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DNA Replication, Repair, and Recombination (Chapter 17) – Becker's World of the Cell

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DNA Replication, Repair, and Recombination

Introduction

Accurate duplication and maintenance of genetic material are essential for cell division and organismal survival. DNA replication ensures that each daughter cell receives a complete set of genetic instructions, while DNA repair mechanisms correct damage and errors. This chapter focuses on the molecular details of DNA replication, the experimental evidence supporting its mechanisms, and the proteins involved in the process.

17.1 DNA Replication

Learning Objectives

  • Describe the cell cycle and the importance of the S phase.

  • Summarize experimental evidence for semiconservative replication (Meselson-Stahl experiment).

  • Identify key proteins required for DNA synthesis.

  • Explain the roles of leading and lagging strands in DNA replication.

  • Discuss the importance of telomerase and telomeres in aging.

Key Terms

  • Mitosis: Nuclear division resulting in two identical daughter nuclei.

  • Cytokinesis: Division of the cytoplasm following mitosis.

  • Interphase: Period between cell divisions; includes G1, S, and G2 phases.

  • S phase: Phase of the cell cycle where DNA synthesis occurs.

  • Gap Phases (G1 and G2): Periods before and after S phase, respectively.

  • Replication Fork: Y-shaped region where DNA is actively being unwound and replicated.

  • Theta Replication: Replication of circular DNA molecules, typical in bacteria.

  • Replicons: Units of DNA replicated from a single origin.

  • DNA Helicase: Enzyme that unwinds the DNA double helix.

  • Single-Stranded Binding Proteins (SSB): Stabilize unwound DNA strands.

  • DNA Polymerase: Enzyme that synthesizes new DNA strands.

  • Primase: Enzyme that synthesizes RNA primers.

  • DNA Ligase: Enzyme that joins Okazaki fragments.

  • Topoisomerase: Enzyme that relieves supercoiling during replication.

  • Leading Strand: DNA strand synthesized continuously.

  • Lagging Strand: DNA strand synthesized discontinuously as Okazaki fragments.

  • Okazaki Fragments: Short DNA fragments synthesized on the lagging strand.

  • Primers: Short nucleic acid sequences that provide a starting point for DNA synthesis.

  • Telomeres: Repetitive DNA sequences at chromosome ends.

  • Telomerase: Enzyme that extends telomeres.

DNA Replication and Cell Division

All DNA in the nucleus of a parent cell must be duplicated and distributed to daughter cells. This involves:

  • Mitosis: Division of the nucleus.

  • Cytokinesis: Division of the cytoplasm.

  • Sister chromatids: Duplicated chromosomes attached together until separated during mitosis.

Separation of Sister Chromatids

  • Microtubules of the mitotic spindle separate sister chromatids.

  • Each chromatid becomes a full-fledged chromosome and moves to opposite poles.

  • New nuclear envelopes form around the two sets of daughter chromosomes.

The Eukaryotic Cell Cycle

The cell cycle consists of interphase (G1, S, G2) and M phase (mitosis and cytokinesis). DNA synthesis occurs during S phase, doubling the amount of nuclear DNA.

  • G1 phase: Gap before DNA synthesis.

  • S phase: DNA replication.

  • G2 phase: Gap after DNA synthesis, before mitosis.

Semiconservative DNA Replication

The Watson and Crick model proposed that DNA replication is semiconservative: each new DNA molecule consists of one parental strand and one newly synthesized strand.

  • Semiconservative replication: Each daughter DNA has one old and one new strand.

  • Conservative model: Parent molecule remains intact; new molecule is entirely new.

  • Dispersive model: Each strand is a mix of old and new segments.

Experimental Evidence: Meselson-Stahl Experiment

Meselson and Stahl demonstrated semiconservative replication using isotopic labeling and equilibrium density centrifugation.

  • Bacteria grown in 15N medium incorporated heavy nitrogen into DNA, then transferred to 14N medium.

  • After one replication cycle, DNA showed intermediate density; after two cycles, two bands appeared (hybrid and light).

  • Results supported semiconservative replication.

Bidirectional DNA Replication

DNA replication proceeds in both directions from the origin. In Escherichia coli, this was visualized by John Cairns using autoradiography.

  • Replication forks form at the origin and move away in both directions.

  • Theta (θ) replication is observed in circular DNA molecules.

  • Cell divides by binary fission after replication.

Eukaryotic DNA Replication

Replication of linear chromosomes initiates at multiple sites, forming replicons. Each replicon is a unit of DNA replicated from a single origin.

  • Thousands of replicons per chromosome (50,000–300,000 bp each).

  • Replication bubbles form at each origin, with two forks synthesizing DNA in opposite directions.

Origins of Replication and Consensus Sequences

Replication initiates at specialized DNA elements called origins of replication. In E. coli, the origin (oriC) is AT-rich and contains tandem repeats. Conserved sequences are called consensus sequences.

Replication Initiation in Bacteria

  • Enzymes DnaA, DnaB, and DnaC bind oriC to initiate replication.

  • DnaA binding unwinds DNA at specific sites.

  • SSB proteins stabilize unwound DNA.

  • DnaB acts as a DNA helicase.

Replication Initiation in Eukaryotes

  • Origin recognition complex (ORC) binds the replication origin.

