MBG PART TWO: Lecture 2

Question 2

What doe DNA replication require?

Answer 2

• DNA dependent DNA polymerase
• Four deoxyribonucleoside triphosphates (dNTPs)
• A single stranded template of DNA to be copied
• An RNA primer (provides the 3’-OH end to initiate DNA synthesis by DNA
  polymerase)

Question 3

What direction is DNA synthesized in?

Answer 3

5’ to 3’ direction

Question 4

What are the characteristics of the newly synthesized DNA strand?

Answer 4

Complementary and Anti-parallel to parent strand.

Question 5

What are DNA strands held together by?

Answer 5

Hydrogen bonds between complementary bases

Question 6

What happens since DNA is antiparallel and synthesis proceeds in the 5’ to 3’ direction?

Answer 6

DNA replication must proceed n opposite directions on each strand.

Question 7

What happens on the leading strand?

Answer 7

DNA synthesis is continuous (same direction as unwinding)

Question 8

What happens on the lagging strand?

Answer 8

DNA Synthesis is discontinuous (opposite direction as unwinding)

Question 9

What does the lagging strand create?

Answer 9

Okazaki fragments

Question 10

What is the Eukaryote size of Okazaki fragments?

Answer 10

0.1 - 0.2kb

Question 11

Where does all DNA replication start?

Answer 11

At an origin (Note the differences in origin between Theta, Rolling Stone and Linear Eukaryotic)

Question 12

For prokaryotic DNA where does all replication begin at?

Answer 12

An “origin”

Question 13

What is the replication region called for E.coli?

Answer 13

OriC

Question 14

What is the Tandem 13-mer sequences?

Answer 14

13 base pairs that houses the AT-rich region

Question 15

What is the Four 9-mer initiation protein binding sites?

Answer 15

The 4-DnaA binding sites

Question 16

What is the function of the DnaA proteins?

Answer 16

They are initiator proteins that bind to oriC (replication origin) and cause a short stretch of DNA to unwind

Question 17

What are the first three steps involved in Prokaryotic DNA replication?

Answer 17

1.) Initiator proteins (DnaA) bind to orC (origin of replication)

2.) They unwind a small strand of DNA

3.) The unwinding allows helices and Single Stranded Binding Proteins to attach to the single stranded DNA

Question 18

Where does Helicase only bind to?

Answer 18

Single Stranded DNA (this is unique to prokaryotes) and disrupts the hydrogen bonding between complementary pairs

Question 19

What is DNA helicase (prokaryote)?

Answer 19

Unwinds the DNA in the 5’ to 3’ direction: it travels only on the lagging strand ahead of the replication machinery.

Question 20

What is Single-Strand Binding Proteins?

Answer 20

Protect and keep DNA single stranded (avoiding hairpin)

Question 21

What is DNA gyrase?

Answer 21

A topoisomerase that makes double stranded breaks in the DNA to relieve the torsional strain that builds up from helicase.

Question 22

What are the second three steps involved in prokaryotic DNA replication?

Answer 22

1.) DNA helicase binds to the lagging strand template at each replication fork and moves in the 5’ to 3’ direction, breaking hydrogen bonds and moving the replication fork.

2.) SSBP stabilize the exposed single stranded DNA

3.) DNA gyrase relieves the strain ahead of the replication fork.

Question 23

What is Primase (RNA polymerase)?

Answer 23

Synthesizes a short RNA primer that provides the 3’ OH end for DNA polymerase to begin DNA synthesis

Question 24

Where does primate bind to initiate synthesis?

Answer 24

Helicase

Question 25

How does the replication fork proceed?

Answer 25

Bidirectionally

Question 26

What is DNA polymerase?

Answer 26

Elongates the new nucleotide strand by catalyzing DNA polymerization. Requires a primer.

Question 27

How many DNA polymerases are there in E.coli?

Answer 27

5

Question 28

DNA Pol III

Answer 28

Main DNA polymerase. Has 5’ to 3’ polymerase activity and 3’ to 5’ exoncuclease activity.

It can add a dNTP on the 3’ end but can also back up to remove a nucleotide that has been mis-incorporated.

Question 29

DNA Pol I

Answer 29

Similar to DNA Pol III but has 5’ to 3’ exonuclease activity

Question 30

What does DNA Pol I 5’ to 3’ exonuclease activity mean?

Answer 30

It can remove the RNA primer and replace it with DNA

Question 31

What happens at the leading strand?

Answer 31

DNA Pol III synthesizes continuously making a new strand

Question 32

What happens at the lagging strand?

Answer 32

DNA synthesis by polymerase III occurs in “sections” - discontinuous

Question 33

What happens to DNA Pol III on the lagging strand when it reaches the 5’ end?

Answer 33

It is swapped for DNA Pol I.

Question 34

What does DNA ligase do?

Answer 34

Makes a phosphodiester bond between the 5’ phosphate and the 3’ OH group joining Okazaki fragments (replaces nicks)

Question 35

E.coli polA mutants have a defective DNA polymerase that lacks 5’ to 3’ exonuclease activity. How will this affect replication?

Answer 35

DNA polymerase I will not be able to excise the RNA primers.