DNA replication Flashcards
what does dna repliation require
activated substrated known as dNTPS (deoxyribonucleoside 5/-triphosphates) i.e. dCTP, dATP, dUTP, dGTP
double stranded DNA plus dNTPs reacted with polymerases makes
2 double stranded DNA
what are the generic four steps that in vivo occur simultaneously during DNA replication
initiation (starting the replication)
priming (primer=rna)
synthesis (replication happens)
termination
t/f
eukaryotes have circular chromosomes
false
prokaryotes have circular chromosomes
how many start sites for dna replication are there in prokaryotes & what are they called
one.
initiation site
t/f
DNA is replication is bidirectional
true
t/f
a second round of replication cannot begin until the first is completed
false
second round starts before first is done
where does initation of dna replication in prokaryotes start
OriC (origin of replication)
what are the OriC recognized by
the initatior protein known as DnaA
what type of repeats are adjacent to OriC
AT rich repeats
t/f
only in prokaryotes there are AT rich sequences near the replication initiation site
false
this is also true in eukaryotes
why are AT rich repeats important
they’re less stable that native DNA so can be used to form the replication bubble
what is the helicase in prokaryotes
DnaB
what is the role of SS binding proteins
they bind to SS regions of replication bubble and stop it from making secondary structures (bc secondary will hinder replication)
t/f
dna polymerase can initatiate new strand synthesis
f
no DNA poly can initiate, it needs to have a primer
what is needed to add dNTP DNA polymerases
need a 3’ hydroxyl group at the end of a base paired primer strand
in what direction is RNA primer created
5’ to 3’
all of dna synthesis happens in this direction
why is a primer needed in each fork i.e. 2
bc synthesis of dna is bidirectional
describe rna primer synthesis
primase DnaG is part of the primosome
primosome binds to both strands of the replication bubble –>makes short RNA primer
what is the role of DNA poly 1 in prokaryotes
remove the RNA primer (primer excision)
and DNA repair
role of dna poly 3 in prokary
replication –> chain elongation during DNA synthesis
and DNA repair
diff bn 3’ and 5’ exonucleases
3’ removes prna primers in 3’ to 5’ direction
5’ removes it 5’ to 3’ direction
why is dna replication the most accurate process
bc it has proof reading
3/ exonuclease checks it
3’ exonucleases are found in which dna polymerases
5’ found in which
poly 1 and 3 have 3’ exonucleases
only poly 1 has 5’ exonucleases
what is the processivity of dna poly 1 and 3
poly 1 = low to moderate
poly 3 = high processitivity
which polymerase removes rna primer
dna poly 1
which poly is involved in replicating dna
dna poly 3
what is processivity
measure of how many nucleotides can be added to dna strand as it’s growing before it disengages
what subunit serves as a sliding clamp that encircles DNA
beta
what is the role of the gamma complex
it is a clamp loader so helps get beta (sliding clamp) loaded onto dna
which subunit is a structural protein that holds things together
tau
what is contained within epsilon
3’ exonuclease used for proof reading
what is the role of theta
needed for 3’ exonuclease activity
t/f
each replication fork has one DNA poly 3 holoenzyme
true
what charge is the interior of the beta sliding clamp and why
neutral or negative bc for clamp to move on the dna needs to be repulsive
-dna is negative
t/f both strands of the replication fork are synthesized simultaneously
true
what is the difference bn how the leading and lagging strands are synthesized
the leading strand synthesized continuously
lagging synthesized discontinuously
in what direction in relation to the fork is dna synthesis occuring in the lagging strand
its being synthesized away from the fork
what is special of the backbone on the lagging strand
it has a nick on it
okozaki fragments are made on which strand of the replication fork
lagging strand
t/f
for the leading strand the fork has to open before new synthesis can be initiated
false
the LAGGING strand needs the fork to be open
since both strands are made simultaneously how do both strands become in the same orientation
a loop is added on the lagging strand which puts the 2nd okozaki fragment in same orientation as the leading strand so the polymerase can move smoothly
as the DS helix is being opened, helical tension is formed and ___ is needed to regulate it
topoisomerase
dna poly 3 can recognize
mismatched base pairs (aka AG or CT)
what is the role of epsilon subunit
removes mismatched pairs with it’s 3’ to 5’ exonuclease activity
how is the chain extended after epsilon subunits remove mismatched base pairs
extended by dna poly 3
where is the termination region in relation to OriC
180 degrees from it
what are ter sites
termination sites in prokaryotes
has 6 sites oriented in opposite directions on either side of the 180 degree point
why is the ter orientation in opp dir’s on either side of the 180 degrees
bc binds to the TUS protein
in how many directions can the TUS protein go
one
the fork can pass right past it but can’t pass it in other direction
what is the process of rna primers being removed by dna poly 1 called
nick translation
how does nick translation work
