DNA Replication

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