Chapter 34

Question 1

DNA Replication

Answer 1

DNA replication takes place w/ the activity of several polymerases

In E.coli, there are over 20 different protein, 5 of which are DNA polymerases, which catalyze the synthesis of new DNA

DNA polymerases are the enzymes

1. Four deoxynucleoside triphosphates (dATP, dGTP, dCTP, TTP) and MG2+ are required
2. A DNA template strand is sued to direct DNA synthesis
3. A primer from which the new stand grows must be present
   - Primer is RNA made my “primase”
   - DNA polymerase needs 3’-OH to add (builds 5’ to 3’)
4. Many DNA polymerases have nuclease activity that allows for the removal of mismatched bases
   - Exonucleases remove mismatches

Polymerase only works in 5’ to 3’ direction, thus resulting in a leading strand and a lagging strand (built backwards)

Question 2

DNA Replication – Replication Fork

Answer 2

 Replication fork = site of replication

• Fork is created by multiple different enzymes that will allow for unwinding of the double stranded DNA, stabilization of the unwound segment, and subsequent polymerization of the new DNA strand

1. Helicase is the enzyme that will unwind the double stranded DNA and allow for replication to take place

• Single-stranded binding proteins will help to stabilize the unwound double stranded DNA

2. Primase will next synthesize the RNA primers
3. Next, DNA polymerase III will make the DNA
4. DNA polymerase I will remove the RNA primer
5. Ligase will help join the fragments together

 Fork moves in one direction ~ demonstrating that both strands are copied simultaneously

 All DNA polymerases synthesize DNA in the 5’ to 3’ direction

 At replication fork, one strand is synthesized discontinuously as small prices, called Okazaki fragments, in the 5’ to 3’ direction

 The discontinuous assembly allows fork movement in the 3’ to 5’ direction while fragments are made in the 5’ to 3’ direction

 The leading and lagging strands are synthesized in a coordinated fashion

 The lagging strand is looped so that it passed through the polymerase active site in the 3’ to 5’ direction, allowing synthesis to occur in the 5’ to 3’ direction

 After DNA of ~ 1000 nucleotides in length is synthesized, the loop is released and a new loop is formed, process call trombone model

Question 3

DNA Replication – Holoenzyme & Trombone Model

Answer 3

Holoenzyme
- Contains DnaB helicase ring: unwinds DNA
- 2 polymerase cores: synthesizes DNA
- Coordination occurs due to holoenzyme structure
- Each of the DNA core polymerase enzymes are attached to a central structure

DNA replication structure exists as a trombone-like structure

Question 4

DNA Replication -Repair

Answer 4

 Many polymerases proofread the newly added bases and excise (remove) errors
 Many polymerases also have nuclease activity that’s used to remove mismatched nucleotides
 The E.coli enzymes DNA polymerase I, which is used in replication and DNA repair, has exonuclease activity that removes mismatched nucleotides from the 3’ end of the DNA
 An incorrectly inserted nucleotide moves the polymerase active site to the nearby exonuclease active site, where it is hydrolytically cleaved

Question 5

DNA Replication – Free Ends

Answer 5

Once replication reaches the ends, the free ends of the linear DNA molecules present two biochemical difficulties

1. They are susceptible to damage by nucleases
2. Due to the nature of DNA synthesis, with the lagging and leading strand, one strand will shorten upon each round of DNA synthesis