The Replicon Flashcards
synonymous mutation
A mutation in the genetic code that changes the DNA nucleotide sequence and RNA nucleotide sequence but does not ultimately change the encoded amino acid
nonsynonymous mutation
A mutation in the genetic code that changes the DNA and RNA nucleotide sequences as well as the encoded amino acid
the replicon
A region of a chromosome that replicates as a single unit starting at a specific origin of replication
- a unit of DNA in which individual acts of replication occur; contains an origin and may have a terminus
- can be linear or circular
origin of replication
The start point of DNA replication
unidirectional replication
Form of DNA replication in which the replication fork proceeds in one direction only
- replication fork created at the origin
bidirectional replication
Form of DNA replication in which the replication fork proceeds in both directions
- when origin creates two replication forks that move in opposite directions
plasmid
Small extrachromosomal circular DNA molecule that can replicate independently of the main cellular chromosome
- autonomous circular DNA genome that constitutes a separate replicon
extrachromosomal
Nucleic acids present in a cell, that are not a part of the main chromosome
in vivo
Preforming an experiment within a living organism
in vitro
Preforming an experiment under highly controlled conditions outside of a living organism.
how many replicons to bacteria have? how many do humans have?
- bacteria usually only has one replicon
- humans typically have thousands of replicons in each chromosome
- ours typically has 10,000 replicons
what is the bacterial genome?
- the bacterial genome is the replicon
what are some examples of replicons?
- bacterial chromosome
- eukaryotic chromosomes
- plasmids
- mitochondrial genome
- phage
- virus
how do bacterial chromosomes terminate?
- they may or may not have a terminus
- most common way is for DNA polymerases to crash into one another (humans do this)
- E. coli have a different mechanism
what is the termination mechanism E. Coli have?
- there are sequences that designate the termination point
- in bacteria you do not want DNA replication to go in the opposite direction because the opposite is for transcription - especially for ribosomes
what organelles contain origins of replications?
- nucleus
- chloroplasts
- mitochondria
what does the initiation of DNA replication commit the cell to?
- commits the cell to division
- division must not occur until after replication is completed
- replication is controlled at the stage of initiation and is highly regulated
what are the ways you can map replication?
- autoradiography
- fluorescent labelling
- electrophoresis
- electron microscopy
what are the types of replication control?
single copy and multicopy
single copy
- replicates once alongside bacterial chromosome
- Usually when the bacterial chromosome itself replicates
- Have a mechanism to make sure each copy is segregated to each daughter cell
multicopy
- present in more copies than bacterial chromosome
- small plasmids do this
unidirectional
- replication in only one direction
- mitochondria
bidirectional
- replication is two directions
- bacterial and eukaryotic chromosomes
replication fork
- separation of the two strands of DNA
- at each end of the replication eye
- also called a theta structure
- initiated at the origin
- moves sequentially along DNA
replication eye
opening of the two strands to enable the reading
- also called a theta structure in circular DNA
prokaryotic replication
- single and circular
- competitive inhibition between DNA A and seq A
- at the origin seq A binds to the hemimethylated state on the new strand and blocks the binding of DNA A and slows the rate of DNA replication, causing a 13 minute delay
- when Seq A binds to DNA is has an affinity for the membrane and pulls the origin to the membrane to hide it from DAM methylase
- this also stops DAM methylase from binding as well
- Seq A recognizes the origin because it has an affinity for DNA A sites called high affinity binding sites - DNA A binds to these sites but there are not enough for actually replication
- bacterial oriC contains 11 GATC/CTAG repeats that are methylated on adenine on both strands
- DAM methylates the A on each strand on each strand of the GATC sequence
- the new strand contains a non methylated strand
- once methylated by DAM methylase, DNA A is able to replicate - it takes 20-40 DNA A proteins to binds at the origin to pull the strands apart
what is the role of termination sites and Tus proteins in bacterial replication?
- bacterial chromosome is a single replicon that normally terminates when the replication forks meet
- Ter sites serve as a backup to prevent replication from going too far
- transcription of major genes is in the same direction as replication
- ter sites are pointed in opposite directions
- ter sites are pointed in opposite directions and binds to the Tus proteins that blocks helicase thus stopping DNA polymerase
- Tus proteins are bounded asymmetrically - one side is bounded alot tighter than the other - if the helicase is going the right way it’ll just bounce off but if it’s going the wrong way the Tus protein stops
why do you need ter sites?
- one side of the DNA pol may be faster than another
- this is to prevent replication from crossing into transcription
bacteria number of replicons, length, and movement
replicons: 1
length: 4,200 kb
movement: 50,000 bp/min
yeast number of replicons, length, and movement
replicons: 500
length: 20 kb
movement: 3,600 bp/min
fruit fly number of replicons, length, and movement
replicons: 3,500
length: 40 kb
movement: 2,600 bp/min
toad number of replicons, length, and movement
replicons: 15,000
length: 200 kb
movement: 500 bp/min
mouse number of replicons, length, and movement
replicons: 25,000
length: 150 kb
movement: 2,200 bp/min
plant number of replicons, length, and movement
replicons: 35,000
length: 300 kb
what are the length of eukaryotic replicons?
