Exam I - Lecture 6-9 Flashcards
Meselsson & Stahl’s Experiments
The mode of replication
transfer of information
cell division
perpetuate
make (something) continue indefinitely; preserve(something valued) from oblivon or extinction
DNA replication
The duplication of the cellular genome in which the stored genomic information is handed down to the next generation
DNA structure carries information needed to perpetuate
Each parental strand: template for one daughter strand
semi-conservative
one new strand, one old
conservative replication
One original strand, one completely new
Mehelson and Stahl demonstrated that replication is _______
semi conservative
dispersive replication
Original strand would break into chunks, and from there replicate those new strands
Meselson and Stahl experiment
- Used two isotopes of N to change the DNA density
- Grew bacteria in media contianing only 15 N (heavy) or 14N (light nitrogen)
- Extracted DNA from bacteria and used density gradient centrifugation to separate “heavy” DNA from “light”
After the 1st generation of the Meselson and Stahl experiment, only ____ could be eliminated, because it did not form a hybrid
conservative
After the 2nd generation of the Meselson and Stahl experiment, ___ could be eliminated
dispersive ( because there were no heavy 15N)
Replicon
a unit of genome in which DNA is replicated
each replicon, has an
origin of replication; a DNA sequence where replication initiates
Any DNA molecule that contains an origin can be replicated in the cell
Replication is controlled at the initiation stage
at a replication fork
the DNA of both new daughter strands is synthesized by a multi enzyme complex that contains DNA polymerase
replication fork
point at which replication is occurring
replication is initiated at ORIGINS and proceeds
BIDIRECTIONALLLY
bacterial chromosome: Theta Form replication
Origin - a sequence that can support replication of any DNA joined to it (replicon - DNA under a control of one origin
OriC - 245 bp
General feature: rich in A and T
E coli -> single origin, bidirectional replication (approx 30 proteins needed)
Rate: 1000 nucleotides per second
MOST of bacterial, viral and extrachromosomal eukaryotic genomes are circular
OriC
replication originin bacteria
245bp
**bidirectional replication
is the most common form of replication, but not completely universal
Eukaryotic chromosomes: multiple replicons
Each chromosome is composed of multiple replicons (40-100)
Many origins necessary because of slower replication and more DNA present (100 nucleotides per second)
ARS (autonomously replication sequence) elements from yeast. (Similar to OriC of E.coli
Any sequence containing ARS can be replicated within a yeast cell
The start of S phase (replication)
Activation of first replicon(s)
Not all replicons are activated at the same time
All of the DNA must be replicated ONLY ONCE prior to cell division
Replication is semi-discontinuous
only one daughter strand is synthesized continuously; the other is made as a series of discontinuous fragments
OKAZAKI fragments
What strand always contains the okazaki fragments
Lagging strand
Okazaki fragments
1-2 kb in bacteria
100 to 200 nucleotides in eukaryotes
DNA polymerase requires:
A template strand
A primer (to provide 3’-OH to add to new nucleotide)
DNA polymerase elongates in
5’ to 3’ direction always
proofreading
done by DNA polymerase in a 3’-5’ exonuclease fashion
polymerase and nucleases activities
reside in different sites
removal of exonculeases (in e.coli)
increases mutation rates (mistakes) x 100
chemistry of polymerizsation
1) New DNA is synthesized from dNTPs
2) in replication, the 3’-OH group of the last nucleotide on the strand attacks the 5’ phosphate group of the incoming dNTPs
3) two phosphates cleaved off
4) a phosphodiester bond forms between two nucleotides
5) and phosphate ions are released
dNTP binding site
fingers
polymerase active site
palm
3-5 exonucleaise activity site
palm
Pol I - open form
dNTPs can bind to the finger domain
the accuracy of the polymerase functions at the level of
shape recognition
dNTP enter between
thumb and fingers
base pairing with template causes fingers to
close, positioning substrates in the catalytic site (in palm)
the conformational change of the finger domain after dNTPs have been bound
a conformational change brings dNTPs and primer into correct orientation with metal ions
DNA polymerase active site (palm) contains
two divalent metal ions (cofactors) that are required for catalysis
Mg2+
mg2+ (or divalent atom)
deprotonates the primers 3’-OH group
AND
binds the incoming dNTP and facilitates departure of the pyrophosphate
the formation of ____ leads to the opening of the fingers domain and ___
phosphodiester bond
and movement of template/primer by one basepair
proofreading by DNA polymerase
slow or no DNA synthesis
“wrong” geometry of mismatched pair reduces its affinity for polymerase active site
DNA slides down the exonuclease active site
removal of mismatched nucleotide(s)
removed by 3-5’ exonuclease activity
the slow incorporation yet rapid removal of a mispaired dNTP underlies the inherent accuracy of DNA polymerases. Accuracy is further enhanced by a vastly diminished rate of dNTP incorporation at a mismatched 3’ terminus
error rate in DNA replication is less than
1 in 10^9 (a billion)
- nucleotide selection by DNA polymerase
errors are 1 in 10^4-^5
proofreasing by DNA polymerase increases fidelity by
100 fold
mismatch repair system
increases fidelity another 100 -1000 fold
all DNA synthesis
5-3’ direction
all DNA polymerases link the alpha C-5 phosphate a new dNTP, to the 3’ position of the nucleotide reside in the end of the chain
DNA synthesis is
semidiscontinious
lagging strand is opened
3-5’
SSB - single stranded binding proteins
unwind DNA< synthesize primers and keep strands apart
helicase function
slides 5-3 on the TEMPLATE for the lagging strand - it uses ATP to separate strands.
