dna replication Flashcards
dna pol III
carries out genomic replication and DNA repair functions
note: high polymerization rate, high processivity, and proofreading
dna pol I
has roles in replication, recombination, and DNA repair
note: 3’->5’ proofreading and 5’->3’ exonuclease activities (repair)
pol I and pol III have _______________________
high fidelity= they don’t make a lot of mistakes
mutations
permanent errors in replication that will be inherited by subsequent daughter cells
what are the 3 mechanisms to avoid mistakes during replication?
presynthetic error control, proofreading, and mismatch repair
presynthetic error control
demands correct base pairing
presynthetic= before phosphodiester bond formation
geometry of watson and crick base pairs allows them to fit into the catalytic site of DNA polymerase
mispaired base pairs are excluded
errors can occur due to spontaneous tautomerism
proofreading
postsynthetic error control
recognizes and corrects mistakes
high-fidelity DNA polymerases have 2 active sites: catalytic site for DNA synthesis & 3’->5’ exonuclease site for proofreading
prokaryote DNA pol I, II, and III are all high-fidelity enzymes
tautomers
tautomeric base pairs result from chemical rearrangements of the bonds within the base
- occur spontaneously
- can participate in forming alternative base pairs
- isomerization of the tautomeric base to a normal base results in a mismatched base pair
bidirectional synthesis
replication forks proceed in both directions, away from the origin of replication
DNA synthesis begins at origin of replication
yields two DNA molecules with identical nucleotide sequences
replication fork
where parent DNA is being unwound (by helicase) and the separated strands are replicated by DNA polymerase
leading strand
primed only once
synthesis occurs continuously from 5’-> 3’
lagging strand
synthesis occurs discontinuously from 5’->3’
okazaki fragments
each fragment must be primed
fragments must later be joined
what is true about replication in prokaryotes?
occurs only once per cell cycle
initiation is the regulated step in control of DNA synthesis
oriC (origin of replication)
has binding sites for DnaA protein
DNA unwinding element (DUE) is an AT-rich segment where strand separation occurs (energy requirement is much lower and hydrogen bonds are easier to break)
what causes localized strand separation at the DUE (dna unwinding element)? (DNA replication initiation in prokaryotes)
binding of DnaA proteins to the R and I sites stresses the DNA helix
DnaC loads a DnaB helicase at both ends of the bubble on what will be the lagging strand. What does this lead to?
loading of DnaB helicase commits the cell to replication and division
DnaB helicase leads the replication fork and separates DNA strands 5’-> 3’
what happens after DnaC is released?
DNA pol III’s are assembled onto both DnaB helicases
DnaA is then released from the DNA
DNA pol III is ready to replicate the strand
holoenzyme
core polymerase + accesory subunits
core polymerase (pol III)
catalyzes DNA synthesis
there are 2 - one for leading strand and one for lagging strand
keeps lagging and leading moving together
clamp loader serves as…
-a scaffold for DNA polymerase III complex
-to assemble the B clamp onto DNA using ATP
-coordinates the replication fork by interacting with DnaB helicase through tau subunits
B clamp (aka sliding clamp)
tethers the core polymerase to DNA
- decreases the polymerase dissociation from DNA -> increases processivity
primase
DNA template-dependent and primer-independent RNA polymerase
- synthesizes a RNA primer at the beginning of the leading strand and each okazaki fragment
loading the DNA pol III on B clamp on DNA
B clamp encircles DNA- provides means for high processivity and rapid DNA polymerization
-ATP binding to the clamp loader causes it to open and bind to the clamp
-once open, it can associate with the DNA strand
- the clamp loader will add the clamp at site of the primer
-ATP hydrolysis causes the clamp to close around DNA
what generates the leading strand?
DNA synthesized as a continuous polynucleotide chain by one core polymerase
how is the lagging strand synthesized?
as a series of okazaki fragments
_________ functions at the replication fork just as core polymerase near completion of an okazaki fragment
- _________ begins synthesis of a new RNA primer
primase
single strand binding protein (SSB)
protects gaps in DNA double helix
what is loaded onto the lagging strand at each new RNA primer by the clamp loader?
