Lecture 30: DNA Replication

Question 1

DNA Polymerase

Answer 1

replicates DNA

Question 2

how does DNA synth occur?

Answer 2

5’ to 3’

Question 3

linkage of what to what for DNA sytnh

Answer 3

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

Question 4

what is in the replication fork?

Answer 4

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

Question 5

how does lagging strand work?

Answer 5

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

Question 6

How do we avoid errors in DNA so well

Answer 6

polymerizing reaction
proofreading activity
DNA repair mechanisms to remove mismatched nucleotides

Question 7

replisome

Answer 7

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

Question 8

why mutate?

Answer 8

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

Question 9

replisome in the replication fork

Answer 9

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

Question 10

topoisomerase

Answer 10

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

Question 11

LISTEN TO SLIDE 3 for VIDEO

Answer 11

LISTEN TO SLIDE 3 for VIDEO

Question 12

Messelson Stahl Experiment

Answer 12

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???

Question 13

conservative model

Answer 13

one brand new strand

one original strand

Question 14

Messelson Stahl Experiment (the experiment)

Answer 14

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 `

Question 15

see slide 5 to make sure you understand the bands

Answer 15

see slide 5 to make sure you understand the bands

Question 16

so what did Messelson Stahl Experiment find?

Answer 16

that its the semi-conservative model

Question 17

discovery of lagging strand synthesis

Answer 17

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

Question 18

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

Answer 18

because they’re not jointed together

Question 19

DNA Polymerases

Answer 19

all are template and primer depedent for DNA synth

see table on slide 8

Question 20

DNA Polymerase 1

Answer 20

repair enzyme

works on lagging strand

Question 21

DNA Polymerase 3

Answer 21

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

Question 22

Polymerization: Process

Answer 22

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

Question 23

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

Answer 23

hydrolysis of pyrophosphate

Question 24

How does it know which nucleotide to put in? Fitting

Answer 24

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

Question 25

what about tautomeric forms of nucleotide bases?

Answer 25

sometimes wrong base can get in this way

when it is the normal form, the enzyme goes backwards and cuts off the wrong nucleotide

Question 26

Two ways to avoid the wrong base

Answer 26

1. see if it fits

2. if wrong tautemer accidentally gets put in, enzyme goes back and fixes it

Question 27

what direction is profreading in all three pols

Answer 27

3’ to 5’

makes sense because it removes whatever it just put in

Question 28

which pol has 5’ to 3’ exonuclease activity. why?

Answer 28

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

Question 29

When would DNA Pol 1 need to remove RNA?

Answer 29

okazaki fragments

during lagging strand synth

Question 30

What enzyme repairs the nick left behind?

Answer 30

ligase

Question 31

DNA Pol 3

Answer 31

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

Question 32

DNA Pol 3 dimer

Answer 32

catalyzes synth of leading and lagging strand

LOOP lagging around so that its in the right orientation

Question 33

DNA Helicase

Answer 33

opens rep fork so DNA Pol 3 can synth nascent DNA on both strands
topoisomerase cleaves supercoils

Question 34

Trombone Model

Answer 34

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

Question 35

Replication Initiation and termination in E coli

Answer 35

slide 17 and 18