primase
initaiates on ssDNA containing a specific trimer (GTA in e coli); actvity increases when associated with a helicase
primase size
10-13nt
SSBs
stabilize ssDNA prior to replication
cooperative binding ensures quick coverage of exposed template
topoisomerase
removes positive supercoils
repliosome
primase and helicase
Pol III holoenzyme
22 subunits
1,068 mass
DNA helicase (DnaB in prokaryotes)
slides on the template for the lagging strand (then in the 5-3’) it uses ATP to separate strands
6 subunits in a ring shape to unzip the double helix
Topoisomerase (gyrase or II)
removes positive supercoils.
untwists DNA by cutting one OR both strands of DNA to unwind it, then reaeals it.
acts on duplex DNA ahead of the replication fork
primase - specialzed RNS polymerases
RNA primer in Ecoli come from DnaG primase; initiate each ot the thousands of okazaki fragments on the lagging strand; the leading strand is initiated by primase at a replication origin
In Ecoli - RNA primer is synthesized from
DnaG
E coli - DnaG
must be bound to the helicase for activity
DNA pol I
remove RNA primers at the end of each okazaki fragments and replaces with DNA.
*RNaseH can also remove RNA primer, but not the last rNMP
ligase
seals the nick in the phospodiester bonds
SSB
protect the DNA from endonucleases;
stimulates DNA polymerase activity by melting small DNA hairpin structures (i.e. separating base pairs ) in ssDNA
DNA pol III
exonuclease 3-5 YES
exonuclease 5-3 NO
E coli, Pol III, holoenzyme
22 subunits
DNA sliding clamps greatly
increase speed and processivity of replication
clamp loader
assembles beta clamp onto DNA
Beta clamp is loaded onto DNA by clamp loader by using ATP
the beta clamp allows the DNA polymerase to have higher processivity, meaning it can add more dNTPs to the daughter DNA strand
clamp loader
five subunits ( gap between two of the five subunits) is where the beta clamp attaches to
DnaB helicase connect to the Pol III holoenzyme
increase helicase actibity
repliosome
POL III, DnaB, helicase, primase
leading strand
POL III - Beta clamp moves continiously with DnaB helicase
lagging strand
POL III - beta clamp repeadetely moves on and off the DNA to extend multiple primers
trombone model
looping DNA grows and shortens during lagging strand synthesis
finishing lagging stradn
okazaki fragments require removal of RNA primer and ligase mediates joining of DNA
Nick translation , activity by
DNA POL I
5-3”
primer removal
RNase activity
5-3’
degrades both DNA and RNA
DNA synthesis
DNA pol
3-5’ exonuclease for proof reading
replication fork in eukaryoes
30 - 50 nucleotides per second
CMG complex -
functional helicase
CDC45, Mcm2-7, GINS
3-5’
(opposite of ecoli - 5-3)
PCNA
proliferating cell nuclear antigen (DNA sliding clamp)
RFC
replication factor C ( clamp loader)
RPA
replication protein A ( equivalent to SSB in E.coli)
E Coli - 3 Pol III enzymes at the replication fork
in Eukaryotes
pol alpha - primase -> extends each primer
pol epsilon - synthesizes leading strand
pol delta - synthesizes lagging strand
a specific sequence (replicator or origin) is bound by
initiator protein
initiator proteins
origin - CIS acting element
Initiator - TRANS
cis acting DNA element
a short DNA sequence that acts as a binding site for a protein that has an affinity for that specific sequence
prokaryotic initiator protein
DnaA - 9 bp
the open complex
DnaA-ATP-OriC-HU
HU
small basic histone like protein
control of initiation: binding of DnaA at oriC
DNA methylation
only methylated origins are functional
GATC sequence in oriC
N6 methylation
11 GATC within 245 bp in oriC
SeqA
binds hemimethylated DNA, preventing DnaA binding
DnaA- ADP
cannot destabilze A=T regions mainting the open complex, forming a closed complex
disenrangle the two daughter chromosomes
topoisomerase
MutS
recognizes mismatched base pair
MutS-MutL
scands bidirectionally
MutH
site specific endonuclease that cleaves unmethylated GATC sites
Mut S - L
recruits helicase II (UvrD). exonuclease degrades the newly replicated DNA
