Unit 3: DNA Replication

Question 1

Why is our DNA semi-conservitive?

Answer 1

Each DNA strand serves as a template strand to produce a new complementary one

Question 2

What do the 2 Mg2+ ions held in Pol do?

Answer 2

1st Mg2+: Deprotonates the primer 3’-OH hydroxly to 3’-O- nucleophile
2nd Mg2+: Binds PPi and facilitates its departure

Question 3

What are the DNA polymerase reaction steps?

Answer 3

1. Synthesis of the RNA primer
2. Incorporation of the 1st nucleotide
3. Translocation of polymerase
4. Incorporation of 2nd nucleotide

Question 4

How does the DNA polymerase know which dNTP to incorporate?

Answer 4

Binding of a dNTP causes a conformational change of Pol Active site in closed form

Question 5

What kind of proofreading activity does DNA polymerase have?

Answer 5

A 3’ - 5’ exonuclease to get rid of incorrect nucleotides added

Question 6

What does Pol III contain?

Answer 6

Pol III core x3
Sliding Clamp x3
Clamp loader

Question 7

What is the role of the Pol III core?

Answer 7

Add the nucleotides & proofreading

Question 8

What is the role of the Pol III beta sliding clamp?

Answer 8

Increase speed of replication by not letting polymerase fall off of DNA

Question 9

What is the role of the clamp loader?

Answer 9

Loads beta sliding clamp onto DNA

Question 10

What does the DNA helicase do? Where is it located?

Answer 10

DNA “unzipping”
In bacteria: located on lagging strand
In eukaryotes: located on leading strand

Question 11

What does the Topoisomerase do?

Answer 11

Works ahead of the Helicase to untwist DNA

Question 12

What does the Primase do?

Answer 12

RNA polymerase that adds RNA primer as the first nucleotides

Question 13

What does a ligase do?

Answer 13

Seals DNA nicks, “glues the Okazaki fragments together”

Question 14

What is a nick in DNA?

Answer 14

A place where the phosphdiestar bond is not made between fragments

Question 15

What occurs during nick translation?

Answer 15

The Pol I exonuclease works 5’ to 3’ to remove RNA primers and replace with DNA

Question 16

What happens after Pol I is done?

Answer 16

After Pol I, beta clamp stays on to recruit ligase

Question 17

How is the beta clamp removed?

Answer 17

Delta subunit of clamp loader

Question 18

What is 1 difference between bacteria and eukaryotic DNA replication structures?

Answer 18

Eukaryotic replisome has 2 DNA polymerases (1 for each strand) as opposed to 3 so it is much slower

Question 19

How does replication initiation begin in bacteria?

Answer 19

A long bp sequence with repeating bp sites is recognized by DnaA and the DnaA binds to these R-repeats. An A&T rich repeat unwinds the DNA

Question 20

What happens after the A-T region is destabilized?

Answer 20

DnaC loads helicase onto each strand

Question 21

What prevents the origin from initiating again?

Answer 21

DAM sites are methylated in old strands, but new strands are not allowing SeqA to bind to the unmethylated strand and prevent DnaA from binding to origin.

Question 22

How do eukaryotes orchestrate chromosomal replication? What are the steps?

Answer 22

Through cell cycle phases

• # 1 G1: Assembly of pre-replication complexes
• # 2 S: Degradation of preRC
• # 3 S: Replication
• # 4: S-G2-M: no pre-RC assembly = no initiation