Exam 2 (Ppt 2)

Question 1

What are the two substrates for DNA polymerase

Answer 1

1) DNTP
2) Primer:Template junction*

*3’ end that’s base-paired

Question 2

What is the difference in DNA residues

Answer 2

2’ deoxygenated sugar

Question 3

in what direction is DNA made

Answer 3

Made 5’ -> 3’
read 3’ -> 5’

*3’ exonuclease activity is opposite

Question 4

What are the two enzymatic steps of replication (include enzyme and what it does for the DNA replication process

Answer 4

DNA polymerase: performs catalysis at the primer:template jxn joining the incoming base pair to the template (3’ OH on the template bonds to the 5’ on the incoming base.

pyrophosphatase: removes 2 of the phosphate residues on the incoming base if it is correctly paired to pay for the reaction. Makes it more favorable to proceed forward and continue replication

Question 5

Why is the pyrophosphatase reaction important?

Answer 5

“Only when a correct base pair is formed, pyrophosphatase began to cut βγ off. Incorrect match will not continue..” Prof Ye

Question 6

What are the parts of DNA polymerase and what are their fxns?

Answer 6

Thumb: Positioning
Fingers: catalysis
Palm: catalysis

Question 7

What is the fxn of the thumb region of DNA polymerase?

Answer 7

Thumb:

1) Maintain the correct position of the primer and the active site
2) Maintain a strong association between the DNA polymerase and its substrate

Question 8

What purpose do metal ions serve for the DNA replication process?

Answer 8

“Ion A: Mostly interacts with the 3’ OH group (allowing the O- to attack the phosphate group)Ion B: Mostly interacts with the O- s attached to the triphosphate – neutralizing its negative charge”

• Prof Ye

Question 9

Degree of processivity

Answer 9

“the average number of nucleotides added each time the enzyme binds a primer:template junction.” Prof Ye

Question 10

what forces are allowing DNA to be replicated?

Answer 10

Stacking forces
metal ion interactions (ionic?)
hydrogen bonds
van der waal forces

Question 11

What happens in the palm region?

Answer 11

The template is bent at a 90 deg angle then the incoming nucleotide binds the template base. The DNA polymerase then moves toward the 5’ end of template DNA one base pair at a time.

Question 12

nonprocessive vs processive

Answer 12

Non-processive vs highly processive – It’s a matter of how many bp can be added before the enzyme “falls off” the DNA molecule.

Processive: many dNTPs added

nonprocessive: one dNTP added

Question 13

Tautomeric pairing

Answer 13

mismatched pairing due to tautomeric conformation. Instead of keto-guanine pairing to cysteine, its enol form pairs with thymidine

“makes PROOFREADING EXONUCLEASE necessary!

Question 14

Explain DNA polymerase’s proofreading activity

Answer 14

=3’ exonuclease

• Degrades DNA from 3’ end
• removes mismatched nucleotide but it can only touch the last nucleotide laid down.

so if dATP was mismatched and then somehow dTTP was mismatched, then only dTTP could be corrected (?)

Question 15

What is the basic steps of DNA proofreading. In what DNA polymerase region does this activity occur?

Answer 15

slow DNA synthesis –> nucleotide removal –> synthesis continues

exonuclease active site located in the finger region near the palm catalytic site

Question 16

True or False: Proofreading can improve accuracy to 10^-10

Answer 16

False; it can improve accuracy to 10 ^-7

*additional mech. must be at play

Question 17

DNA polymerase does what?

Answer 17

“adds nucleotides to an existing primer:template junction” Prof Ye

Question 18

Where does the energy for DNA replication come from?

Answer 18

“From the HYDROLYSIS of the incoming dNTPs”

Prof Ye

Question 19

DNA pol has a ___-___ structure that contains _ to _ pol activity as well as _ ____ _____

Answer 19

DNA polhave a THREE-DOMAIN STRUCTURE that contains 5′-to-3′ pol activity as well as 3′-to-5′exonuclease activity.

Question 20

What in the active site catalyzes the rxn?