dna poly 1 5’ exonuclease hydrolyses the RNA primers
simultaneously the 3’ end of the okazaki fragment (DNA) is extended by the incorporation of deoxyribonucleotides
role of dna ligase
seals the nick
what is the last step in termination of dna synthesis in prokaryotes
type 2 topoisomerase separated the 2 interlinking strands of the replicated circles and refolds the dna into supercoils
what are the 4 similarities bn pro and euk dna replication
- semi conservative
- bidirectional
- short RNA primers req’d to initiate new strand synthesis
- leading strand made continuously
lagging made discontinuously
in eukaryotes
what is te role of dna poly gamma
replication of mitochondrial dna
in euk
whats role of dna poly delta and epsilon
poly delta replicated the lagging strand
poly epsilon replicates the leading strand
which strand does dna poly alpha replicate
both leading and lagging
what is the inherent processivity of all 5 eukaryotic dna polymerases
alpha-moderate beta-low gamma-high delta-low epsilon-high
which eukaryotic dna poly has high processivity with PCNA
delta
in the tertiary structure, where are nucleosome dissociated and where are they reassembled
dissociated ahead of the replication fork
reassembled on newly synthesized DNA
what is the speed and size of the tertiary structure in replication
10 times slower
size of genome is massive
what are present to accomodate the large size of the genome
multiple initation sites of replication known as ARS
what are the highly repetitive sequences at the ends of linear chromosomes that protect the chromosome from being damaged
telomeres
what is the origin of replication
in prok and euk
pro: OriC
Euk: ARS
what is the bind origin of replication (i.e. what protein recognizes the origin of replication in each)
in prok and euk
pro: DnaA complex
euk: ORC
what is the helicase
in prok and euk
pro: DnaB/C complex
euk: MCM
what is used to stabilize SS DNA
in prok and euk
pro: SSBs
Euk: RPA
what is used to increase processivity
in prok and euk
pro: Beta-subunit poly 3
euk: PCNA for DNA poly delta
what is the clamp loader
in prok and euk
pro: gamma complexx
euk: RFC (replication factor C)
what makes RNA primer
in prok and euk
Pro: DnaG
euk: DNA poly alpha
what synthesizes DNA
in prok and euk
pro: DNA poly 3
Euk: DNA poly epsilon (leading strand)
DNA poly delta (lagging strand)
DNA poly gamma (mitochondria)
what is used for the removal of RNA primers
in prok and euk
pro: DNA poly 1 (nick translation)
euk: RNAse H and Fen1 (flap endonuclease 1)
what does RNAse H do
destroys DNA/RNA hybrids and removes the RNA from it
diff bn exonuclease and endo
exo cuts from the ends
endo makes cut from within
what genetic defect causes Aicardi-goutieres syndrome
RNAse H which acts on RNA/DNA hybrids and is involved in removal of RNA primers in eukaryotic replication
during eukaryotic replication, a pre-replication complex (pre-RC) assembles on what
on the ARS which includes ORC(origin of replication), MCMs (inactive helicases) and other proteins
how are helicases activated
by the addition of addtl proteins
-thats when it can start unwinding the DNA bc it;s active
what is ARS rich in and what does that allow it to do
ARS is AT rich –> helps it open up inititial replication bubble
t/f
more pre-RC are assembled (licensing) than used (firing)
true
in order for replication in eukaryotes to start the helicase needs to be
activated so it can unwind the dna
what keeps things regulated during eukaryotic replication
topoisomerase
-which work ahead of the replication fork to keep the DNA untangled
what makes the leading and lagging strand into the same orientation
a loop
what is RNaseH / FEN 1 doing during eukaryotic replication
taking out RNA primers
what causes meier-gorlin syndrome (failure in growth)
mutations in genes that encode the proteins needed for the Pre-RC complex including: ORC
-bc of impaired replication licensing (aka assembly)
what are the highly repetitive seq’s at the 3’ ends of linear chromosomes
telomeres
what are telomeres added by
telomerase
what is the protein compnent enzyme and the RNA component of telomerase
protein: hTERT = human telomerase reverse transcriptase
RNA: hTR = human telomerase RNA
what does the telomerase use it’s rna component (hTR) for
to add repeats
-telomerase repositions itself to add each repeat
how is the daughter strand synthesized
by unsing complementary base pairing by DNA poly alpha (puts RNA primer to synthesize the DNA)
what is left when telomerase moves away
a single stranded 3’ overhang
the telomere ends are protected by loops that are stabilized by binding of telomere-binding proteins called
TRF1 and TRF2
telomere length is maintained by
telomerase
what would happen in a dividing cell with little or no telomerase
not much short term
telomeres would shorten after rounds of replication
cells would die
cells would stop dividing
telomeres shorten with each round of dna replication unless maintained by
telomerase
what mutation causes Dyskeratosis congenita (which worsens with successive generations)
mutations in the catalytic domain (hTERT) or rna component of telomerase (hTR)
diseases of telomere shortening show anticipation because
loss or partial loss of telomerase function leads to telomere shortening n the germ cells
