DNA Replication Flashcards
Which type of DNA is biologically relevant?
double helix B DNA
Prime Carbons?
on the deoxyribose sugar, not the base
Which phosphates get removed during replication?
gamma and beta
How many bonds between G and C?
2
How many bonds between A and T?
3
Mechanism of forming DNA helix?
- 3’ hydroxyl attacks alpha phosphate
- pyrophospate is released (net zero energy)
- breaking the pyrophosphate makes it irreversible
What speed is the E.coli genome replicated at?
2000 bps-1
How is the E.coli genome replicated?
origin of replication –> bidirectional replication –> region of termination
Problems to overcome in replication?
- antiparallel nature
- strand coiling
- circular bacterial genomes
- stacking of bases in helix
DNA Polymerase I basics?
400 molecules per E.coli cell.
Requires dNTP, template strand and primer.
Polymerase is processive, doesn’t dissociate so 10-100 bases are added at a time.
Growth is 5’ to 3’.
Binds to nicked or gapped DNA, not intact ds/ss DNA.
Activities of DNA Pol I?
- 5’ to 3’ polymerase as normal (C terminal - Klenow fragment)
- 3’ to 5’ exonuclease, backtracks as a proofreading mechanism (N terminal)
- 5’ to 3’ exonuclease replaces DNA in front of the nick (C terminal - Klenow fragment)
Why are no dRTPs added?
Due to steric clash with 2’OH on sugar
Pol I mutants?
Accumulate small DNA fragments and the cells are UV sensitive with high rate of mutation.
How do polymerases proofread?
Addition to a mismatched base is slow and allows time for the strand to contact the exonuclease site.
Which is the principle Polymerase in prokaryotes?
DNA Polymerase III
How processive is DNA Pol III?
50,000 nucleotides added per binding event
Mw of DNA Pol III holoenzyme?
10^6 Mw
Alpha subunit in DNA Pol III?
Polymerisation
Epsilon subunit in DNA Pol III?
3’-exonuclease
Quick stop mutants?
Immediately halt DNA replication
Slow stop mutants?
Allow replication to finish but can’t begin a second round.
Conditionally lethal mutants?
Temperature sensative mutants. Powerful tool for studying loss of function phenotypes in particular essential genes.
Why does DNA Replication require so many enzymes?
- DNA strands are antiparallel
- All DNA polymerases are 5’ to 3’
- Not self priming
- DNA strands are plectonemically coiled
Outline Okazaki’s experiment
E.coli culture infected with phage T4 > add 3H thymidine > take samples at intervals > lyse with alkali into ssDNA > alkaline sucrose density gradient
Results from Okazaki’s experiment
Large number of radioactive short units > likely discontinuous method. Further supported with the discovery of polynucleotide ligase (links short DNA strands together).
After a longer time, more long DNA fragments but short are still present. Can “chase” the short fragments into the long fragments.
Pulse chase experiment?
Radioactive for short period of time, then follow with non radioactive substance.
The radioactivity appears in the short fragments, then the long fragments.
How is the lagging strand synthesised?
In short pieces, which are subsequently joined together by DNA ligase. Requires a 3’-OH and 5’-P at adjacent complementary base pairs. Also requires ATP to join the nick.
Why does the leading strand appear to be made in sections?
U is being removed from the newly synthesised DNA - results in transient breaks, giving short fragments
True semi discontinuous replication is evident if removal of U is prevented.
V short fragments if more Us are removed at the replication fork
Why does uracil appear in DNA?
- U is incorporated in place of T, opposite A. Non offensive as U has same base pairing properties as T
- U can arise in situ from the spontaneous deamination of C. Offensive as causes a mutation. GC -> GU -> AU
How is uracil removed?
uracil-N-glycosylase and AP endonuclease act to remove it.
The gap is then filled with DNA Pol I and sealed with DNA ligase.
describe primase?
