DNA and Genomics I (DNA Replication)

Where does DNA replication begin?

• Origin of Replication (ori) on the parental DNA molecule

• Enzymes like helices and proteins are required to initiate replication → Bind to ori

What does helicase do?

• Unzips the double helix → Separates 2 parental strands → Creates replication forks in both directions → Replication bubble

• Breaks H bonds between complementary base pairs with the help of ATP

How are parental DNA strands kept apart in the replication bubble?

Single-strand binding proteins bind to separated DNA strands to keep them apart → Prevention from reannealing

What does topoisomerase do?

Relieves overwinding strain ahead of replication forks by breaking, swiveling and rejoining DNA strands

What does DNA polymerase do?

• Initiates DNA synthesis → Adds nucleotides in the 5’ to 3’ direction to a pre-existing chain which provides the free 3’OH end → Elongation of the new complementary strand

• Catalyses phosphodiester bonds between the 3’OH end and the incoming free nucleotide

• Part of DNA polymerase proof reads the previous region as it moves along the strand

  • Ensures proper base pairing

  • Swiftly removes any incorrect DNA nucleotide + Replacement with the correct one

• A different DNA polymerase removes the RNA primer eventually and replaces it with DNA nucleotides

What are the conditions for DNA polymerase to work?

• A pre-existing chain in the form of an RNA primer

• A template in the form of parental DNA strands

What does primase do?

Adds short RNA primer on each of the parental DNA strands

What do the free dNTPs do?

Use the parental DNA strand as a template → Align in a sequence complementary to that of the parental strand

What does DNA ligase do?

Forms a phosphodiester bond between 2 DNA fragments to seal the nick

What is the difference between the leading and lagging strand?

Leading → Synthesized continuously in the 5’ to 3’ direction

Lagging → Synthesized discontinuously in the 5’ to 3’ direction?

Why can DNA polymerase only synthesize in the 5’ to 3’ direction?

DNA polymerase only interacts with the free 3’OH group attached to the end of the growing DNA strand, not the phosphate group attached to the 5’ end

What are Okazaki fragments?

• Fragments produced by discontinuous synthesis on the lagging strand

• Synthesis of each Okazaki fragment is initiated by an RNA primer before addition of nucleotides

How is a continuous DNA strand produced from the joining of many Okazaki fragments?

1. DNA polymerase excises the RNA primer → Replace with DNA

2. DNA ligase joins the 3’ end of each new fragment to the 5’ end of the growing chain with a phosphodiester bond

How can DNA replication be sped up in eukaryotes?

Multiple ori in a single DNA molecule → Many DNA polymerases can work simultaneously

*Prokaryotes only have a single ori

What properties of DNA make it suitable for storing information? (4)

• DNA can be replicated accurately

• DNA is a stable molecule

• There is a backup of DNA code

• Coded information can be readily utilized and accessed

Why is DNA able to replicate accurately and what is the implication?

• Weak H bonding between strands → Easy separation for templates

• Daughter cells have identical copies of DNA as the parent cell

How is DNA a stable molecule?

• Numerous H bonds hold the 2 strands together

• Strong phosphodiester bonds between nucleotides

How is there code backup in DNA?

• Double-stranded molecule

• Mutations may occur spontaneously on either strand → One strand = Template for repair

How is coded information readily utilized and accessed in DNA?

• Weak H bonding → Template strand can separate from non-template strand → Transcription

• Complementary base pairing → Faithful transfer of information during transcription → Subsequent translation

What are the differences between DNA replication in eukaryotes and prokaryotes?

• Eukaryotes → S phase of interphase at the nucleus, Prokaryotes → Prior to cell division by binary fission at the cytoplasm

• Eukaryotes → Multiple ori, Prokaryotes → 1 per circular strand of DNA

• Eukaryotes → Ends at telomeres at the ends of linear chromosomes, Prokaryotes → Ends at the terminus region

• Eukaryotes → 50 nucleotides/s, Prokaryotes → 500 nucleotides/s

What are telomeres?

• Both ends of eukaryotic chromosomes

• Non-coding regions of DNA

• Series of tandem repeat sequences (TTAGGG in humans)

• 3’ overhang (single-stranded region at the 3’ end)

• Extended by telomerase

What do telomeres do?

• Ensure genes are not eroded with each round of DNA replication due to the end replication problem

• Prevents the loss of vital genetic information with each replication cycle

What is the end replication problem?

• 5’ end of newly synthesized strand will have the RNA primer removed without DNA replacement

• Chromosome shortening

What happens when a shortening chromosome reaches critical length?

Cell undergoes apoptosis in response to the apparent damage to the chromosome before genes get eroded