DNA Replication, Translation, Transcription

• DNA Replication:

  • Semi-conservative
  • Requires primers
  • Semi-discontinuous
  • Usually bidirectional
  • Ordered, sequential and highly accurate
  • Occurs in the nucleus
  • Takes place by separation of the strands of the double helix, and synthesis of two daughter strands complementary to the two parental templates.
• DNA polymerase requires a primer which provides the 3’ hydroxyl terminus on which to add new nucleotides.
• DNA polymerase is responsible for synthesizing new DNA strands from a DNA template.
• Polymerization occurs in the 5’-to-3’ direction.
• Core polymerase consists of three subunits:

  α-specifies the polymerase activity

  ε-3’-5’ exonuclease activity

  θ-stimulates 3’-5’ exonuclease acitivty
• The DNApolymerase III holoenzyme is a multisubunit complex.
• Subunit τ is responsible for dimerization of the core DNA polymerase.
• The sliding clamp β provides the ring structure that is needed for processivity.
• Five subunits have clamp-loader functions — γ, δ, δ', χ and ψ.
• – One strand (the leading strand) is replicated continuously in the direction of the movement of the replicating fork.
• – The other strand (the lagging strand) is replicated discontinuously as 1-2 kb Okazaki fragments in the opposite direction.
• – This allows both strands to be replicated in the 5’ 3’ direction.
• • Both daughter strands are synthesized simultaneously.
• • The leading strand (in the direction of the replication fork movement) is synthesized continuously.
• • The lagging strand (in the opposite direction of the replication fork movement) is synthesized discontinuously.
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• Replication starts at the origin site, where a number of proteins bind to initiate replication.
• • Replication forks are points where a pair of replicating segments come together and join the nonreplicated segments.
• A circular bacterial chromosome has a single origin of replication.
• oriC : origin of replication of E. coli

  1. Recognition of origin of replication by specific proteins
  2. Formation of replication fork
• Proteins involved in the formation of replication fork in E. coli
• 9-bp repeats, five copies of which are dispersed throughout oriC, is the binding site for a protein called DnaA.
• DnaA binding results to “melting” of the double helix at the tandem array of three AT-rich, 13-bp repeats.
• Helicase (DnaB) and single-stranded DNA-binding (SSB) proteins unwind the parental duplex and separate the two strands.
• Primase (RNA polymerase that assembles short RNA primers) and helicase form a “primosome”, which processively moves along the lagging-strand template.
• Tension is built up as DNA begins the unwinding process, and the DNA becomes positively supercoiled.
• DNA gyrase(topoisomerase II) relieves the tension by changing the DNA into negatively supercoiled DNA; uses ATP hydrolysis.
• The two tethered polymerases can replicate both strands by looping the DNA of the lagging-strand template back on itself, causing this template to have the same orientation as the leading-strand template.
• Once the polymerase assembling the lagging strand reaches the 5’ end of the Okazaki fragment synthesized during the previous round, the lagging-strand template is released and the polymerase begins work at the 3’ end of the next RNA primer toward the fork (trombone model).
• Excision of RNA primers by DNA pol I– 5’-3’ exonuclease function removes approximately 10 nucleotides from the 5’ end of a single strand nick. This activity plays a key role in removing the RNA primer.
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• DNA Polymerase I: Removal of RNA primers

  1. Excision of RNA primers by DNA pol I
  2. Filling up of the gaps by DNA pol I
  3. Nick ligation by DNA ligase
• Careful selection of the nucleotide, proofreading, and mismatch repair account for low error rates in replication (about 10–9).
• Replication is rapid (~103 nucleotides/sec).
• 3’ 🡪 5’ exonuclease activity (of DNA pol I and III) removes mispaired nucleotides from the 3’ end of the growing DNA. This function is key in maintaining the accuracy of DNA synthesis.
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• Terminator sequences: recognition site for a sequence-specific DNA binding protein called Tus.– Orientation of bound Tus relies on the orientation of the termination sequences.
• Tusallows a replication fork to pass if the fork is moving in one direction, but blocks progress if the fork is moving in the opposite direction around the genome.
• Eukaryotic chromosome contains multiple origins of replication.
• ARSs : Autonomously replicating sequences (about 400 in yeast, an 11bp A-T rich region)