RNA polymerase self priming 5'-3' direction no editing/proofreading functions 5-10 nucleotides in length activity is increased in presence of helicase
okazaki fragment composition?
begin with 50-100 bp of RNA then 2000 bp of DNA in prokaryotes
how is RNA removed from okazaki fragments?
when the newly synthesised strand meets the previous RNA primer, POL I takes over and removes the RNA primer by nick translation (5’-3’ exonuclease)
how are okazaki fragments joined together?
DNA ligase seals the “nick” in the phosphodiester backbone between a 3’ OH and a 5’ phosphate.
Energy - ATP
alternative energy source for okazaki fragment joining?
NAD in some organisms
how is the fork opened?
DNA is unwound by helicases
which is the main helicase?
product of dnaB gene
DNA dependent ATPase - needs 1 per bp unwound
Processive, moves from 5’-3’ on lagging strand.
Bloom’s and Werner’s syndrome?
due to defective helicases - not as active as they should be
DNA helicase structure?
6 subunits - hexamer
not symmetric - slightly squished
which subunits bind what in DNA helicase?
two - ATP
two - ADP
two - empty
opposing are same
alternate between each configuration as ATP is hydrolysed –> ripple effect
mechanism of DNA helicase?
ripple effect causes the loops in the middle of the ring to oscillate.
this allows DNA to pass through, unwinding DNA in the process
how is the unwound DNA stabilised?
ssb - single strand binding protein
product of DnaT gene
binds cooperatively, mainly on lagging strand
why is the cooperativity of ssb important?
so that the end proteins can be removed more easily when necessary for DNA replication
how long for POL III to add a nucleotide?
1 msec
how does one POL III enzyme synthesise both strands in prokaryotes?
the lagging strand is looped out before synthesis in the 5’ to 3’ direction. Acts as a dimer
subunits of POL III holoenzyme?
alpha, epsilon, delta are the polymerase
tau is responsible for dimerisation - holding two halves together
beta clamp forms a ring around the DNA - processivity
gamma, delta, si, chi use ATP to load the beta clamp on the DNA
slowest process?
putting the clamp on the DNA
how long are okazaki fragments in prokaryotes?
1000-2000 nts in length
what is the beta clamp known as?
processivity factor
how does the clamp loader work?
gamma, delta, si, chi have ATP binding and hydrolysis which catalyse ring opening and loading
how does the clamp interact with DNA?
hole in the centre is positively charged to interact with negative DNA. filled with water to prevent v strong ionic interactions - larger distance
one unwind of the DNA duplex is required for how many bp to be replicated?
10
speeds involved with unwinding?
POL III - 1,600 nt/sec
helix - 10,000 r.p.m
what are topoisomerases?
enzymes that relieve the superhelical stress that is produced around the replication fork
also separates the two daughter molecules after the cycle is complete
what is the linking number?
no of times that one strand passes around the other
LK = N/h N = no of base pairs h = helical repeat (10.5)
must be an integer
when can LK not change?
for a circular genome when the circle remains closed
what happens to circular DNA before closing the circle?
overwound, or normally UNDERWOUND, decreasing LK
typically change in LK/N = -0.06
results in coiling the DNA molecule
which equation describes supercoiling?
LK = T + W
T - twist
W - writhe
what is twist?
coiling of strands around helical axis
what is writhe?
coiling of helical axis in space
how can the DNA be put into a relaxed state?
create a double stranded break and untwist the circle
type I topoisomerases?
odd numbers
catalyse the relaxation of supercoiled DNA - thermodynamically favoured
only cuts one strand
LK - 1
type II topoisomerases?
even numbers
utilise energy from ATP hydrolysis to add negative supercoils
cuts both strands
LK - 2
how do topoisomerases work?
cleave one/both strands,holds by phosphodiester linkage to tyrosine
passage of a DNA segment through the break (swivels)
resealing
what is DNA gyrase?
introduces negative supercoils in the first place (assembling DNA)
requires ATP
A2B2 structure A gyr (nalA) - nick closing activity B gyrB (cou)- DNA dependent ATPase
LK - 2
where does initiation of replication take place?
at a fixed sequence (OriC) - then the forks move bidirectionally until they reach terC - termination sequence
describe OriC?