Answer 20

Two metal ions

Question 21

What happens to incorrectly paired dNTPs?

Answer 21

They are excluded from the growing chain because they are poorly positioned in the active site.

Question 22

DNA needs to be ____ into a ____ ____ in order to proceed with replication

Answer 22

needs to be UNWOUND into single strand in order to proceed with replication .

Question 23

How is DNA unwound?

Answer 23

DNA helicase:

A hexamer ring that slides down one strand 5’-3’ unwinding the DNA

Question 24

What two processes are occuring at the replication site?

Answer 24

Replication of the..

1) Leading strand
2) Lagging strand

*lagging strand => okazaki fragments

Question 25

How are okazaki fragments linked together?

Answer 25

RNAse H

=RNA:DNA hybrid

Question 26

RNAse H

Answer 26

removes most primer nts (along with 5’ exonuclease to finish it of) then DNA polymerase extends the strand at the primer:template jxn. FInally DNA ligase fills in the nick

Question 27

Does RNAse remove all RNA nts?

Answer 27

No, but most.

5’ exonuclease helps out with the last RNA nt

Question 28

What other proteins fxn at the replication fork?

Answer 28

• Topoisomerases
• SSB
• Primase
• DNA helicase

Question 29

Primase

Answer 29

“an enzyme that synthesizes short RNA sequences called primers.” -Prof Ye

*starting point of synthesis

Question 30

SSB

Answer 30

=single-strand binding protein

• coats unwound unstable ssDNA QUICK!
• sequence INDEPENDENT
• Binds DNA in cooperative fashion

Question 31

what does it mean when SSBs bind DNA in a _____ _____?

Answer 31

Cooperative Fashion

-“ 1 binds, and makes it much more likely that others will bind neighboring nts”

Question 32

DNA poly III vs I

Answer 32

III

• main one used for most replication,
• found in huge complex
• very highly processive – has 3’ exo proofreading capability.

I
– Specialized for removal of RNA PRIMERS
-has a 5’ exonuclease activity.

Question 33

What strand does DNA poly 1 mostly fxn on?

Answer 33

lagging strand

Question 34

Prokaryote DNA polymerases

Answer 34

I : RNA primer removal + DNA repair
II : Chromos. replic.
III: Chromos. replic. (Holoenzyme)

Question 35

Eukaryotic DNA polymerases

Answer 35

a : primer synthesis during DNA replic.
d : lagging strand DNA synthesis + nucleotide and base excision repair
e: leading strand synthesis + nt and base excision repair

Question 36

Polymerase Switch

Answer 36

Replication involves switching from LESS PROCESSIVE priming enzymes to HIGHLY PROCESSIVE polymerization enzymes.

Question 37

Polymerase Switch in Eukaryotes

Answer 37

primase →pol a→ pol d / pol e

Question 38

DNA Pol processivity is further _____ by the _____ _____

Answer 38

DNA pol processivity is further INCREASED by the SLIDING CLAMP

Question 39

Sliding clamp of E.coli is a ____ of __ _____

Answer 39

E. coli -dimer of b protein
Euk - Trimer of PCNA

PCNA: proliferating cell nuclear antigen

Question 40

How does a sliding clamp attach to DNA

Answer 40

-loaded onto, and removed from, DNA by SLIDING CLAMP LOADER
-(ATP driven to open clamp)
-occurs by CONFORMATIONAL CHANGE @ ssDNA:dsDNA jxns
-moves in the direction of replic.
-

Question 41

Why does DNA pol III have HIGH processivity

Answer 41

DNA “sliding clamp” helps keep the DNA poly from “falling off” the ssDNA

w/ clamp: 20-100 bp
w/o ^ ^ : 1000/s

Question 42

DNA IS DOUBLE HELIX, so how does polymerases perform replication simultaneously on individual strand that are uncoiled at the same time?

Answer 42

Pol II Holoenzyme

• -> 3 Pol III cores, sliding clamp, clamp loader, tau proteins
• -> replicates LEADING and LAGGING strands