DNArep Flashcards
origins of replication
Location where replication begins
1 in prokaryotes
many in eukaryotes
oriC
prokaryotic origin of replication
consist of repeating AT-rich sequences (9mers and 13mers)
9mers & 13mers less stable, so easy to break apart, enabling unwinding
Bidirectional
Replication occurs in both directions away from the origin
ter
The termination sequence for prokaryotes undergoing DNA synthesis
forks meet here and replicon gets popped out
Replicon
replisome
The DNA, the DNApol, and the enzymes involved in preparing DNA
Eukaryotic DNA prep enzymes
Helicase
SSBPs
Topoisomerase
Prokaryotic DNA prep enzymes
DNA helicase
SSBP
DNA gyrase
Prokaryotic unwinding process
protein DnaA binds to a region of 9mers.
The 9mer-DnaA complex associates with a region of 13mers.
This puts strain on the DNA, causing the helix to destabilize.
This exposes an area of ssDNA.
DnaA
protein that binds to a region of 9mers to begin unwinding
9mer-DnaA complex associates with 13mers, causing helix to destabilize
9mers and 13mers
repeating sequences of base pairs
AT-rich, less stable
9mer binds to DnaA and forms a complex to put strain on DNA and begin unwinding
DNA Helicase
made of several DnaB subunits
Recruits the holoenzyme DNApol3 to bind to replication fork
Moves along ssDNA, unzipping helix
DNA gyrase
Toposiomerase in prokaryotes
Makes “cuts” along ssDNA and dsDNA to relieve supercoiling
Reseals the cuts in non-coiled conformation
SSBP
bind to ssDNA to prevent its reassociation
Primase
creates short RNA sequences along the template strand
This allows DNApol to do DNA synthesis, as it gives them a starting point to work from
DNApol synthesis initiation issue
DNApol can’t initiate DNA synthesis
It can only bond phosphate groups to hydroxyl groups
So it must have a 3’ end to work off of
RNA primer
short RNA sequences along the template strand added by primase
serve as a base for DNApol to begin adding nucleotides to new daughter strand
runs antiparallel to template strand
Ligase
After RNA primer has been replaced by DNA by DNApol, ligase rejoins the replacement DNA with the other nucleotides
Basic DNA synthesis process
Primase adds primer
DNApol uses primer as a base to begin synthesizing off the primer’s 3’ end
Another DNApol removes the primer and replaces it with DNA nucleotides
ligase rejoins replacements with existing bases
Chain elongation direction
5’ to 3’ (concerned with daughter)
DNApol wants to attach phosphate groups to a hydroxyl group
The hydroxyl group of the primer is at its 3’ end
Prokaryote DNApol enzymes
DNApol1 (directs & repairs, removes & replaces primer)
DNApol3 (synthesizes new DNA segments)
DNApol1
directs DNA repair and synthesis
require the presence of all four dNTPs, and template DNA
Unlike other DNApols, it has a 5’ to 3’ exonuclease activity: removes the RNA primers and fills the gaps after primer removal
DNApol3
synthesizes new DNA segments off of the primer
holoenzyme (core enzyme, clamp loader, sliding clamp)
5’ to 3’ exonuclease activity
unique to DNApol1
Ability to remove RNA primers while going forward
exonuclease
enzymes that work by cleaving nucleotides out of a nucleotide chain
3’ to 5’ exonuclease activity
a “backwards” function of all DNApol
the ability to polymerize in one direction, pause, reverse direction, and cut out nucleotides just added
activates when an incorrect nucleotide is inserted, allowing repair
holoenzyme
Enzyme has multiple subunits and is a complex of all of the,
DNApol3 core enzyme
The largest subunit type
can be multiple in a holoenzyme
Necessary for catalytic activity (the reaction the holoenzyme exists to catalyze)
consists of alpha, epsilon, and theta
DNApol3 alpha subunit
Responsible for DNA synthesis along template strands
part of core enzyme
DNApol3 epsilon subunit
Possesses 3’ to 5’ exonuclease capability (allows backwards proofreading)
DNApol3 sliding clamp loader
attaches to the core enzyme
Facilitates the function of the Sliding DNA Clamp
DNApol3 sliding DNA clamp
Many of its subunits have a donut shape; these can open and shut to encircle the DNA strand
Opening and closing depends on phosphorylation
Leads the way during synthesis; maintains binding of core enzyme to the template
This increases processivity
Physical mechanism of synthesis in prokaryotes
coordinated synthesis means both strands produced at same time
concurrent DNA synthesis
both the lagging and leading strand are produced simultaneously by the same holoenzyme
lagging strand template is spooled out to form a loop
allows the physical direction of replication to change
Biochemically the direction of DNA synthesis is the same.
