E. coli DNA Replication Origin
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Questions and Answers

What is the benefit of tightly coupling DNA replication to the cell cycle?

  • To increase the activity of RNaseH
  • To increase the rate of DNA replication
  • To reduce the activity of topoisomerases
  • To avoid aneuploidy (correct)

    • What is the function of ORC in the process of DNA replication?

  • To remove primers from the newly synthesized DNA strand
  • To seal the nick between the Okazaki fragments
  • To load the MCM2-7 helicase onto DNA (correct)
  • To synthesize primers for DNA replication

    • What is the function of FLAP ENDONUCLEASE Fen1 in DNA replication?

  • To synthesize DNA in the 5' to 3' direction
  • To remove RNA primers from the newly synthesized DNA strand
  • To create a flap structure during lagging strand synthesis
  • To recognize and cut the flap structure during lagging strand synthesis (correct)

    • What is the function of PCNA in DNA replication?

    <p>To act as a sliding clamp to coordinate DNA replication</p> Signup and view all the answers

    What is the effect of fluoroquinolones on bacterial DNA synthesis?

    <p>They inhibit the activity of topoisomerase</p> Signup and view all the answers

    What is the function of DnaA protein in E. coli?

    <p>Localising to the origin of replication</p> Signup and view all the answers

    What is the function of DnaB helicase?

    <p>Unwinding of double-stranded DNA</p> Signup and view all the answers

    What is the role of single-stranded DNA-binding protein (SSB)?

    <p>Preventing reannealing of DNA</p> Signup and view all the answers

    What is the main replicative enzyme in E. coli?

    <p>DNA pol III</p> Signup and view all the answers

    What is the function of DNA polymerase I?

    <p>Nick-translation during Okazaki fragment processing</p> Signup and view all the answers

    What is the function of RNA primer?

    <p>Providing a starting point for DNA synthesis</p> Signup and view all the answers

    What is the function of beta clamp?

    <p>Processive synthesis of DNA</p> Signup and view all the answers

    What is the result of replication from multiple origins of replication?

    <p>Multiple replication bubbles</p> Signup and view all the answers

    What is the purpose of RNaseH in DNA replication?

    <p>To remove primers from RNA-DNA hybrids</p> Signup and view all the answers

    What is the role of MCM2-7 helicase in DNA replication?

    <p>To load onto DNA by ORC</p> Signup and view all the answers

    What is the function of type 1 topoisomerases in DNA replication?

    <p>To pass ssDNA around the other strand before resealing</p> Signup and view all the answers

    What is the function of DNA ligase in DNA replication?

    <p>To seal nicks in DNA</p> Signup and view all the answers

    What is the effect of inhibiting type 2 topoisomerases on bacterial DNA synthesis?

    <p>It disrupts DNA synthesis</p> Signup and view all the answers

    Where does DNA replication begin in E. coli?

    <p>Origin of replication</p> Signup and view all the answers

    What is the function of DnaC loader protein?

    <p>Opening and loading of helicase ring</p> Signup and view all the answers

    What is the purpose of single-stranded DNA-binding protein (SSB)?

    <p>To prevent reannealing of single-stranded DNA</p> Signup and view all the answers

    What is the name of the structure formed during bi-directional replication?

    <p>Replication bubble</p> Signup and view all the answers

    What is the function of DNA polymerase III?

    <p>To synthesize new DNA strand</p> Signup and view all the answers

    What is the function of primase?

    <p>To synthesize RNA primer</p> Signup and view all the answers

    What is the function of beta clamp?

    <p>To allow processive synthesis</p> Signup and view all the answers

    What is the result of replication from multiple origins of replication?

    <p>Creation of multiple replication bubbles</p> Signup and view all the answers

    Study Notes

    Origin of Replication

    • E. coli origin of replication is OriC, a 250bp sequence
    • DnaA initiator protein binds to DnaA boxes, which are tandem repeats
    • Binding of DnaA to DnaA boxes spreads to A-T rich region, leading to DNA unwinding element (DUE) causing initial separation of strands

    DNA Unwinding and Replication

    • DnaB helicase unwinds dsDNA, using ATP and forming a hexameric ring
    • DnaB helicase causes unwinding by translocating on ssDNA, using steric exclusion to separate it from other strand
    • DnaC loader protein opens and loads helicase ring
    • Single-stranded DNA-binding protein (SSB) prevents reannealing behind helicase, stabilizing ssDNA

    Replication Fork and DNA Polymerases

    • Creation of replication bubble from bi-directional replication, leading to Y-shaped replication forks
    • E. coli has 5 DNA polymerases (I-V), with DNA pol III being the main replicative enzyme
    • DNA pol I has a specialized role in nick-translation during Okazaki fragment processing