  • Minichromosome maintenance (MCM) proteins (including DNA helicases) bind the origin.

  • Helicase loaders recruit MCM proteins to the ORC.

  • Formation of the pre-replication complex is called licensing; only licensed origins can replicate.

DNA Polymerases and Elongation

DNA polymerase catalyzes the addition of nucleotides to the 3' hydroxyl end of the growing DNA chain, so elongation occurs in the 5' → 3' direction.

  • Arthur Kornberg discovered DNA polymerase I.

  • DNA polymerase III is the main replicative enzyme in bacteria (about 50,000 bp/min).

Table: Some Important DNA Replication Proteins in Bacteria and Eukaryotes

Protein

Cell Type

Main Activities and/or Functions

Initiator proteins

Both

Bind origin of replication and initiate unwinding of DNA double helix

DNA polymerase I

Bacteria

DNA synthesis; 5'→3' exonuclease (for primer removal); 3'→5' exonuclease (for proofreading)

DNA polymerase III

Bacteria

Main replicative DNA polymerase; 3'→5' exonuclease (proofreading)

DNA polymerase α

Eukaryotes

Initiates DNA synthesis; tightly associated with primase

DNA polymerase δ, ε

Eukaryotes

Main replicative DNA polymerases; 3'→5' exonuclease (proofreading)

Primase

Both

RNA synthesis; creates RNA primers for DNA synthesis

DNA helicase

Both

Unwinds double-stranded DNA

SSB protein

Both

Stabilizes single-stranded DNA

Topoisomerase

Both

Relieves supercoiling ahead of replication fork

DNA ligase

Both

Joins Okazaki fragments on lagging strand

Telomerase

Eukaryotes

Extends telomeres at chromosome ends

Leading and Lagging Strand Synthesis

  • DNA polymerase synthesizes DNA only in the 5' → 3' direction.

  • Leading strand: Synthesized continuously.

  • Lagging strand: Synthesized discontinuously as Okazaki fragments (1000–2000 nucleotides in bacteria; shorter in eukaryotes).

  • Okazaki fragments are joined by DNA ligase.

Proofreading and Error Correction

  • DNA polymerases have 3' → 5' exonuclease activity for proofreading.

  • Exonucleases: Remove nucleotides from ends of DNA.

  • Endonucleases: Make internal cuts in DNA.

  • Error rate is only a few per billion base pairs due to proofreading.

RNA Primers and Initiation of DNA Synthesis

  • DNA polymerase requires a primer with a free 3' hydroxyl group.

  • Primase synthesizes short RNA primers (~10 bases) using DNA as a template.

  • In bacteria, primase is part of a complex called the primosome.

  • In eukaryotes, primase is tightly bound to DNA polymerase α.

Steps in RNA Primer Function

  1. Primase synthesizes RNA primers using a single DNA strand as template.

  2. DNA polymerase extends the primer, synthesizing DNA.

  3. RNA primers are removed (by DNA polymerase I in bacteria), and DNA fills the gap.

  4. DNA ligase joins adjacent fragments.

Unwinding the DNA Double Helix

  • DNA helicases unwind the double helix using ATP hydrolysis.

  • SSB proteins stabilize unwound DNA.

  • Topoisomerases relieve supercoiling ahead of the replication fork.

Summary of DNA Replication in Bacteria

  • Initiation at oriC by initiator proteins.

  • Unwinding by helicase, stabilization by SSB.

  • Primer synthesis by primase.

  • Elongation by DNA polymerase III (leading and lagging strands).

  • Primer removal and gap filling by DNA polymerase I.

  • Fragment joining by DNA ligase.

Telomeres and the End-Replication Problem

Linear chromosomes face the end-replication problem: lagging strand synthesis cannot complete the very end, leading to progressive shortening. Telomeres are repetitive, noncoding sequences at chromosome ends that protect genetic information. Telomerase extends telomeres using an RNA template, maintaining chromosome integrity in germ cells and some stem cells.

  • Human telomeres: 100–1500 copies of TTAGGG.

  • Telomerase is a ribonucleoprotein (protein + RNA template).

  • Telomere shortening is associated with cellular aging.

  • Telomerase activity is detected in most cancer cells.

Telomeres, Aging, and Disease

  • Normal somatic cells have limited telomerase activity, leading to telomere shortening and eventual cell senescence.

  • Immortalized cell lines (e.g., HeLa cells) express telomerase and can divide indefinitely.

  • Defects in telomere capping proteins (e.g., WRN in Werner syndrome) lead to premature aging and disease.

  • Telomerase-based therapies are being explored for aging and cancer treatment.

Example: Meselson-Stahl Experiment

Bacterial cells grown in 15N medium, then transferred to 14N medium, showed intermediate DNA density after one replication cycle, and two bands after two cycles, supporting semiconservative replication.

Example: Okazaki Fragments

On the lagging strand, DNA is synthesized in short fragments (Okazaki fragments), which are later joined by DNA ligase to form a continuous strand.

Formula: Directionality of DNA Synthesis

DNA polymerase adds nucleotides to the 3' end:

Elongation occurs in the 5' → 3' direction.

Additional info:

  • Some details on telomere structure and telomerase function were expanded for clarity.

  • Table entries were inferred and summarized from textbook context.

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