates DnaA activity?
Only ATP-bound DnaA can initiate replication at oriC. (I, t , DUE)
How is ATP-DnaA inactivated after initiation?
Loading of β sliding clamp (DnaN) causes DnaA to hydrolyze ATP → inactive ADP-bound form (replisome intiation is inactie immediatley after replication starts)
How is oriC temporarily prevented from reinitiating replication?
By hemi-methylation and SeqA binding.
Which enzyme methylates DNA in E. coli?
Dam methylase (at 5′-GATC-3′ sites).
How does SeqA work?
Binds to 11 hemi-methylated GATC sites in oriC right after replication, preventing DnaA-ATP binding and intiation of DNA rep
How long does this sequestration last?
Until Dam methylase fully methylates DNA, then SeqA falls off and oriC becomes accessible to DnA-ATP again.
does the damn methylase travel with the dnap replisome (rep fork)?
no, methylates dna minutes after replication