Molecular Biology Lecture 7 - DNA Replication

Question 1

DNA synthesized in the ________ direction by ________

Answer 1

5’ → 3’ , DNA polymerase

Question 2

Proofreading is done by ______ in the _______ direction

Answer 2

exonuclease, 3’ → 5’

Question 3

What are nucleoside triphosphates (dNTPs)?

Answer 3

Nucleoside triphosphate (NTP) is a molecule containing a nucleoside bound to three phosphates. These include the four bases needed for DNA replication.

Question 4

Primer

Answer 4

A particular arrangement of single-stranded DNA (ssDNA)

Question 5

Correct base pairing provides specificity

Answer 5

3’ OH and α Phosphate correctly positioned.

Question 6

Right hand glove model for DNA pol

Answer 6

Draw and label

Question 7

DNA polymerase form processive reaction. This means?

Answer 7

Processivity is a characteristic of enzymes that operate on polymeric substrates. In the case of DNA processivity is defined as the average number of nucleotides added each time the enzyme binds a primer:template juction

1. Binding of pol to DNA is the rate-limiting step
2. Polymerization is very fast
3. Processive – multiple nucleotide additions per polymerase binding

Question 8

Proofreading of DNA….

Answer 8

….is done by the exonuclease in the 3’ to 5’ direction. Mispaired DNA alters the geometry of the 3’-OH. This will decrease the rate of addition and increase the rat of exonucleases

1. Unpaired (SS) DNA slows down the polymerase
2. Exonuclease removes mismatched base
3. 10-5 vs. 10-7 errors
4. Post-replication repair reduces errors

Question 9

Replication fork

Answer 9

DNA synthesis on two strands
Two DNA polymerase molecules
Leading and lagging strands - Okazaki fragments

Question 10

RNA primer

Answer 10

Primase are short RNA primers (5-10 nucleotides long) on an ssDNA template.

DNA polymerase requires a primer to copy ssDNA, RNA primer can be created de novo (the method of creating a transcriptome without a reference genome)

Question 11

RNA primer Con’t…..

Answer 11

1. RNA not DNA primer
   
   • 5-10 bases
2. Primase
   
   • RNA polymerase
   • Does not require a primer
3. Recognizes simple sequence (GTA in E. coli)
4. Stimulated when bound by helicase
   
   • restricts activity to replication fork

Question 12

Removal of primer

Answer 12

To RNA primers with DNA, an enzyme RNaseH recognizes and removes most of each RNA primer. RNaseH recognizes and degrades RNA base paired with DNA.

RNaseH can only cleave bonds between RNA:DNA

The removal of RNA leaves a gap in the dsDNA.

DNA polymerase I fills this up from the 5’-3’ end

DNA ligase – covalent linkage, requires ATP

Question 13

DNA helicase

Answer 13

1. DNA polymerase does not unwind DNA efficiently
2. Helicase moves in front of polymerase
   Moves along SS and denatures DS
3. Requires ATP
4. Hexameric protein - circles DNA
5. Polarity of movement
   5’ → 3’ (lagging strand)
6. Requires DNA to be SS first (initiation).

Question 14

ssDNA-binding proteins

Answer 14

After DNA helicase passes, the newly separated ssDNA must remain free for DNA polymerase to use a template for DNA synthesis.

ssDNA proteins (SSB’s) rapidly bind to the separated strands

Question 15

SSBs characteristics

Answer 15

1. Cooperative binding
2. Cannot denature DNA
3. Stabilize ssDNA
4. Non-specific binding: electrostatic interactions
5. Easily displaced

Question 16

Topoisomerase II

Answer 16

As the strands of DNA are uncoiled by DNA Helicase on the replication fork it increasingly becomes super-coiled.

1. Topoisomerase II relieves supercoiling
2. DNA gyrase (+1 to -1 in figure)
3. Remember that the E. coli chromosome starts out negatively supercoiled (DNA gyrase).

Question 17

Prokaryotes and eukaryotes have multiple DNA polymerases. In E.coli ……

Answer 17

1. E. coli
   
   • DNA pol I – primer removal, gap filling
     
     • accurate, not processive
   • DNA pol III – main replication pol
     
     • accurate, highly processive
   • Other DNA pols involved in DNA repair

Question 18

DNA polymerases in Eukaryotes

Answer 18

Eukaryotes

- DNA pol α/primase – primer synthesis, initial DNA 	synthesis
- DNA pol δ – lagging strand synthesis
- DNA pol ε – leading strand 
- Other DNA pols involved in DNA repair or mit DNA replication

Question 19

DNA polymerase switching

Answer 19

??

Question 20

Overview of DNA replication

Answer 20

1. DNA synthesis
2. Proofreading
3. Primer removal
4. Filling in gap and covalently sealing
5. Producing SS template
6. Relieve positive supercoils
7. Processivity - sliding clamp and clamp loader
8. Regulation
   
   • initiation of replication
   • cell-cycle regulation

Question 21

Sliding clamps

Answer 21

These are proteins composed of multiple identical subunits that assemble in the shape of a donut.

in the absence of a replication fork, DNA Polymerase dissociates and diffuses away from the template DNA on average one every 20-100bp synthesized.

In the presence of the sliding clamp DNA polymerase may still disengage but will not diffuse away from the DNA

Question 22

Sliding clamp mechanism

Answer 22

Draw & label

Question 23

How do you put a sliding clamp on DNA?

Sliding clamp loaders

Answer 23

A sliding clamp is a closed ing in solution but most open to encircle DNA.

Sliding clamp loaders catalyze the opening and placement of sliding clamps onto DNA