Basic Rules of Replication

• Semi-conservative: both strands will serve as a template for new strand synthesis

• Starts at an origin

• Synthesis always in the 5🡪3’ direction b/c the DNA polymerase needs a 3’ hydroxyl end

• Can be unidirectional but as a rule it’s bidirectional

• Semi-discontinuous: one DNA strand is replicated in fragments

• RNA primers required

Origin of Replication

• Origin of replication: provides an opening called a replication bubble that forms two replication forks.

  • DNA replication proceeds outward from forks

Role of DNA Polymerase

• DNA polymerase: covalently links nucleotides together

  • Deoxynucleoside triphosphates: free nucleotides with three phosphate groups

    • Breaks covalent bonds to release pyrophosphate (two phosphates) and provides energy to connect nucleotides

Features of DNA Polymerase

• DNA polymerase cannot begin synthesis on a bare template strand

  • Requires a primer to get started

  • DNA primase makes the primer from RNA

  • The RNA primer is removed and replaced with DNA later

• DNA polymerase only works 5’ to 3’

Leading Strand vs Lagging Strand

• Leading strand

  • DNA synthesized in as one long molecule (continuous)

  • DNA primase makes a single RNA primer

  • DNA polymerase adds nucleotides in a 5’ to 3’ direction as it slides forward

• Lagging strand

  • DNA synthesized 5’ to 3’ but as Okazaki fragments (discontinuous)

  • Okazaki fragments consist of RNA primers plus DNA

• In both strands

  • RNA primers are removed by DNA polymerase and replaced with DNA

  • DNA ligase joins adjacent DNA fragments

Core Proteins At the Replication Fork

• Topoisomerases: prevents torsion by DNA breaks

• Helicases: separates 2 strands

• Primase: RNA primer synthesis

• Single-strand binding proteins: prevent reannealing of single strands

• DNA Polymerase: synthesis of new strand

• Clamp: stabilizes polymerase

• DNA ligase: seals nick via phosphodiester linkage

Accuracy of DNA Replication

• Three mechanisms for accuracy

  • Hydrogen bonding between A and T, and between G and C is more stable than mismatched combinations

  • Active site of DNA polymerase is unlikely to form bonds if pairs mismatched

  • DNA polymerase can proofread to remove mismatched pairs

    • DNA polymerase backs up and digests linkages

    • Other DNA repair enzymes as well

DNA Polymerases

• E. coli has 5 DNA polymerases

  • DNA Polymerase II: multiple subunits, responsible for majority of replication

  • DNA Polymerase I: a single subunit, rapidly removes RNA primers and fills in DNA

  • DNA Polymerase II, IV, and V: DNA repair and can replicate damaged DNA

    • DNA polymerases I and III stall at DNA damage

    • DNA polymerases II, IV, and V don’t stall but go slower and make sure replication is complete

• Humans have 12 or more DNA polymerases

  • Designated with Greek letters

  • DNA polymerase α*:* its own built in primase subunit

  • DNA polymerase δ and 𝜀: extend DNA at a faster rate

  • DNA polymerase 𝛾: replicates mitochondrial DNA

  • When DNA polymerases α, δ, and 𝜀 encounter abnormalities, they may be unable to replicate

  • Lesion-replicating polymerases may be able to synthesize complementary strands to the damaged area

Telomeres

• Series of short nucleotide sequences repeated at the ends of chromosomes in eukaryotes

• Specialized form of DNA replication only in eukaryotes in the telomeres

• Telomere at 3’ does not have a complementary strand and is called a 3’ overhang

Telomerase Functions

• Shortening of telomeres is correlated with cellular senescence

• Telomerase function is reduced as an organism ages

• 99% of all types of human cancers have high levels of telomerase