• Enzymes that catalyze the synthesis of DNA through a phosphory group transfer.
• E. coli makes at least five DNA Polymerases.

• Principal replication enzyme in E. Coli
• At least 10 subunits
• Fast
• High Processivity
• Function: chromosome Replication.

• Template
  • Structure that would allow molecules to be lined up in a specific order and joined
• Primer:
  • Strand segment (complementary to the template) with a free 3'-hydroxyl group to which a nucleotide can be added.

• Extended at the 3' terminus
  • The nucleophile in the group transfer reaction is the 3' -OH of the primer
  • Identity of the incoming dNTP is determined by the complementary base in the template strand.
  • Proper base-pair geometry is important to fidelity.

• Number of nucleotides incorporated into DNA before the DNA polymerase dissociates from the primer/template.
• Varies between a few nucleotides per binding event up to thousands.

• Hydrogen bonding between base pairs
  • Specifies the correct pairing between complementary bases.
• Geometry of base pairs
  • DNA Polymerase active site can only accommodate base pairs with a specific geometry.
• Incorrect bases can be rejected before the phosphodiester bond is formed.

• Proofreading: 3' →5' exonuclease activity.

DNA_n + H₂O → DNA_n-1 + dNMP

• A separate active site integral to the DNA polymerase.
• Activated by a mismatch at the 3' OH of the primer/template. Continues to act until the 3'OH is properly base-paired with the template.
• Functions to remove nucleotides incorporated in error.

• Accuracy of DNA synthesis: 1 error per 10⁵ nucleotides incorporated into DNA.
• Accuracy of DNA polymerase action (synthesis and proofreading): 1 error per 10² nucleotides incorporated into DNA.
• Overall accuracy replication: 1 error per 10⁹ or 10¹⁰ nucleotides incorporated into DNA.

• Helicases
• Topoisomerases
• DNA binding proteins:
  • dsDNA binding protein (HU)
  • ssDNA binding protein (SSB)
• Primase (DNA-dependent RNA synthase)
• DNA polymerases I and III
• DNA ligase (covalent joining of DNA strands)

• Complex of DnaB*DnaG.
• Functions to:
  • Unwind the parental duplex ahead of the DNA polymerase.
  • Synthesize RNA primers (primase enzyme)

• Meeting of two replication forks.
• Resolution of the two linked circles.
• Segregation of daughter chromosomes into separate cells.

• Replicates the leading and lagging strands of the fork.
• Contains both polymerase and 3' →5' exonuclease activities.
• Employs a "sliding clamp" protein called PCNA, which ensures high processivity. PCNA is functionally homologous to the β subunit of DNA polymerase III

• Synthesizes RNA primers for the lagging strands of the fork.
• Contains DNA and RNA polymerase activities on separate subunits.
• Has no 3'-5' or 5'-3' nuclease activities.

• May remove primers by nick translation.
• Contains DNA polymerase activity
• Has both 3'-5' and 5'-3' nuclease activities.

• Substitution mutation: replacement of one base pair with another.
• Insertion or deletion mutations: addition or deletion of one or more base pairs.

• Deaminating agents
• Base analogs
• UV radiation
• Alkylating agents
• Hydroxylating agents
• Intercalating agents
• Ionizing radiation (x-rays, gamma-rays)
• Transposable elements including retroviruses
• DNA replication / cell division

• Lesions (unrepaired DNA) that are repaired by this pathway include:
  • Base deamination( e.g. cytosine deamination to yield uracil)
  • Oxidized and alkylated bases
  • Spontaneous loss of a base.
• DNA glycosylases specific for a particular lesion hydrolyze the N-glyosidic bond. (e.g. Uracil DNA glycosylase)
• An apurinic or apyrimidinic (AP) site is created as the first step in repair.