DNA Replication, Repair, and Cell Cycle Regulation

Overview

This chapter explores the mechanisms by which cells replicate their DNA, repair genetic damage, and regulate the cell cycle. Accurate DNA replication and repair are essential for maintaining genetic integrity and preventing disease.

DNA Replication

Semiconservative Model of DNA Replication

DNA replication is the process by which a cell duplicates its genetic material before cell division. The semiconservative model, proposed by Watson and Crick, states that each new DNA molecule consists of one parental strand and one newly synthesized strand.

• Semiconservative replication: Each daughter DNA molecule contains one original (parental) strand and one new strand.

• Meselson & Stahl experiment: Used density isotopes in E. coli to confirm the semiconservative model.

• Alternative models: Conservative (both strands new) and dispersive (mixed segments).

Example: After one round of replication, DNA molecules are hybrids of old and new strands.

Origins of Replication and Replicons

Replication begins at specific DNA sequences called origins of replication. In eukaryotes, multiple origins create replication units called replicons.

• Origin Recognition Complex (ORC): Recognizes origins in eukaryotes.

• Replication bubble: Formed as DNA unwinds at the origin.

• Replicons: Each origin initiates a replicon; thousands per chromosome in eukaryotes.

• Bacteria: Typically have a single origin per chromosome.

Key Enzymes and Proteins in DNA Replication

DNA replication requires a coordinated set of enzymes and proteins:

• DNA polymerase: Catalyzes DNA chain elongation in the 5' → 3' direction. Requires a primer.

• Primase: Synthesizes short RNA primers to initiate DNA synthesis.

• Helicase: Unwinds the DNA double helix using ATP hydrolysis.

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

• Topoisomerase: Relieves supercoiling by creating temporary breaks.

• Ligase: Joins Okazaki fragments on the lagging strand.

• Telomerase: Extends telomeres at chromosome ends.

Leading and Lagging Strand Synthesis

DNA polymerase synthesizes DNA continuously on the leading strand and discontinuously on the lagging strand.

• Leading strand: Synthesized continuously in the direction of the replication fork.

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

• Okazaki fragments: Joined by DNA ligase to form a continuous strand.

RNA Primers and Initiation

DNA polymerase requires a primer to start synthesis. Primase synthesizes short RNA primers.

• Primase: An RNA polymerase that initiates primer synthesis.

• RNA primers: Provide a 3' hydroxyl group for DNA polymerase.

• Removal: RNA primers are removed and replaced with DNA.

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.

Unwinding the DNA Double Helix

Unwinding is essential for replication and is facilitated by helicases, topoisomerases, and SSBs.

• Helicase: Breaks hydrogen bonds between DNA strands.

• Topoisomerase: Relieves supercoiling.

• SSBs: Prevent re-annealing of single strands.

The Replisome and Trombone Model

The replisome is a large protein complex that coordinates DNA synthesis on both strands. The trombone model describes the looping of the lagging strand template.

• Replisome: Contains all necessary replication proteins.

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

Chromatin Remodeling During Replication

Chromatin remodeling proteins facilitate the unfolding and reassembly of nucleosomes during DNA replication.

• Replication factories: Immobile structures where DNA is synthesized.

• Chromatin assembly proteins: Nap-1 and CAF-1 help reassemble nucleosomes.

Telomeres and Telomerase

Telomeres are repetitive DNA sequences at chromosome ends that protect genetic information. Telomerase extends telomeres to solve the end-replication problem.

• Telomeres: Repeated sequences (e.g., TTAGGG in humans).

• Telomerase: Enzyme with RNA template that adds repeats to telomeres.

• Telomere capping proteins: Protect chromosome ends.

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

• Telomerase in cancer: Enables unlimited cell division.

DNA Damage and Repair

Types of DNA Damage

DNA can be damaged spontaneously or by environmental mutagens.

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

• Induced mutations: Caused by chemicals (base analogues, intercalating agents) or radiation (UV, X-rays).

• Trinucleotide repeat disorders: Expansion of repeats leads to diseases like Huntington's.

DNA Repair Mechanisms

Cells employ multiple repair systems to maintain genetic integrity.

• Proofreading: DNA polymerase corrects errors during replication.

• Photoactive repair: Photolyase repairs UV-induced pyrimidine dimers using visible light.

• Base Excision Repair (BER): Removes single damaged bases.

• Nucleotide Excision Repair (NER): Removes bulky lesions and helix distortions.

• Mismatch Repair (MMR): Corrects replication errors not fixed by proofreading.

• Error-prone repair (SOS response): Last-resort mechanism for severe damage.

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

Base Excision Repair (BER)

BER corrects single damaged bases, such as deaminated cytosine.

• DNA glycosylase: Removes damaged base.

• AP endonuclease: Cuts DNA backbone.

• DNA polymerase: Fills gap.

• DNA ligase: Seals nick.

Nucleotide Excision Repair (NER)

NER removes bulky DNA lesions, such as thymine dimers.

• Excinuclease: Cuts DNA on both sides of lesion.

• Helicase: Removes damaged segment.

• DNA polymerase and ligase: Fill and seal gap.

• Transcription-coupled repair: NER recruited to stalled transcription sites.

Mismatch Repair (MMR)

MMR corrects errors that escape proofreading during replication.

• MutS and MutL: Recognize mismatches in bacteria.

• Methylation: Distinguishes old (methylated) from new (unmethylated) DNA strands.

Double-Strand Break Repair

Double-strand breaks are repaired by NHEJ or HR.

• NHEJ: Joins broken ends; error-prone.

• HR: Uses homologous template for accurate repair.

• BRCA1/BRCA2: Genes involved in HR; defects lead to cancer susceptibility.

Cell Cycle Regulation

Phases of the Cell Cycle

The cell cycle consists of interphase (G1, S, G2) and mitosis (M phase).

• G1 phase: Cell growth and preparation for DNA synthesis.

• S phase: DNA synthesis (replication).

• G2 phase: Preparation for mitosis.

• M phase: Mitosis and cytokinesis.

• G0 phase: Quiescent state; cells exit the cycle.

Checkpoints and Cell Cycle Control

Checkpoints ensure proper progression and integrity of the cell cycle.

• G1 checkpoint: Checks for DNA damage before S phase.

• G2 checkpoint: Ensures DNA replication is complete.

• M checkpoint: Ensures proper chromosome segregation.

• MPF (Maturation Promoting Factor): Cyclin-dependent kinase (CDK) complex regulates entry into M phase.

• Cyclins: Regulate CDK activity and cell cycle progression.

• Tumor suppressor genes: Rb and p53 regulate checkpoints; mutations promote cancer.

Key DNA Replication Proteins Table

Comparison of important proteins involved in DNA replication in bacteria and eukaryotes.

Key Equations

DNA polymerization reaction:

Energy for DNA synthesis is provided by hydrolysis of dNTPs:

Summary Table: DNA Repair Mechanisms

Additional info:

• DNA replication occurs during S phase of the cell cycle.

• Telomerase activity is restricted to germ cells, stem cells, and cancer cells in multicellular organisms.

• Defects in DNA repair pathways are associated with diseases such as xeroderma pigmentosum (NER), Lynch syndrome (MMR), and Huntington's disease (trinucleotide repeat expansion).

• Cell cycle checkpoints are regulated by cyclins and CDKs; tumor suppressors like Rb and p53 are critical for preventing cancer.