DNA Replication, Repair, and Recombination

Overview

Accurate duplication and maintenance of genetic material are essential for cell division and survival. This chapter explores the mechanisms of DNA replication, the processes that repair DNA damage, and the recombination events that contribute to genetic diversity.

DNA Replication

The Eukaryotic Cell Cycle and DNA Synthesis

DNA replication occurs during the S phase of the cell cycle, ensuring that each daughter cell receives a complete set of genetic material.

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

• S Phase: DNA content doubles as replication occurs.

• Mitosis (M phase): Nuclear division, followed by cytokinesis.

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

Semiconservative Replication

DNA replication follows the semiconservative model, where each new DNA molecule contains one parental and one newly synthesized strand.

• Watson–Crick Model: Suggested semiconservative mechanism based on base pairing.

• Alternative Models: Conservative (parental DNA remains intact) and dispersive (mixed segments).

• Meselson–Stahl Experiment: Used isotopic labeling (14N and 15N) to demonstrate semiconservative replication.

• Replication is bidirectional: Replication forks move away from the origin in both directions.

Origins of Replication

Replication begins at specific DNA sequences called origins of replication.

• Bacterial origin (oriC): AT-rich, contains consensus sequences.

• Eukaryotic origins: Multiple per chromosome; in yeast, called ARS (autonomously replicating sequence).

• Replication units (replicons): Each origin defines a replicon; thousands per eukaryotic chromosome.

Initiation of Replication

Initiation involves the assembly of protein complexes at replication origins.

• Bacteria: DnaA, DnaB (helicase), DnaC, and SSB proteins initiate unwinding.

• Eukaryotes: Origin Recognition Complex (ORC), MCM helicases, and licensing factors ensure replication occurs only once per cell cycle.

• Licensing: Formation of pre-replication complex; only licensed origins can initiate replication.

DNA Polymerases and Elongation

DNA polymerases catalyze the synthesis of new DNA strands in the 5′ → 3′ direction.

• Arthur Kornberg: Discovered DNA polymerase I.

• Bacterial DNA polymerases: I (repair), II, III (main replication enzyme), IV, V (repair).

• Eukaryotic DNA polymerases: α, δ, ε (nuclear replication); γ (mitochondrial replication); others for repair.

• Elongation: Nucleotides added to the 3′ hydroxyl end.

Leading and Lagging Strands

DNA synthesis occurs continuously on the leading strand and discontinuously on the lagging strand.

• Leading strand: Synthesized continuously toward the replication fork.

• Lagging strand: Synthesized in short fragments (Okazaki fragments) away from the fork.

• Okazaki fragments: Joined by DNA ligase.

Proofreading and Fidelity

DNA polymerases possess proofreading activity to correct errors during replication.

• 3′ → 5′ exonuclease activity: Removes incorrectly paired nucleotides.

• Error rate: Reduced to a few per billion base pairs.

RNA Primers and Initiation

DNA polymerases require a primer to begin synthesis.

• Primase: Synthesizes short RNA primers (~10 bases).

• Primosome: Complex of primase and other proteins in bacteria.

• Primer removal: In bacteria, DNA polymerase I (5′ → 3′ exonuclease); in eukaryotes, RNAse H and FEN1.

Unwinding the DNA Helix

Several proteins facilitate the unwinding of DNA for replication.

• Helicases: Unwind the double helix using ATP.

• Single-stranded binding proteins (SSB): Stabilize unwound DNA.

• Topoisomerases: Relieve supercoiling; gyrase is key in bacteria.

The Replisome and Trombone Model

The replisome is a large protein complex coordinating DNA synthesis on both strands.

• Clamp loader and sliding clamp: Keep DNA polymerase attached to DNA.

• Trombone model: Lagging strand forms a loop to allow coordinated synthesis.

Chromatin Remodeling in Eukaryotes

Replication requires disassembly and reassembly of nucleosomes.

• Replication factories: Immobile structures where DNA is synthesized.

• Chromatin remodeling proteins: Nap-1, CAF-1 facilitate nucleosome assembly.