B clamp
DNA pol III-dependent synthesis of an okazaki fragment is complete when….
the enzyme reaches the previous primer
what happens when the lagging strand core polymerase pauses?
it releases its B-clamp; a new B-clamp is loaded and the lagging strand is transferred (old clamp is abandoned)
lagging strand core polymerase starts the synthesis of the next okazaki fragment
B-clamp loader acquires a new clamp and opens it in preparation for loading into the next primer
____________ pauses at an okazaki primer, then abandons its B sliding clamp and transfers to a newly loaded clamp (completing the lagging strand)
DNA pol III
__________ enters and uses its 5’->3’ exonuclease to remove the primer
- as RNA primer is being digested, _________ synthesizes DNA filling the gap
DNA pol I
________ repairs the nick linking the okazaki fragments into a single DNA strand
- in bacteria, ____________ uses NAD+
DNA ligase
after replication, circular chromosomes are linked together like links in a chain which is called _________________
catenated state
torsional stress from unwinding DNA at the replication fork results in over-winding of the DNA ahead of the fork causing __________________
positive supercoiling
enzymes that relieve both negative and positive supercoils in DNA
topoisomerases
topoisomerase type I
single-strand break, pass the intact strand through the break, and seal the nick
topoisomerase type II
double-strand break, pass an intact segment of DNA through the break, and then rejoin broken strands
eukaryotic DNA polymerases
- dna synthesis is semiconservative an bidirectional
- mechanism is template & primer dependent
- synthesized from 5’ -> 3’
- there are about 15 DNA polymerases with specialized functions
- replisome carries out genome replication (instead of DNA pol III complex in prokaryotes)
differences in eukaryotic replication
-eukaryotic chromosomes are much longer and linear
- genomic replisome (DNA polymerase in eukaryotic cells) is constituted by DNA Pol delta gamma epsilon
- primase is still an RNA polymerase but now is in a complex with DNA pol alpha
initiation (eukaryotes)
-very different compared to prokaryotic
-eukaryotic chromosomes have thousands of origins of replication whereas prokaryotes have 1
- coordination requires licensing
elongation (eukaryotes)
-rate is much slower
- okazaki fragments are shorter
2 different core enzymes are used by replicase
- removal of RNA primer and DNA replacement is different
termination (eukaryotes)
-telomeres are unique to eukaryotes
- different details
each of our 46 chromosomes contains 1000s origins of replication. Origins of replication on all chromosomes must be activated once and only once during __________________________
S phase of the cell cycle
_________________ bind tightly to DNA in early G1 phase
origin of replication complexes
during mid-G1, cell division cycle 6 joins the ________, following by Cdt1
ORC
_______________ joins the ORC in late G1
mini chromosome maintenance (MCM)
in S phase, replication is initiated by phosphorylation of complex proteins by a _______________ and another protein kinase (DDK)
cell cycle-dependent kinase (cdk)
what coordinates replication initiation?
licensing
since replication initiation is an ____________________ event, so many origins can be activated very rapidly.
enzyme catalyzed
what is the core polymerase for the leading strand (eukaryotes)?
DNA pol epsilon
what is the core polymerase for the lagging strand (eukaryotes)?
DNA pol delta
what is a complex that contains primase for RNA primer synthesis and a separate DNA synthesis activity? (eukaryotes)
DNA pol alpha-primase
what is the helicase? (eukaryotes)
MCM (mini chromosome maintenance)
what is the clamp loader? (eukaryotes)
RFC
what is the clamp? (eukaryotes)
PCNA
what is a single-strand DNA binding protein (SSB)? (eukaryotes)
RPA
what is the DNAse? (eukaryotes)
FEN1
replisome uses two different core enzymes:
_________ synthesizes the leading strand
_________ makes the lagging strand
DNA pol epsilon
DNA pol delta
what is different in the completion of the lagging strand in eukaryotes compared to prokaryotes?
DNA pol delta is a “strand-displacing” DNA polymerase
FEN1 is flap endonuclease-1 that clips off the overhang
DNA ligase repairs the nick left in the sugar-phosphate backbone to join the two okazaki fragments
what are specialized DNA structures at the ends of linear eukaryotic chromosomes tht consist of thousands of repeats of a short sequence?
telomeres
proteins are bound to the telomere sequence to form “________” DNA structures
T loop
what are proteins that protect the single-stranded 3’ end within a DNA duplex?
shelterin
what do telomeres do?
protect the chromosome from cellular exonucleases and DNA repair enzymes
what has an integral template RNA with 1 1/2 copies of the CA repeat?
telomerase
template RNA anneals to existing TG sequence at a telomere
- telomerase catalyzes 5’->3’ DNA synthesis of the TG synthesis
- following each round of TG synthesis, telomerase shifts so that the integral template RNA anneals to newly synthesized DNA
process repeated many times
the complimentary CA strand of a telomere is synthesized by ______________ acting as a DNA polymerase
DNA pol alpha-primase
