Lecture 30: DNA Replication Flashcards

1
Q

DNA Polymerase

A

replicates DNA

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2
Q

how does DNA synth occur?

A

5’ to 3’

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3
Q

linkage of what to what for DNA sytnh

A

base-paried dNTP to the nascent strand
at 3’OH
realeases PPi, hydrolyze to 2Pi

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4
Q

what is in the replication fork?

A

leading and lagging strand (associated primase and ligase)
helicase
topoisomerase
signle strand binding protein (SSB)

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5
Q

how does lagging strand work?

A

discontinuous synth
so RNA primers are made by primase to make Okazaki fragments
Okazaki fragments are linked by liagase

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6
Q

How do we avoid errors in DNA so well

A

polymerizing reaction
proofreading activity
DNA repair mechanisms to remove mismatched nucleotides

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7
Q

replisome

A

both strands move in same direction.
its a loop
like trombone model

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8
Q

why mutate?

A

we need them! so that not everyone dies! mutations can equal survival

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9
Q

replisome in the replication fork

A

protein complex
travlels along DNA to unwind it
synths leading and laggings strands (okazaki fragments)

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10
Q

topoisomerase

A

prevents DNA from getting totally kinked
it cuts so that the DNA can swivvle
then seals it back up

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11
Q

LISTEN TO SLIDE 3 for VIDEO

A

LISTEN TO SLIDE 3 for VIDEO

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12
Q

Messelson Stahl Experiment

A

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???

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13
Q

conservative model

A

one brand new strand

one original strand

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14
Q

Messelson Stahl Experiment (the experiment)

A

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 `

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15
Q

see slide 5 to make sure you understand the bands

A

see slide 5 to make sure you understand the bands

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16
Q

so what did Messelson Stahl Experiment find?

A

that its the semi-conservative model

17
Q

discovery of lagging strand synthesis

A

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

18
Q

why are there only short Okazaki fragments in bacteria with ligase mutations

A

because they’re not jointed together

19
Q

DNA Polymerases

A

all are template and primer depedent for DNA synth

see table on slide 8

20
Q

DNA Polymerase 1

A

repair enzyme

works on lagging strand

21
Q

DNA Polymerase 3

A

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

22
Q

Polymerization: Process

A

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

23
Q

one phosphate bond is broken, and one new bond is formed, so what delta G component helps “pull” DNA synth to the right?

A

hydrolysis of pyrophosphate

24
Q

How does it know which nucleotide to put in? Fitting

A
  1. 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

25
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
26
Two ways to avoid the wrong base
1. see if it fits | 2. if wrong tautemer accidentally gets put in, enzyme goes back and fixes it
27
what direction is profreading in all three pols
3' to 5' | makes sense because it removes whatever it just put in
28
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
29
When would DNA Pol 1 need to remove RNA?
okazaki fragments | during lagging strand synth
30
What enzyme repairs the nick left behind?
ligase
31
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
32
DNA Pol 3 dimer
catalyzes synth of leading and lagging strand | LOOP lagging around so that its in the right orientation
33
DNA Helicase
opens rep fork so DNA Pol 3 can synth nascent DNA on both strands topoisomerase cleaves supercoils
34
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
35
Replication Initiation and termination in E coli
slide 17 and 18