Lecture 30: DNA Replication Flashcards
DNA Polymerase
replicates DNA
how does DNA synth occur?
5’ to 3’
linkage of what to what for DNA sytnh
base-paried dNTP to the nascent strand
at 3’OH
realeases PPi, hydrolyze to 2Pi
what is in the replication fork?
leading and lagging strand (associated primase and ligase)
helicase
topoisomerase
signle strand binding protein (SSB)
how does lagging strand work?
discontinuous synth
so RNA primers are made by primase to make Okazaki fragments
Okazaki fragments are linked by liagase
How do we avoid errors in DNA so well
polymerizing reaction
proofreading activity
DNA repair mechanisms to remove mismatched nucleotides
replisome
both strands move in same direction.
its a loop
like trombone model
why mutate?
we need them! so that not everyone dies! mutations can equal survival
replisome in the replication fork
protein complex
travlels along DNA to unwind it
synths leading and laggings strands (okazaki fragments)
topoisomerase
prevents DNA from getting totally kinked
it cuts so that the DNA can swivvle
then seals it back up
LISTEN TO SLIDE 3 for VIDEO
LISTEN TO SLIDE 3 for VIDEO
Messelson Stahl Experiment
To answer: How do you replcate?
need to test which mechanism is used to make the complimentary nascent strand from the template strand.
mixture
1 strand is template for one strand, other is template for other
conservative model
WHICH ONE???
conservative model
one brand new strand
one original strand
Messelson Stahl Experiment (the experiment)
grew “heavy” bacteria, isolate DNA
looked at the made DNA after 1 and 2 generations
get 2 hybrids (with one heavy strand each), and 2 completely new strands `
see slide 5 to make sure you understand the bands
see slide 5 to make sure you understand the bands
so what did Messelson Stahl Experiment find?
that its the semi-conservative model
discovery of lagging strand synthesis
radioactively labeled bacteria
looked at the short over okazaki fragments over time
after not too long, the okazaki fragments appeared to be linking b/c the fragments got longer
did it again in cells without ligase, proved that ligase was really needed
why are there only short Okazaki fragments in bacteria with ligase mutations
because they’re not jointed together
DNA Polymerases
all are template and primer depedent for DNA synth
see table on slide 8
DNA Polymerase 1
repair enzyme
works on lagging strand
DNA Polymerase 3
works at DNA at the replication fork
has highest polymeration rate (up to 1000/sec)
highest processivity value
can polymerize half a mill nucleotides before it falls off
Polymerization: Process
3’ OH of nascent strand attacks alpha phosphate on incmojng dNTP (new base)
nucleotide addition and release of PPi result
cleavage of pyrophosphate drives reaction
one phosphate bond is broken, and one new bond is formed, so what delta G component helps “pull” DNA synth to the right?
hydrolysis of pyrophosphate
How does it know which nucleotide to put in? Fitting
- nucleotide has to fit into active site
this is due to hydrogen bonding between nucleotide bases on the strands
incoming strands must have the right geometry, other wise it dissociates
what about tautomeric forms of nucleotide bases?
sometimes wrong base can get in this way
when it is the normal form, the enzyme goes backwards and cuts off the wrong nucleotide
Two ways to avoid the wrong base
- see if it fits
2. if wrong tautemer accidentally gets put in, enzyme goes back and fixes it
what direction is profreading in all three pols
3’ to 5’
makes sense because it removes whatever it just put in
which pol has 5’ to 3’ exonuclease activity. why?
DNA Pol 1
used for removal and repair of DNA strands
causes a “nick”, removes (5’ to 3’) the DNA or RNA in front, synths new strand behind it
nick is covalently linked, theres a new nick, ligase seals it
When would DNA Pol 1 need to remove RNA?
okazaki fragments
during lagging strand synth
What enzyme repairs the nick left behind?
ligase
DNA Pol 3
catalyzes synth of nascent DNA on leading and lagging strands at replication fork
beta clamp is one of most important tupes
stabalize alpha subunit to max procesivity
DNA Pol 3 dimer
catalyzes synth of leading and lagging strand
LOOP lagging around so that its in the right orientation
DNA Helicase
opens rep fork so DNA Pol 3 can synth nascent DNA on both strands
topoisomerase cleaves supercoils
Trombone Model
coordination of leading and lagging strands
by RNA priming and synth of Okazaki frgas
loop formed, fed into DNA Pol 3, synth Okazaki
Beta clamp releases once this is complete so new loop can be formed
Replication Initiation and termination in E coli
slide 17 and 18
