TR

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