    RNA Primer Synthesis and Processing

    • Primase synthesizes RNA primer on template DNA, using DNA-dependent RNA polymerase
    • Okazaki fragments in bacteria are 1000bp, while in eukaryotes they are 100-200bp
    • DNA pol III stalls when it encounters RNA, and DNA pol I has 5’-3’ exonuclease activity to chew up RNA primer and replace with DNA

    Replication Process

    • Processivity is the ability of polymerase to synthesize without stopping
    • DNA pol III relies on beta clamp for processive synthesis
    • Two DNA pols work together at replication fork, forming replisome, with leading and lagging strand synthesis coupled in trombone model
    • Multiple origins of replication, with yeast having defined origins called autonomously replicating sequences (ARSs)

    Eukaryotic DNA Replication

    • Eukaryotic DNA replication is tightly coupled to cell cycle to avoid aneuploidy
    • Origin licensing in G1, when MCM2-7 helicase is loaded onto DNA by ORC
    • Origin firing at beginning of S-phase
    • Primers removed by RNaseH, which digests RNA base paired to DNA
    • Replicative DNA pol displaces residual primer, creating flap, then recognized and cut by flap endonuclease Fen1, nick sealed by DNA ligase

    Topoisomerases

    • Topoisomerases relieve topological tension in DNA and untangle
    • Type 1 nick ssDNA and pass it around the other strand before resealing
    • Type 2 cut both strands of one duplex, allowing another duplex through the gap, re-sealing
    • Reactions proceed via covalent protein-DNA attachment

    Cellular Proliferation

    • MCM used as markers for cellular proliferation
    • Fluoroquinolones inhibit type 2 topoisomerases and disrupt bacterial DNA synthesis

    Origin of Replication

    • E. coli origin of replication is OriC, a 250bp sequence
    • DnaA initiator protein binds to DnaA boxes, which are tandem repeats
    • Binding of DnaA to DnaA boxes spreads to A-T rich region, leading to DNA unwinding element (DUE) causing initial separation of strands

    DNA Unwinding and Replication

    • DnaB helicase unwinds dsDNA, using ATP and forming a hexameric ring
    • DnaB helicase causes unwinding by translocating on ssDNA, using steric exclusion to separate it from other strand
    • DnaC loader protein opens and loads helicase ring
    • Single-stranded DNA-binding protein (SSB) prevents reannealing behind helicase, stabilizing ssDNA

    Replication Fork and DNA Polymerases

    • Creation of replication bubble from bi-directional replication, leading to Y-shaped replication forks
    • E. coli has 5 DNA polymerases (I-V), with DNA pol III being the main replicative enzyme
    • DNA pol I has a specialized role in nick-translation during Okazaki fragment processing

    RNA Primer Synthesis and Processing

    • Primase synthesizes RNA primer on template DNA, using DNA-dependent RNA polymerase
    • Okazaki fragments in bacteria are 1000bp, while in eukaryotes they are 100-200bp
    • DNA pol III stalls when it encounters RNA, and DNA pol I has 5’-3’ exonuclease activity to chew up RNA primer and replace with DNA

    Replication Process

    • Processivity is the ability of polymerase to synthesize without stopping
    • DNA pol III relies on beta clamp for processive synthesis
    • Two DNA pols work together at replication fork, forming replisome, with leading and lagging strand synthesis coupled in trombone model
    • Multiple origins of replication, with yeast having defined origins called autonomously replicating sequences (ARSs)

    Eukaryotic DNA Replication

    • Eukaryotic DNA replication is tightly coupled to cell cycle to avoid aneuploidy
    • Origin licensing in G1, when MCM2-7 helicase is loaded onto DNA by ORC
    • Origin firing at beginning of S-phase
    • Primers removed by RNaseH, which digests RNA base paired to DNA
    • Replicative DNA pol displaces residual primer, creating flap, then recognized and cut by flap endonuclease Fen1, nick sealed by DNA ligase

    Topoisomerases

    • Topoisomerases relieve topological tension in DNA and untangle
    • Type 1 nick ssDNA and pass it around the other strand before resealing
    • Type 2 cut both strands of one duplex, allowing another duplex through the gap, re-sealing
    • Reactions proceed via covalent protein-DNA attachment

    Cellular Proliferation

    • MCM used as markers for cellular proliferation
    • Fluoroquinolones inhibit type 2 topoisomerases and disrupt bacterial DNA synthesis

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    Learn about the origin of DNA replication in E. coli, including the role of DnaA initiator protein, DnaB helicase, and DnaC loader protein.

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