Topic 2 Molecular Genetics: DNA Replication

Question 1

• Recall that the S phase of interphase is when DNA is replicated. Therefore, in order to do so, DNA is unzipped and each strand serves a template for complementary replication
• DNA is replicated via semiconservative replication, in which one the two strands of DNA is always old (blue strand below) and one is always new (shown in green)

Answer 1

1. A second chromatid containing a copy of DNA is assembled during interphase

Question 2

• Once DNA is unwound, we introduce several new enzymes to carry out replication
  a. Single stranded binding proteins (SSBPs) attach to each strand of uncoiled DNA to keep them separate
  b. Topoisomerases (like DNA gyrase) break and rejoin the DNA double helix of the replication fork, preventing knots
  c. To visualize this, imagine unwinding a twist by pulling the ends apart. As you pull, the ends get really tight and begin knotting up. A topoisomerase would break and rejoin these knots so that you have an unknotted piece of string

Answer 2

2. Helicase is the enzyme that unwinds DNA, forming a Y shaped replication fork

Question 3

• Leading strand - works continuously as more DNA unzips (synthesized 5’ → 3’)
• Lagging strand - for the 5’ → 3’ template strand, the DNA polymerase has to go back to the replication fork and work away from it. It produces fragments piece by piece, and these fragments are called Okazaki fragments
  a. DNA ligase connects Okazaki fragments

Answer 3

3. DNA polymerase moves from the 3’ -> 5’ direction only, and synthesizes a new strand that is antiparallel (5’ -> 3’)

Question 4

• DNA replication requires an RNA primer
• Every Okazaki fragment has an RNA primer, and these RNA strips are later replaced with DNA by DNA polymerase I
  a. DNA polymerase I replaces base pairs from the RNA primers and functions in DNA repair
  b. DNA polymerase III is mainly for replication
  c. Polymerases I and III have 3’ → 5’ exonuclease function, meaning that they can break the phosphodiester backbone on a single strand of DNA and remove a nucleotide. An exonuclease can only remove from the end of the chain.
  d. Polymerase III also has some proofreading function; if there is a mistake during replication, polymerase III will go back and replace a nucleotide
  e. Polymerase I also has 5’ → 3’ exonuclease function to remove the primer; polymerase I can also proofread in the 3’ → 5’ direction when laying down a new nucleotide strand

Answer 4

4. Primase is an enzyme that creates a small strip of RNA off of which DNA polymerase can work since it can only add to an existing strand

Question 5

• DNA Polymerases I, II, III are in prokaryotes, while eukaryotes have different polymerases (alpha, gamma, epsilon, etc.) with similar function, but don’t worry about the eukaryotic polymerases for the OAT
• DNA Replication is almost 20x faster in prokaryotes than in eukaryotes, primarily because eukaryotes have more complex genomes

Answer 5

Note

Question 6

• Basically, Polymerase III mainly replicates the DNA in the 5’ → 3’ direction, but can also proofread in the 3’ → 5’ direction, via its exonuclease function.
• Polymerase I primarily breaks down the RNA primer in the 5’ → 3’ direction via exonuclease function (this is unique to polymerase I), and replaces the primer with DNA nucleotides.
• These nucleotides are mainly laid down between Okazaki fragments in the 5’ → 3’ direction and proofread as polymerase I travels. Note that polymerase I can proofread in the 3’ → 5’ direction via exonuclease function as well.
• In prokaryotes, the strand without any errors is methylated after it has been successfully replicated, so it doesn’t become accidentally repaired as proofreading is done.
• In all cases of repair, DNA ligase must come in to seal the backbone after replication is done
• The energy for DNA elongation is provided by two additional phosphates that are attached to each new nucleotide. When the bonds holding the two extra phosphates are broken, the breakage provides chemical energy for the process, which is the same for DNA

Answer 6

Summary

Question 7

1. Helicase - unzips DNA to form replication fork
2. Single Stranded Binding Proteins (SSBPs) - keep DNA strands separate
3. Topoisomerase - ease tension of coiled DNA by introducing nicks and cuts into the DNA strand
4. DNA polymerase - synthesizes the new strand of DNA
5. Primase - creates a small strip of RNA where DNA polymerase takes over to add DNA nucleotides
6. Ligase - ‘glues’ two strands of DNA together

Answer 7

Recap of DNA Replication Enzymes