245 bp sequence containing
4 x 9 bp repeats
3 x 13 bp repeats
Both AT rich
how does initiation of replication take place?
DnaA binds cooperatively to the 9 bp repeats (requires ATP)
DnaA interacts with 13 bp repeats, melting the strands
DnaB moves along the lagging srand opening the fork
DnaG (primase) associates with DnaB and an RNA primer is made at each fork
what is dnaA?
AAA+ protein - ATPase associated with diverse cellular activities. 3D structure mimics DNAs
why does initiation only occur once per cycle?
OriC contains a large number of GATC sequences - substrate for dam ( DNA adenosine methylase) which methylates N6 of adenine.
Immediately after replication - hemimethylated - inhibits initiation
Needs to be methylated before inititation can occur again
GATC sites are methylated v slowly (13mins) which occurs at the membrane and the DNA is spooled through the membrane bound replication apparatus
what does the ter region contain?
six homologous 23 bp sequences - 3 sites oriented in each direction
tus?
binds to ter region. prevents fork movement in one direction only.
how do the daughter chromosomes get separated?
hemimethylated DNA at OriC has a strong affinity for the cell lipid membrane - possibly to assist.
separated by topoisomerase IV
differences of dna replication in eukaryotes?
- nucleus
- more genetic material
- more than one chromosome
- not circular
- additional packaging (histones, nucleosomes, etc)
- 1000s of replication forks, not a singular origin, but from ars (autonomously replicating sequences)
- polymerases slower (50 bp s-1)
- okazaki fragments are shorter (135bp)
5 main eukaryotic DNA polymerases?
alpha, beta, gamma, delta and epsilon
how long are eukaryotic RNA primers?
10 bp
what is the sliding clamp known as in eukaryotes?
proliferating cell nuclear antigen (PCNA)
which eukaryotic polymerase has primase activity?
alpha - but isn’t processive as doesn’t associate with PCNA and no 3’-5’ exonuclease so no proofreading
which are the main eukaryotic enzymes?
delta - associates with PCNA and does polymerase synthesis on lagging strand.
Polymerase epsilon does polymerase sythesis on leading strand and removes primers
what removes RNA primers in euakryotes?
FEN1 exonuclease - leaves the primer hanging off like a “flap”
what must bind to the origin of replication in order to replicate it?
- ORC (Origin Recognition Complex of proteins)
- Licensing factors
- Cdc-1 and Cdt-1
what happens to ORC?
remain on the DNA throughout the process
what happens to licensing factors?
these accessory proteins accumulate in the nucleus during G1
what are the Cdc-1 and Cdt-1?
bind to ORC and coat the DNA with MCM proteins
why are MCM proteins required?
only DNA coated with MCM proteins (6 types) can be replicated
when do Cdc-1 and Cdt-1 leave the ORC?
once replication begins in S phase
ubiquination and destruction in proteasomes
what is the end replication problem?
RNA primers can be removed by nick translation except the first one so the chromosomes will shorten at each round of replication
Hayflick limit?
In eukaryotes: 40 divisions followed by senescence
why is end problem not a problem in circular DNA?
Rolling circle replication - displacement
End to end joining
how is the end replication problem solved in eukaryotes?
adding repeated units of simple sequences to chromosomal ends (telomeres)
added by telomerase
what composes the telomeres?
TTAGGG (last 50-100 bases at the 3’ end of each chromosome, single stranded)
structure of telomerase?
ribonucleoprotein
contains an RNA strand which has the sequence CUAACCUAAC near its 5’ end. Acts as a template for the synthesis of telomeric repeats
what regulates telomere length?
telomere binding proteins TRF1 and TRF2
when is telomerase inactive?
most differentiated cells
correlation between ageing/senescence and low levels
when are telomerase levels high?
in tumour cells
how are RNA genomes copied?
by RNA dependent RNA polymerase - RNA replicase
plus strand is copied to make a template minus strand
self priming
what is reverse transcriptase used for?
in retroviruses, they have ss RNA genome.
copied into ds DNA by reverse transcriptase to integrate into host genome
RNA/DNA hybrid, has RNA removed by RNase H
