Bacterial Chr. Pt 2 Flashcards
4 characteristics of bacterial chr.
-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
What is the general overview of replication of bacterial chr.
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
How many bps is oriC in DNA? What does the oric site contain?
<260 bp
-binding sites for proteins needed to start replication, including many DnaA, IHF, Fis sites
What are the name of the high affinity sites that are in oriC & the proteins that bind to them?
-DUE, t, I sites: DnaA-P
-R1-5 sites: DnaA (uncharged) - bound all the time
How does DnaA open up DNA for chromosome replication?
-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
What does the DnaC protein do in chr replication?
loads DnaB helicase onto oriC and then falls off
What is the DnaB protein and function in chr replication?
helicase, interacts with DnaA to further unwinds DNA,
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
-primase
- DnaG and DnaB form primosome complex which travels across DNA laying RNA primers
What is RNAP job(s) in chr replication?
can eaither lay down first primer or transcribe through oriC to open up DNA for DnaA protein binding
What are the two possibilities for termination of replication?
1) Replication forks run into ter sites
2) replication forks run into each other, meeting one another cause termination of chr. replication.
What are ter sites? How does it cause replication termination?
-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
What are the two types of ter sites in E.coli?
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
what is chromosome segregation?
the separation of replicated chromosomes prior to partitioning into daughter cells
chromosome segregation is complicated by a couple of things? what are they? (3)
- large size of chromosomes
- possible joining of replicated chromosomes by recombination
- tangling
chromosome segregation studied by which technique?
labelling chr with GFP (DnaA) & imaging dividing cells using fluorescence microscopy
what are chromosome dimers?
-double length circular chromosomes joined by recombination that are common in dividing cells (15%); they prevent segregation of chromosomes
chr. dimers are resolved by what kind of protein? at which sites?
- Xer recombinase at dif sites (different from area of crossover)
How does Xer recombinase resolve chromosome dimers?
XerC/D binds at dif sites and cuts dsDNA & ligates at dif site
How do chromosome dimers form?
-accidental homologous recombination by RecA protein (no DNA added/lost)
How does Xer Site-Specific Recombinase System work?
-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
What is FtsK protein and where does it act? What does it do?
- 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
What is the KOPS sequence?
-FtsK-orienting polar sequence
-5’ GGGNAGGG 3’
What are catenanes?
daughter DNAs that are linked, like links on a chain, formed during DNA replication (two circles wrapped up)
How are Catenanes formed?
-DNA is opened by helicase and causes positive supercoiling of DNA
-supercoiling of DNA form precatananes
-catenanes formed (during DNA replication)
How are Catananes resolved?
-topoisomerase IV cuts both strands one DNA & passes other DNA through to remove catenanes; uses FtsK to center DNA in middle
what is the job of condensins?
-condense newly replicated chromosomes so less chance of entanglement and work with topoisomerases to condense DNA after decatenation
What is MukB protein & what does it look like?
-condensin in E.coli
-dumbbell shaped proteins that binds DNA and holds it in large loops
What is chromosome partitioning? We are still ensure of how it occurs but the systems are similar to that of plasmids.
apportionment of one segregated, replicated chromosome to each daughter cell
what proteins are involved in chr partitioning and what are their function?
DNA binding, actin-like Par proteins (SegA & Seg B)
they pull chromosomes apart by cycling through periods of polymerization & depolymerization
There are two partitioning models with Par proteins, SegA & SegB. What are they?
-Pulling: depolymerizing SegA proteins pull DNA apart
-Pushing: polymerizing SegA proteins push DNA apart
Cell Division & Chromosome Partitioning are coordinated by which two mechanisms?
1- Min Proteins
2- Nuceoid Occlusion
How do Min proteins coordinate cell division and chromosome partitioning?
MinCD proteins prevent the formation of septum where the chromosome is either by localization at the ends of cells or through a oscillation mechanism
How does Nucleoid Occlusion coordinate cell division and chromosome partitioning?
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
What is the Nucleoid occlusion DNA binding protein in B.subtilis? E.coli?
-Noc
-SlmA
What are the 3 mechanisms used to control the timing of chromosome replication?
i) Cell mass
ii) DnaA: Cellular concentration of DnaA and ATP vs. ADP bound state of DnaA
iii) Hemi-methylation & Sequestration
What is the Helmstetter & Cooper 1968 “Baby Machine Experiment” generally?
-they measure DNA content at different stages in cell cycle to determine how chromosome replication & cell division coordinated
How did they do the baby machine experiment?
-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
What were the conclusions of the Baby machine experiment?
-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
How does the cellular concentration of DnaA control the timing of chromosome replication?
-After replication, number of oriC sites doubled so DnaA binding to oriC is slower because of the competition so replisome initiation is inactive
How does the ATP vs ADP bound state of DnaA control the timing of chromosome replication?
- 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
How does Hemi-methylation & Sequestration control the timing of chromosome replication?
-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
After Dam methylase fully methylates DNA, what happens to SeqA?
SeqA falls off oriC, now DnaA-ATP sites exposed again, and DnaA-ATP can bind again and start replication
When does Dam methylase methylates DNA?
-minutes after replication because it does not travel with the DNAP replisome
