The replication of DNA

Substrates required for DNA synthesis

DNA synthesis

XTP + (XMP)n (XMP) n+1 + P-P  G= -3.5 Kcal/mole P-P2 P  G= -3.5 Kcal/mole

The mechanism of DNA polymerase DNA polymerase use a single active site to catalyze DNA synthesis Identical geometry of the A:T and G:C base pairs

Kinetic selectivity An enzyme favors catalysis using one of the several possible substrates by dramatically increasing the rate of bond formation only when the correct substrate is present. The rate of incorporation of incorrect nucleotide is 10,000X lower.

Steric constraining preventing catalysis using rNTPs by DNA polymerase glutamate alanine Results in a DNA polymerase with significantly reduced discriminati on between dNTPs and rNTPs.

DNA polymerase resemble a hand that grips the primer: template junction (  -sheet, catalytic site) (  helix)

Two metal ions bound to DNA polymerase catalyze nucleotide addition Divalent mental ions held in place by interaction with two Asp.

Fingers of DNA polymerase binds to the incoming dNTP (in the finger domain) The closed form stimulates catalysis by moving the incoming nucleotide in close contact with the catalytic mental ions.

The finger domain bends the template to expose only the first template base

Events of DNA polymerization The incoming nucleotide base-pair with the next available template base This interaction cause the ‘fingers’ of the polymerase to ‘close’ around the new base-pair dNTP This conformation of the DNA polymerase place the critical mental ions in a position to catalyze formation of the next phosphodiester bond

DNA polymerases are processive enzymes Processivity: the average number of nucleotides added each time the enzyme binds a template-primer junction The processivity of DNA polymerase range from only a few nucleotide to more than 50,000 bases

DNA polymerase synthesize DNA in a processive manner.

Increased processivity is facilitated by the ability of DNA polymerase to slide along the DNA template. Once bound to DNA, polymerase interacts with DNA in a sequence- non-specific manner, including electrostatic interaction between phosphate backbone and the “thumb”, and interaction between the minor groove and the ‘palm’ domain. The sequence-independent nature of DNA-polymerase interaction permits the easy movement of the DNA to polymerase. Processivity of DNA polymerase

Proofreading exonuclease activity of DNA polymerase 1 mistake in every base pairs added Rate of DNA synthesis Is reduced, the affinity of 3’ end of the primer for the polymerase active site is diminished. When mismatched, the 3’ end has increased affinity for the exonuclease active site.

The replication fork Okazaki fragment: 1000 to 2000 in bacteria, NT in eukaryotes RNA primer: primase (specialized RNA polymerase to make short RNA primer 5-10 NT) Primase is activated only when it associates with other DNA replication proteins like DNA helicase

Removal of RNA primer

DNA helicases separate two strands in advance of the replication fork Hexameric 5’-3’ polarity processivity

Single-stranded binding proteins (SSBs) stabilize single strand DNA Binding of one SSB promotes the binding of another SSB to the immediately adjacent ssDA : cooperative binding Cooperative binding SSBs hold the ssDNA in a elongated state that facilitates its use as a template

Action of topoisomerase at the replication fork: to remove supercoils produced by DNA unwinding at the replication fork Simple Linear DNA rotate along its length to dissipate the supercoiled

Enzymes that function at the replication fork E. coli S. cerevisiae Human Primase DnaG primase Primase (PRI1/PRI2) DNA helicase DnaB Mcm complex Mcm complex SSB SSB RPA RPA Topoisomerase Gyrase, TopoI Topo I, II Topo I, II

DNA polymerases are specialized for different roles in the cell Prokaryotes (E. coli has at least five DNA polymerase) Pol I (RNA removal and repair), Pol III holoenzyme (high processivity) Eukaryotes (with a typical cell having more than 15) Pol  (including primase), Pol  and Pol  (high processivity)

Polymerase Switching: the process of replacing DNA pol  /primase with DNA Polδor ε (low processivity) Sliding clamps dramatically increase DNA polymerase processivity

Structure of a sliding clamp (multiple identical subunit)

(no ssDNA, conformational change of DNA polymerase reduce affinity to sliding clamp) Sliding clamps increase the processivity

Sliding clamp loaders Box 8-2

DNA polymerase III holoenzyme Holoenzyme is a general name for a multiprotein complex in which a core enzyme activity is associated with additional components that enhance function.

The “trombone” model for coordinating replication Interacts with both helicase and polymerase

(associating with helicase to synthesize RNA primer)

Interaction between replication fork proteins form a replisome Helicase interacts with polymerase through clamp loader. Preventing helicase from running away from the pol III Weak helicase and primase interaction. This interaction could determine the size of Okazaki fragment.

Initiation of DNA replication Replicator: entire set of DNA sequences that is sufficient to direct the initiation of replication. Origin of replication: the specific sites at which DNA unwinding and initiation of replication occur Replicon: DNA replicated from a particular origin Initiator: specifically recognizes a DNA element in the replicator and activator the initiation of replication

The structure of replicators Green: initiator binding site; blue: A-T rich DNA that unwinds readily Red: site of first DNA synthesis Initiator: DnaA Multicellular organisms have replicators longer than 1000 bp. ORC (origin recognition complex, 6-protein complex) binds to B1 Replicator sequences include initiator binding sites and easily unwound DNA

Three functions of initiator proteins (when DnaA bound with ATP) Origin Recognition Complex (ORC) (in Eukaryotes) does not direct strand separation.

E. coli initiation of DNA replication Protein-protein and protein-DNA interactions direct the initiation process ssDNA and DnaA recruit DNA helicase(DnaB) and helicase loader (DnaC) helicase recruits primase (6 copies)

template;primer junction helicase moves 1000 bp, 2nd primer is synthesized on lagging strand. E. coli initiation of DNA replication New primer:template junctions are targeted by the clamp loaders. Clamps are recognized by the second DNA Pol III core enzyme pol III.

Chromosomes are replicated exactly once per cell cycle To achieve this task, two criteria must be met: 1.Enough origins must be activated to ensure that each chromosome is fully replicated during each S phase.

2. No origin of replication can initiate after it has been replicated. Chromosomes are replicated exactly once per cell cycle To achieve this task, two criteria must be met:

Two steps in the initiation of replication 1.Replicator selection: identifying replicator sequence, and assembly of a multiprotein complex (pre-replicative complexpre-RC). This occurs at G1 phase. 2. Origin activation: triggering the pre-RC to initiate DNA unwinding and polymerase recruitment. This occurs at S phase.

Formation of pre-replicative complex (pre-RC) directs the initation of replication in Eukaryotes initiator Helicase loading proteins eukaryotic replication fork helicase

Activation of pre-RC Kinase activated when cells enter S phase. Template: primer junction is recognized by sliding clamp loader

Effect of Cdk activity on pre-RC formation and activation

Cell cycle regulation of Cdk activity and pre-RC formation Pre-RCs are disassembled after the DNA is replicated.

Finishing replication

end replication problem

Protein priming as a solution to the end replication problem In linear chromosomes of certain species of bacteria

(RT) Replication of telomeres by telomerase Telomerase is a novel DNA polymerase that does not required an exogenous template