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BIOC2004 > 7 - DNA Polymerases > Flashcards

7 - DNA Polymerases Flashcards

1
Q

How are prokaryotic DNA polymerases classed?

A

Polymerase type I-V

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2
Q

What is DNA poly I responsible for?

A

Primer Removal

DNA Repair

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3
Q

What is DNA poly II responsible for?

A

DNA Repair

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4
Q

What is DNA poly III responsible for?

A

Chromosome Repair

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5
Q

What is DNA poly IV responsible for?

A

DNA Repair

Translesion Synthesis

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6
Q

What is DNA poly V responsible for?

A

Translesion Synthesis

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7
Q

How are DNA polymerases classed across eukaryotes and prokaryotes?

A

Family A-D, X, Y and RT

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8
Q

What are family A DNA polymerases responsible for?

A

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

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9
Q

What are family B DNA polymerases responsible for?

A

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

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10
Q

What are family C DNA polymerases responsible for?

A

Replication
Prokaryotes only
Includes Pol II in prokaryotes

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11
Q

What are family D DNA polymerases responsible for?

A

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

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12
Q

What are family X DNA polymerases responsible for?

A

Replication
Base Excision repair
Eukaryotic only
Contain lyase domain

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13
Q

What are family Y DNA polymerases responsible for?

A

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

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14
Q

What are RT family DNA polymerases responsible for?

A

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

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15
Q

What is the error rate of DNA polymerases?

A

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

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16
Q

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

A

No.

17
Q

How is it thought that DNA polymerases increase fidelity geometrically?

A

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

18
Q

How is it thought that DNA polymerases increase fidelity energetically?

A

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

19
Q

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

A

Klentaq1

20
Q

What is the first step in the DNA polymerisation cycle?

A

DNA polymerase binding to the primer/template strand dimer.

21
Q

What is the second step in the DNA polymerisation cycle?

A

Binding the dNTP by the DNA polymerase.

22
Q

What is the third step in the DNA polymerisation cycle?

A

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

23
Q

What is the fourth step in the DNA polymerisation cycle?

A

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

24
Q

What is the fifth step in the DNA polymerisation cycle?

A

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

25
Q

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

A

Distributive and Processive

26
Q

What is involved in the distributive step?

A

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

27
Q

What is involved in the precessive step?

A

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

28
Q

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

A

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

29
Q

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

A

Kpol, rate contant of the polymerisation reaction.

30
Q

What is Klentaq1?

A

A Klenow fragment of Taq polymerase.

31
Q

What is the general structure of Klentaq1?

A

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

32
Q

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

A

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

33
Q

How was the structure of the dNTP bound enzyme elucidated?

A

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

34
Q

Where does the dNTP bind to the DNA polymerase?

A

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.

35
Q

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

A

The ternary complex.

36
Q

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

A

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

37
Q

What structural change characterises the ternary complex?

A

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.

38
Q

At what stage is the fidelity of the enzyme determined?

A

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

39
Q

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

A

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.

BIOC2004 (10 decks)

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