- 40-200 kb in length
- replicons only work once before cell division
- a chromosome is divided into many replicons
what portion of DNA replication did eukaryotes inherit from archaea?
- Orc1 and Cdc6
what are the differences between replication termination for prokaryotes and eukaryotes?
- eukaryotes are terminated by the fusion of replication eyes
- no ter sites or Tus proteins
what is the cell cycle?
- mother cells divides by mitosis
- two daughter cells form and grow
- interphase occurs
- gap 1 in synthesis
- synthesis
- gap 2 in synthesis
foci
- active replicons are clustered (foci)
- 300-500 active sites at any given time
- eukaryotic nucleus contains 100-300 foci
- one focus can contain more than 300 replicons
ORC
- origin recognition complex
- a complex of sic proteins that binds to an ARS at the A and B1 sites
- 6 protein
- about 400 kD (roughly the size of DNA polymerase)
how does licensing factor components work?
- necessary for initiation at each origin
- is present in the nucleus prior to replication but is activated or destroyed by replication
- initiation of another replication cycle becomes possible only after licensing factor reenters the nucleus after mitosis
- ORC is already there and bound there
- ORC has to bind to ATP, then recruits cdc6 and cdc6 recruits cdt1
- in turn cdt1 recruits MCM 2-7 to the origin site
- then DNA synthesis can initiated
cdc6
- part of the licensing factor that binds to the orc
- recruits cdt1
cdt1
- cannot arrive until cdc6 arrives
- recruits the helicase
- irreversibly committed to replication
geminin
- orc normally gets too many helicases, so geminin is recruited and bind to cdt1 so it no longer recruits the helicases
MCM 2-7
- a helicase
- minichromosome maintenance
- 6 subunits
- moves in opposite polarity from the direction of the bacterial helicase
what is the percentage of eukaryotic replicons active at any given time?
about 15%
- if all replicons were acrive, replication could be completed in 1 hour, but the S phase lasts about 6 hours which explains that only 15% of replicons are active at any given time
what are the general rules for replication?
- initiation of DNA replication commits cells to division
- division must not occur until after replication is completed
what does the bacterial origin support?
- initiation of replication
- control frequency
- segregates replicated chromosomes - not in eukarotes
what is controlling reinitiation?
- physical sequestration of oriC
- delay in remethylation
- inhibition of DNAA binding
- repression of DNAA transcription
what can the beginning of eukaryotic DNA replication be traced back to?
- archaeal chromosomes
- orc1/cdc6 are the same proteins found in archaea and eukaryotes
what is the system controlling reinitiation?
- physical sequestration of OriC
- delay in remethylation
- inhibition of DnaA binding
- repression of DnaA transcription
oriC
- 245 bp fragment
- smallest fragment that can support replication
- very AT rich
how is eukaryotic replication terminated?
- fusion of replication etes - no ter sites
eukaryotic replication
- The A domain is where ORC attaches and is very AT rich
- the B domain is where transcription factors attach
- once ATP binds to ORC initiation can begin
- at the B domain ABF1 stimulates initiation by opening the two strands
- then cdc6 binds to ORC which in turn recruits cdt1
- cdt1 recruits MCM which is a helicase
- when too many MCM binds, geminin binds to cdt1 to stop recruitment of MCM
- now replication can begin
mitotic cell cycle
- G1 is the first growth stage
- then synthesis occurs
- then G2 in another growth phase
- then mitosis can begin and the cell can divide
- interphase is everything in the cycle except mitosis
explain the connection between eukaryotic replication and the cell cycle
- in early G1 ORC is bound
- cdc6 and cdt1 are synthesized and binds
- in late G1 the helicase and possibly geminin are recruited
- then in synthesis, the DNA replication machinery is recruited and synthesizes the DNA to prepare for mitosis
- cdc6 and cdt1 are degraded in S phase to prevent reinitiation
- mcm is phosphorylated at the beginning of S phase and becomes active by leaving the origin
- In the G2 phase all the proteins are degraded
- finally mitosis occurs
where are MCM proteins in metazoans? in budding yeast?
- metazoans = MCM proteins are in the nucleus throughout the cycle
- budding yeast = only after mitosis
what does MCM stand for?
minichromosomal maintenance
how can ORC, cdc6, cdt1, and mcm be downregulated?
by cdk activity
- cell cycle dependent kinase
- phosphorylates enzyme
when is geminin degraded?
during late mitosis and early G1
- this allows cdt1 to bind which results in the recruitment of the MCM helicase in early G1
which of the following proteins or complexes is not a component of licensing factor?
ORC
which of the following proteins blocks binding of mcm to the origin
geminin
