432 Lecture 25

Question 1

What are the three fundamental rules of replication?

Answer 1

1) Replication is semi-conservative 2) Replication begins at an origin and proceeds, bidirectionally 3) Synthesis of new DNA occurs in the 5’ to 3’ direction

Question 2

Semi-conservative model

Answer 2

1) Strand Separation 2) New complementary strands synthesized using each separated strand as a template.

Question 3

Requirements for DNA polymerase Activity (4)

Answer 3

1) Template (Basis for heredity) 2) dNTPs (building blocks) 3) Mg2+ ( promotes reactions) 4) Primer ( anneals to template DNA and is extended w/o DNA pol. can’t start).

Question 4

What are the three functions of DNA pol I?

Answer 4

1) 5’ to 3’ DNA polymerizing activity 2) 5’ to 3’ exonuclease activity 3) 3’ to 5’ exonuclease activity.

Question 5

Mechanism of DNA polymerases (Active site)

Answer 5

1) Two conserved regions of Asp 2) Mg2+ causes 3’ OH to become a nucleophile and negates the charges of the pyrophosphate on the incoming nucleotide 3) O attacks phosphate (a phosphate) and pyrophosphate leaving drives reaction.

Question 6

Mechanism of DNA polymerase

Answer 6

1) Sampling of nucleotides 2) nucleotide base pairs with complementary base 3) removal of H from 3’ OH 4) nucleophillic attack of 3’ OH to a phosphate group of incoming nucleophile 5) displacement of pyrophosphate drives the reaction.

Question 7

Palm domain

Answer 7

1) b-sheet 2) primary elements of catalytic site 3) binds two Mg2+ ions 4) Monitors accuracy.

Question 8

Fingers

Answer 8

1) bind to incoming dNTP 2) Fold over if correct base pair is made. (Lys, arg, and tyr rich) the tyrosine through Van der walls interactions places incoming base to growing chain.

Question 9

How many active sites does DNA polymerase have?

Answer 9

one active site. Useful for ribonucleotide discrimination and nucleotide discrimination

Question 10

How does DNA polymerase know an incorrect base pair has been made?

Answer 10

Distortions in the enzyme’s active site. Enzyme can add up to five bases before they can no longer fix the mistake. Additions are made at a slower rate.

Question 11

Net reaction of DNA polymerase

Answer 11

Based on the reaction between the separate sites of the active site of addition and the 3’ to 5’ exonuclease. Rate of nucleotide addition dominates the net reaction.

Question 12

Nick Translation

Answer 12

5’ to 3’ exonuclease activity; DNA repair and primer removal during replication; cleaves nucleotides in front of others; removes RNA primer

Question 13

Which polymerase has 5’ to 3’ exonuclease activity?

Answer 13

DNA polymerase I

Question 14

For DNA polymerase where and how do you start?

Answer 14

DNA-ATP binds to origins with help of helicase and SSB

Question 15

For DNA polymerase, how do you get a single stranded template?

Answer 15

Helicase

Question 16

For DNA polymerase, how are instabilities in the SS template strand overcome?

Answer 16

Single stranded binding protein; RPA (replication protein A in eukaryotes)

Question 17

Primase

Answer 17

Functions to form a primer

Question 18

For DNA polymerase, how is the lack of a 3’ to 5’ polymerase overcome?

Answer 18

Replication fork

Question 19

For DNA polymerase, how is DNA polymerases speed and processivity issues overcome

Answer 19

SSB and sliding clamp

Question 20

Where does replication in E.Coli occur?

Answer 20

At OriC. DnaA recognizes OriC; promotes the loading of DnaB (helicase) by DnaC (helicase loader)

Question 21

DnaB

Answer 21

helicase

Question 22

DnaC

Answer 22

helicase loader ATP dependent

Question 23

DnaA

Answer 23

ATP dependent, to really get replication going; binds to DNA, causes positive super coiling, opens up DUE region, causes ssDNA formation. Compensating negative supercoils melt the replication bubble at the end

Question 24

Hda

Answer 24

recruited by B clamp desposition; promotes ATP hydrolysis and progressively dissasembles DnaA

Question 25

Hemi-methylated DNA

Answer 25

During replication, 1 strand is methylated and another strand is not methylated (The new strand is NOT methylated)

Question 26

SeqA

Answer 26

Binds to OriC, only when DNA is hemimethylated, (new DNA is NOT methylated) tethers DNA to membrane of a cell, which does not allow DnaA to interact with OriC

Question 27

Dam methylase

Answer 27

adds methyl group to the adenine of a sequence 5’ GATC 3’ after SeqA dissasociates; causes both strands to be methylated. DNA can now interact with DnaA-ATP