7 - DNA Polymerases

Question 1

How are prokaryotic DNA polymerases classed?

Answer 1

Polymerase type I-V

Question 2

What is DNA poly I responsible for?

Answer 2

Primer Removal

DNA Repair

Question 3

What is DNA poly II responsible for?

Answer 3

DNA Repair

Question 4

What is DNA poly III responsible for?

Answer 4

Chromosome Repair

Question 5

What is DNA poly IV responsible for?

Answer 5

DNA Repair

Translesion Synthesis

Question 6

What is DNA poly V responsible for?

Answer 6

Translesion Synthesis

Question 7

How are DNA polymerases classed across eukaryotes and prokaryotes?

Answer 7

Family A-D, X, Y and RT

Question 8

What are family A DNA polymerases responsible for?

Answer 8

Replication/repair
5’-3’ and 3’-5’ proofreading
Found in eukaryotes and prokaryotes
Inc. Pol I in prokaryotes

Question 9

What are family B DNA polymerases responsible for?

Answer 9

Replication/repair
3’-5’ proofreading
Found in eukaryotes and prokaryotes
Inc. Pol II in prokaryotes

Question 10

What are family C DNA polymerases responsible for?

Answer 10

Replication
Prokaryotes only
Includes Pol II in prokaryotes

Question 11

What are family D DNA polymerases responsible for?

Answer 11

V. poorly studied
Thought to be replicative
Found only in euryarchaeota (type of archaea)

Question 12

What are family X DNA polymerases responsible for?

Answer 12

Replication
Base Excision repair
Eukaryotic only
Contain lyase domain

Question 13

What are family Y DNA polymerases responsible for?

Answer 13

Replication
Translesion Repair
Eukaryotic and Prokaryotic
Inc. Pol IV and V in prokaryotes.

Question 14

What are RT family DNA polymerases responsible for?

Answer 14

Reverse transcription
Found in eukaryotes, viruses, retroviruses
Telomerase action
Monomeric unless dimerised with RNaseH

Question 15

What is the error rate of DNA polymerases?

Answer 15

1 error every 10⁹-10¹⁰ bases - very accurate/high fidelity

Question 16

Is watson-crick complementarity sufficient to explain the high fidelity of DNA polymerases?

Answer 16

No.

Question 17

How is it thought that DNA polymerases increase fidelity geometrically?

Answer 17

By responding to the shape of the base on the template stand and altering its complementarity to favour the correct base.

Question 18

How is it thought that DNA polymerases increase fidelity energetically?

Answer 18

By amplifying the favourable difference in free energy between correct and incorrect Watson-Crick base pairing by exclusion of water from the active site.

Question 19

Structural analysis of which enzyme has elucidated the binding/catalysis cycle of DNA polymerases?

Answer 19

Klentaq1

Question 20

What is the first step in the DNA polymerisation cycle?

Answer 20

DNA polymerase binding to the primer/template strand dimer.

Question 21

What is the second step in the DNA polymerisation cycle?

Answer 21

Binding the dNTP by the DNA polymerase.

Question 22

What is the third step in the DNA polymerisation cycle?

Answer 22

Incorporation of the base into the strand, accompanied by a change in the conformation of the DNA polymerase.

Question 23

What is the fourth step in the DNA polymerisation cycle?

Answer 23

Sealing the phosphodiester backbone, leaving a diphosphate bound to the DNA polymerase. This is accompanied by the enzyme reverting back to its normal conformation.

Question 24

What is the fifth step in the DNA polymerisation cycle?

Answer 24

Dissociation of the diphosphate from the DNA polymerase and movement of the enzyme down the strand.

Question 25

What are names of the two possible endings to each run of the DNA polymerisation cycle?

Answer 25

Distributive and Processive

Question 26

What is involved in the distributive step?

Answer 26

Dissociation of the DNA polymerase if replication is finished or translesion synthesis is required.

Question 27

What is involved in the precessive step?

Answer 27

The DNA polymerase incorporating a new dNTP into the newly positioned enzyme:template:primer+n complex - repeating step two essentially.

Question 28

What is the rate-limiting step in the DNA polymerisation cycle?

Answer 28

Step 3, incorporation of the new base due to change in enzyme conformation.

Question 29

What does the rate-limiting step in DNA polymerisation control?

Answer 29

Kpol, rate contant of the polymerisation reaction.

Question 30

What is Klentaq1?

Answer 30

A Klenow fragment of Taq polymerase.

Question 31

What is the general structure of Klentaq1?

Answer 31

It is composed of three active domains; Fingers, Palm and Thumb, as well as a vestigial 3'-5' exonuclease domain.

Question 32

How was the structure of the Enzyme:p/t complex found?

Answer 32

By imaging the enzyme while bound to DNA in a dNTP deficient environment, preventing step 2 from occurring.

Question 33

How was the structure of the dNTP bound enzyme elucidated?

Answer 33

By imaging the DNA when bound to the dNTP only - no DNA strand present, hence preventing the spontaneous step 3.

Question 34

Where does the dNTP bind to the DNA polymerase?

Answer 34

To the O-helix on the fingers domain. This is far too distant from where the strand binds to be incorporated, hence the need to change the enzyme conformation.

Question 35

What is the name of the DNA polymerase conformation produced during base incorporporation?

Answer 35

The ternary complex.

Question 36

How was the E':p/t:dNTP complex imaged with the enzyme in its ternary complex state imaged?

Answer 36

Using chain termination - incubation with a supply of artificial bases that prevent further polymerisation, halting the complex when it incorporates the base.

Question 37

What structural change characterises the ternary complex?

Answer 37

Movement of the dNTP binding O-helix from the open to close position, slotting the base into position. There are also other smaller shifts in position of the rest of the enzyme, especially in the fingers.

Question 38

At what stage is the fidelity of the enzyme determined?

Answer 38

During base incorporation when the enzyme changes to its ternary conformation, slotting the base into position to either be rejected or accepted.

Question 39

What is the general mechanism of phosphodiester backbone sealing in step four?

Answer 39

The 3' hydroxyl of the primer end makes a nucleophilic attack on the gamma phosphate of the dNTP, transferring its hydrogen onto the enzyme and causing the diphosphate oxygen to dissociate.