Telomeres and the End-Replication Problem

Linear chromosomes face the end-replication problem, solved by telomeres and telomerase.

• Telomeres: Repetitive, noncoding sequences at chromosome ends (e.g., TTAGGG in humans).

• Telomerase: Enzyme with RNA template adds repeats to chromosome ends.

• Telomere capping proteins: Protect chromosome ends from degradation.

• Hayflick limit: Maximum number of cell divisions due to telomere shortening.

• Telomerase in disease: Present in cancer cells; absence leads to aging-related degeneration.

DNA Damage and Repair

Sources of DNA Damage

DNA can be damaged spontaneously or by environmental agents.

• Spontaneous mutations: Mispairing due to tautomers, strand slippage, chemical modifications (depurination, deamination).

• Mutagens: Chemicals (base analogues, base-modifying agents, intercalating agents) and radiation (UV, X-rays).

• Trinucleotide repeat disorders: Expansion of repeats leads to diseases (e.g., Huntington's disease).

Types of DNA Damage

• Base analogues: Incorporated during replication, can cause mispairing.

• Base-modifying agents: Chemically alter bases, leading to mispairing.

• Intercalating agents: Insert between bases, causing insertions/deletions.

• Pyrimidine dimers: UV-induced covalent bonds between adjacent pyrimidines.

• Double-strand breaks: Caused by ionizing radiation.

DNA Repair Mechanisms

Cells employ multiple repair pathways to maintain genome integrity.

• Light-dependent repair: Photolyase breaks pyrimidine dimers (not in humans).

• Base excision repair (BER): Removes single damaged bases; DNA glycosylases, AP endonuclease, DNA polymerase, and ligase involved.

• Nucleotide excision repair (NER): Removes bulky lesions; excinuclease, helicase, DNA polymerase, and ligase involved.

• Mismatch repair: Corrects replication errors; MutS, MutH, exonuclease, and DNA polymerase involved.

• Translesion synthesis: Specialized polymerases bypass lesions; error-prone.

• Double-strand break repair: Nonhomologous end-joining (NHEJ) and homologous recombination.

Table: DNA Repair Pathways

Homologous Recombination and Mobile Genetic Elements

Homologous recombination is essential for genetic diversity and accurate repair of double-strand breaks.

• Crossing over: Occurs during meiosis; involves Holliday junctions.

• Resolution: Same-sense (no crossing over) or opposite-sense (crossing over).

• Gene conversion: Repair without exchange; can alter allele frequencies.

Transposons and Retrotransposons

Mobile genetic elements contribute to genome evolution and variability.

• Transposons: DNA-only elements; move via cut-and-paste or copy-and-paste mechanisms.

• Retrotransposons: Move via RNA intermediate; use reverse transcriptase.

• Autonomous vs. nonautonomous: Autonomous encode their own transposase; nonautonomous require help.

• Alu sequences: Common retrotransposons in humans; ~11% of genome.

• L1 element: Encodes its own transcriptase; ~17% of human DNA.

Table: Types of Transposable Elements

Key Equations and Concepts

Directionality of DNA Synthesis

• DNA polymerase reaction:

• Elongation occurs in the 5′ → 3′ direction.

Proofreading by DNA Polymerase

• Exonuclease activity:

Telomerase Extension

• Telomerase adds repeats to chromosome ends:

Summary

• DNA replication is semiconservative, bidirectional, and highly regulated.

• Multiple enzymes and protein complexes ensure fidelity and efficiency.

• DNA repair mechanisms correct a wide range of damage, maintaining genome stability.

• Homologous recombination and mobile genetic elements contribute to genetic diversity and evolution.

Example: The Meselson–Stahl experiment demonstrated semiconservative replication by showing that after one round of replication in 14N medium, DNA molecules had intermediate density, consistent with one old and one new strand.

Example: Xeroderma pigmentosum patients lack functional NER, making them highly sensitive to UV-induced DNA damage.

Additional info: Some details about chromatin remodeling and replication factories were inferred for completeness.