Lecture 3

Question 1

DNA polymerase error rate in vitro

Answer 1

1/10^4 - 10^5

Tautomeric base conformation during addition

Question 2

Proofreading mechanism

Answer 2

Same polymerase has different active site
Exonuclease that unravels 3-5 direction

With base selection : accuracy is 1: 10^6-8

Question 3

Most common dna polymerase in E Coli
Rate of addition

Answer 3

DNA polymerase I (5 total types)

600 bp/min in vitro

But true measurement is 60,000 bp/min

Question 4

Klenow fragment

Answer 4

Protein fragment of DNA polymerase I
Mild protease treatment = exonuclease segment removed which removes primers

Question 5

DNA polymerase I subunits

Answer 5

9 total
Alpha = polymerization
E = proofreading

3x Alpha + e linked together by clamp loading complex

Question 6

DNA polymerase III subunits

Answer 6

16 total
Much greater processivity than dna Pol I

Question 7

Helicases

Answer 7

Strand separation
Use atp

Question 8

Primase

Answer 8

RNA primer synthesizer

Question 9

Dna ligase

Answer 9

Seals nick
Uses atp

Question 10

Functions of Pol I

Answer 10

5-3 exonuclease = nick translation, rna primer removal

3-5 exonuclease - proof reads step by step during addition - induced kinetic pause if mismatch
5-3 polymerase

Question 11

Pol I processivity and rate

Answer 11

20-100 nt

20nt/second

Question 12

Pol III processivity and rate

Answer 12

3-5 exonuclease activity

> 500,000 nucleotides

250-1000nt/s

Question 13

Sequences in Ori C

Answer 13

DUE = at rich
R binding sites
I sites = dna must be complexed w atp to bind

Question 14

DnaA protein

Answer 14

Binds to ori C and starts strand separation

Question 15

DnaB

Answer 15

Primary helicase during replication
5-3

Question 16

What is the only regulated phase of the cell cycle?

Answer 16

Initiation

Question 17

Dam methylase

Answer 17

Methylates n 6 position of adenine

Right after replication, new strand yet to be methylated

All neeeds to be methylated for new round of initiation = DnaA binding

Question 18

Length of rna primer

Answer 18

10-60bps

Question 19

What is DnaB physically associated with during replication

Answer 19

Lagging strand dna POL III

Leading strand dna POL III

Clamp loading complex

Sometimes primase

Question 20

DnaA analog

Answer 20

ORC

Question 21

DnaB analog

Answer 21

MCM2-7

Question 22

Additional initiation reagents

Answer 22

Cdc6
Ctd1
Cdc45
GINS

Question 23

DNA polymerase e

Answer 23

Analog to DNA polymerase III

Question 24

DNA polymerase a

Answer 24

Analog to primase / pol I
Adds rna primers and extends them
No proofreading

Question 25

PCNA

Answer 25

Proliferating cell nuclear agent
Large amounts in proliferating cells
Analog of beta subunit Clamp

Question 26

Clamp loader analog

Answer 26

RFC

Question 27

SSB Analog

Answer 27

RPA

Question 28

Lagging strand in eukaryotes

Answer 28

Polymerase delta

Question 29

Acyclovir

Answer 29

Compound to inhibit herpes viral polymerase

Incomplete ribose ring
No 3’ OH for nucleophilic attack

Question 30

Dogma for information transfer

Answer 30

DNA to rna to protein

Also

RNA to dna

Question 31

Reverse transcriptase

Answer 31

RNA dependent dna polymerase

Uses rna as template
Degrades rna part of double strand product
Replaces with dna

Each have different active site

No proof reading

Retro viruses

Question 32

Retroviruses have 3 genes

Answer 32

Gag
Pol
End

Ends have LTR to facilitate integration

Question 33

tRNA primer

Answer 33

Needs to already be on dna from earlier infection

Question 34

Transposons

Answer 34

Transpose positions via rna intermediate