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Related Concept Videos

Lagging Strand Synthesis01:59

Lagging Strand Synthesis

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During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
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Translesion DNA Polymerases02:10

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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication...
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DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.  After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
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Single-molecule studies contrast ordered DNA replication with stochastic translesion synthesis.

Gengjing Zhao1, Emma S Gleave1, Meindert Hugo Lamers1

  • 1MRC laboratory of Molecular Biology, Cambridge, United Kingdom.

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|December 7, 2017
PubMed
Summary

Cells use translesion DNA polymerases to bypass DNA damage, but how their access is controlled is unclear. This study shows replicative and translesion polymerases alternate binding stochastically, driven by concentration-dependent competition, not lesion recognition.

Keywords:
CoSMoSDNA replicationE. colibiochemistrybiophysicssingle-moleculestructural biologytranslesion DNA synthesis

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • DNA replication requires high-fidelity polymerases, which stall at DNA adducts.
  • Translesion DNA polymerases (TLs) bypass these blocks but are error-prone, linked to mutagenesis and cancer.
  • Mechanisms preventing premature TL access to DNA are poorly understood.

Purpose of the Study:

  • To investigate the exchange dynamics between replicative DNA polymerase Pol IIIcore and translesion polymerases Pol II and Pol IV at the replication fork.
  • To elucidate the regulatory mechanisms controlling translesion polymerase access to DNA during replication.

Main Methods:

  • Co-localization single-molecule spectroscopy (CoSMoS) was employed to track polymerase exchange in E. coli.
  • Real-time observation of polymerase interactions with the DNA clamp was performed.

Main Results:

  • Replicative (Pol IIIcore) and translesion polymerases (Pol II, Pol IV) do not form a stable complex on a single clamp; they alternate binding.
  • Clamp and Pol IIIcore loading are highly organized.
  • Translesion polymerase exchange is stochastic, governed by polymerase concentration-dependent competition, not direct DNA lesion recognition.

Conclusions:

  • The 'toolbelt model' of polymerase complex formation is not supported.
  • Translesion polymerase access is regulated by a competitive, concentration-dependent mechanism rather than lesion-specific recruitment.
  • This stochastic exchange mechanism may influence mutagenesis and cellular response to DNA damage.