5’ end problem
IN EUKARYOTES ONLY (LINEAR CHROMOSOMES)
RNA primer on lagging strand will terminate the daughter 5’ end.
The gap left when the primer is removed cannot be filled; the pairs it represents on the template strand are lost.
the daughter strand loses some DNA with every cycle of replication
DNA synthesis only happens in 5’ to 3’ direction but is done bidirectionally: How?
RNA primer is added in upward increments towards the 5’ end of the daughter strand, and the 3’ template strand. This lets DNA get synthesized “uphill”.
Okazaki fragments
discontinuous segments of DNA made as part of the lagging strand, using “uphill” RNA primers
Semiconservative DNA model of replication
One strand is original
New strand made off of it
50% old, 50% new
for each dsDNA, 1 new strand, 1 old strand
Conservative DNA model of replication
1 old helix, 1 new helix
Dispersive DNA model of replication
Parental strands are dispersed into two new helices
50% old 50% new, but each strand is a mixture of old and new
How do we know about the enzymes implicated in DNA synthesis?
Condition mutations (like ts mutations) are used to observe the loss of function associated with mutations to these enzymes
Then their role in DNA synthesis can be analyzed
DNApol in eukaryotes
alpha, epsilon, delta
all differ in processivities
DNApol alpha
eukaryotes
synthesizes RNA primers
low processivity; undergoes polymerase switching after laying down primer
DNApol epsilon
synthesizes DNA on the leading strand
3’ to 5’ exonuclease activity (proofreading)
good processivity
DNApol delta
synthesizes DNA on the lagging strand
3’ to 5’ exonuclease activity (proofreading)
good processivity
Nucleosomes
DNA is wrapped around these, and must be stripped off before DNA synthesis can begin
made of eight histone proteins
Processivity
ability to stay attached to a template strand
Histones
8 of these make up nucleosomes
new ones are synthesized as DNA is synthesized
CAFs
chromatin assembly factor
assemble new nucleosomes behind the replication forks
Strand invasion
The 3’ G-rich telomere strand end will fold and invade into the doublestranded DNA area, forming a T-loop
Telomerase composition
Made of TERC and TERT
Shelterin complex
Shelterin proteins form the shelterin complex at looped telomeric ends, helping lock the telomeres into this looped position
TERC
RNA template contained within the telomerase, used by TERT to create new telomere
TERT
Reverse transcriptase (can make DNA from RNA) in the telomerase
uses TERC to make new telomere
Meselson-Stahl setup
Grew E. coli in a medium that had N15 as the only source of nitrogen.
After many generations in the N15 medium, the E. coli were immersed in N14.
The E. coli DNA were put into a centrifuge containing CsCl
CsCl creates a density gradient
different “densities” of DNA (N15 and N14) settled along the gradient where their density was equivalent to the medium
Meselson-Stahl results
N15 would be closer to the bottom of the tube than N14
Only 1 band of mixed density, disproving conservative model
when strands were denatured and evaluated for density, there were two bands, showing they did not have a dispersive composition
Meselson-Stahl conclusion
Proved the semi-conservative model in bacteria.
Taylor-Wood-Hughes setup
Taylor-Wood-Hughes results
Taylor-Wood-Hughes conclusion
DNA replication in eukaryotes is semiconservative
Polymerase switching
Once DNApol alpha has laid down primer, it dissociates from the template and is replaced by delta or epsilon
DSBs
double-stranded breaks
recognized by the cell and rejoined
G-rich and C-rich telomere strands
Telomeres in general are G-rich / C-rich (depends on the strand)
G-rich is 3’
C-rich is 5’
G-rich overhangs the C-rich due to 5’ deletions
T-Loop
A telomeric loop. This hides the ends, preventing ends from getting recognized as DSBs.
Telomerase operation
extends the G-rich strand (the 3’ overhang)
normal DNApols add RNA primer at the sister strand’s new end and fill the gap normally
primer is removed
the ends have been lengthened
