D1.1 - DNA Replication

Question 1

DNA Replication

Answer 1

• Process which occurs in all cells before cell
  division (mitosis & meiosis - D2.1)
• Original copy of DNA gets replicated,
  producing two identical DNA molecules
• Process occurs in all living organisms
• Basis for biological inheritance

Question 2

Helicase

Answer 2

• Enzyme that unwinds and unzips DNA helix
• Separates two DNA strands by breaking the
  hydrogen bonds between complementary
  base pairs
• Helicase begins to unwind the DNA at the origin of replication
• Helicase enzymes move in both directions from the point of origin, forming a replication bubble
• At either end of the replication bubble is a replication fork - a Y-shaped region where strands of DNA are elongated

Question 3

DNA Polymerase

Answer 3

• DNA Polymerase is an enzyme that
  polymerises new complementary strands of
  DNA
• Follows complementary base pairing with
  the parent strand
• Collects free-floating nucleotides and
  attaches them to the growing complementary
  strand

*DNA polymerase catalyses the covalent phosphodiester bonds between the sugar and
phosphate group of nucleotides

*Hydrogen bonds form naturally between complementary base pairs

Question 4

Complementary Base Pairs

Answer 4

• Semi-conservative replication is highly
  accurate due to complementary base pairing
• Complementary base pairing ensures that the
  genetic message will be conserved when
  each strand undergoes replication
• In other words, the daughter strands will have
  the same sequence of bases as the original
  parent strand

Question 5

DNA Replication in Labs

Separation of DNA Fragments

Answer 5

Polymerase chain reaction (PCR):

*Technique used in molecular biology to copy and amplify a small segment of DNA (not a whole
genome)

Separation of DNA Fragments

Gel Electrophoresis:

• Technique used to separate and isolate
  proteins or DNA fragments based on size
  and charge
• Electrophoresis involves running a current
  through a gel containing the molecules of
  interest

Question 6

Why Use PCR and Gel Electrophoresis?

Answer 6

• Techniques create a DNA profile, allowing
  DNA samples of individuals to be analyzed
  and compared
• Useful technique for crime scene
  investigations and paternity tests

Forensic Investigations

• Suspects should be a complete match with the DNA sample taken from
  the crime scene if a conviction is to occur

Paternity Testing

• As children inherit half of their chromosomes from each parent, their DNA
  profile should be a combination of both paternal fragments

Question 7

Replication Enzymes

Answer 7

There are many enzymes involved in the replication of DNA, including:

• Helicase
• Single-stranded binding proteins
• DNA gyrase
• DNA primase
• RNA primer
• DNA polymerase I
• DNA polymerase III
• DNA ligase

Question 8

Single-Stranded Binding Proteins

Answer 8

• Bind to and hold the opened strands of DNA apart

Question 9

DNA Gyrase

Answer 9

• As helicase moves along the DNA, it causes the strand to supercoil ahead
• DNA Gyrase works ahead of helicase to release the strain that develops

Question 10

Primase and Primers

Answer 10

• DNA primase is an enzyme that creates RNA primers
• RNA primers are short RNA chains of about 10 bases used as an initiation / attachment point for
  DNA polymerase

Question 11

DNA Polymerase I

Answer 11

• RNA primers are removed and replaced with DNA nucleotides by DNA Polymerase I

Question 12

DNA Ligase

Answer 12

• Joins the Okazaki fragments on the lagging strand together to form a single DNA strand

Question 13

Direction of Replication

Answer 13

• DNA polymerases replicate DNA by moving along a template strand and synthesising a new complementary strand
• Recall: DNA polymerases can only add to the 3’ end of the growing polynucleotide
• DNA is synthesized in a 5’ to 3’ direction, as the 3’ end is being extended

Question 14

Leading & Lagging Strand

Answer 14

• Since the double-stranded DNA is antiparallel, DNA polymerase must move in opposite directions on the two strands
• Leading strand: new strand builds towards the replication fork, copied continuously
• Lagging strand: new strand builds away from the replication fork, copied discontinuously

Question 15

Leading Strand

Answer 15

• Continuous replication:
  DNA polymerase III follows helicase, adding
  nucleotides to 3’ end of new strand
• Only one RNA primer binds to the leading
  strand at the origin of replication

Question 16

Lagging Strand

Answer 16

• Discontinuous replication:
  DNA polymerase III moves away from
  helicase, adding nucleotides to 3’ end of new
  strand
• Creates chunks of DNA, known as Okazaki
  fragments
• Many RNA primers needed at various points
  along the lagging strand
• DNA ligase “glues” Okazaki fragments
  together

Question 17

DNA Proofreading

Answer 17

• Errors in complementary base pairing are
  extremely rare during DNA replication,
  however, they can occur occasionally
• DNA Polymerase III proofreads the growing
  DNA chain and removes any mismatched
  bases, replacing them with the correct
  nucleotides