DNA Replication, Telomeres, Mutations, and DNA Repair Mechanisms

Study Guide - Smart Notes

Tailored notes based on your materials, expanded with key definitions, examples, and context.

DNA Replication

Key Proteins in DNA Replication

DNA replication is a highly coordinated process involving several key proteins that ensure accurate and efficient duplication of genetic material.

DNA Helicases: Enzymes that unwind the DNA double helix ahead of the replication fork by breaking hydrogen bonds, using energy from ATP hydrolysis.
Single-Stranded DNA Binding Proteins (SSB): Bind to exposed single DNA strands to keep them unwound and accessible for replication. SSB proteins are recycled after replication of a segment.
Topoisomerases: Enzymes that prevent excessive supercoiling by creating swivel points in the DNA molecule, making and resealing single- or double-stranded breaks. In E. coli, DNA gyrase is the key topoisomerase for replication.

The Replisome Complex

The replisome is a large protein complex responsible for DNA unwinding and synthesis on both strands. It is about the size of a ribosome and powered by nucleoside triphosphate hydrolysis.

Coordinates synthesis on both leading and lagging strands.
Includes DNA helicase, DNA gyrase, SSB, primase, DNA polymerase, and DNA ligase.

The Trombone Model

The trombone model describes how the lagging strand template is looped during replication, allowing the replisome to synthesize both strands simultaneously.

Lagging strand forms a loop, enabling coordinated synthesis of Okazaki fragments.
The holoenzyme complex includes the clamp loader, sliding clamp, and DNA polymerase linked by tau protein.
The sliding clamp is a ring-shaped protein that keeps DNA polymerase attached to the DNA strand, increasing processivity.

Leading vs. Lagging Strands

The sliding clamp and polymerase remain attached to the leading strand throughout replication, while their association with the lagging strand is more transient due to Okazaki fragment synthesis.

Eukaryotic DNA Replication and Chromatin Remodeling

Nucleosome Disassembly and Reassembly

Eukaryotic DNA replication involves additional complexity due to chromatin structure.

Chromatin remodeling proteins (e.g., SWI/SNF) loosen nucleosome packing ahead of the replication fork.
After replication, nucleosomes are reassembled by proteins such as Nap-1 and CAF-1.
PCNA (proliferating cell nuclear antigen) acts as a DNA clamp to increase processivity of DNA polymerase.
RNA primers are removed by RNase H (endonuclease) and FEN1 (exonuclease).

Telomeres and Telomerase

End-Replication Problem

Linear DNA molecules face a challenge in completing replication at their ends, especially on the lagging strand, due to the requirement for primers.

Each round of replication can result in loss of nucleotides from chromosome ends.
Telomeres are highly repeated, noncoding sequences (e.g., TTAGGG in humans) that protect chromosome ends.

Telomerase Function

Telomerase is a ribonucleoprotein enzyme that adds telomeric repeats to chromosome ends.
Contains an RNA template (e.g., in Tetrahymena, 3'—AACCC C—5') complementary to the telomere repeat.
Telomerase activity is mostly restricted to germ cells and some proliferating cells in multicellular organisms.

Protecting Chromosome Ends

After telomere extension, telomere capping proteins bind to the 3' end to prevent degradation.
Chromosome ends may form a closed loop structure for additional protection.

Telomeres, Cell Culture, and Aging

Absence of telomerase in primary cultured cells limits their lifespan (Hayflick limit).
Shortened telomeres trigger apoptosis.
Immortalized cell lines (e.g., HeLa) produce telomerase and can divide indefinitely.
Telomere shortening is linked to aging and degenerative diseases; telomerase-based therapies are under investigation.
Telomerase is detected in most human cancers.
Werner syndrome is caused by lack of a telomere cap protein (WRN), leading to premature aging.

Telomere Structure

Telomere sequences are G-rich and can form G-quadruplex structures, which are stable four-stranded DNA conformations.

Origins of Mutations

Spontaneous Mutations During Replication

Tautomeric shifts: Rare resonance structures of bases cause mispairing and replication errors.
Strand slippage: Occurs in regions with repetitive DNA, leading to expansion of trinucleotide repeats.
Spontaneous base damage: Includes depurination (loss of purine base) and deamination (removal of amino group).

Trinucleotide Repeat Disorders

Expansion of trinucleotide repeats can cause several human genetic diseases.

Disease	Repeat Sequence	Normal Repeat Number	Disease Repeat Number	Primary Symptoms
Fragile X syndrome	CGG	6–54	200–2000	Mental retardation
Friedreich ataxia	GAA	7–22	200–1700	Loss of motor coordination
Huntington disease	CAG	6–34	36–100	Movement disorders, dementia
Myotonic dystrophy type 1	CTG	5–37	50–1000	Muscle weakness
Spinal and bulbar muscular atrophy	CAG	9–34	38–62	Muscle weakness
Spinocerebellar ataxia	CAG	6–44	45–400	Loss of motor coordination

Chemical Modifications

Depurination: Loss of a purine base (adenine or guanine).
Deamination: Removal of an amino group from a base (e.g., cytosine to uracil).
Failure to repair these changes can result in permanent mutations.

Mutagens and Induced Mutations

Types of Mutagens

Chemical mutagens: Base analogues, base-modifying agents, and intercalating agents.
Radiation: Ultraviolet radiation causes pyrimidine dimer formation; ionizing radiation (X-rays) generates reactive intermediates that damage DNA.
Mobile genetic elements: Viruses and transposons can induce mutations.

Mechanisms of Chemical Mutagenesis

Base analogues: Structurally similar to DNA nucleotides, can be incorporated during replication and cause mispairing.
Base-modifying agents: Chemically alter DNA bases, forming DNA adducts that mispair during replication.
Intercalating agents: Insert between adjacent bases, distorting DNA structure and causing small nicks.

Radiation-Induced DNA Damage

Ultraviolet radiation: Induces covalent bonds between adjacent pyrimidine bases (thymine dimers).
Ionizing radiation: Removes electrons, generating highly reactive intermediates that damage DNA.

DNA Repair Mechanisms

Light-Dependent Repair

Photoreactivation: Pyrimidine dimers are repaired by photolyase enzyme, which uses visible light energy to break the dimer bonds.

Base Excision Repair (BER)

Corrects single damaged bases (e.g., deaminated bases).
DNA glycosylases detect and remove damaged bases, creating an abasic site.
AP endonuclease cuts the DNA backbone at the abasic site; a second enzyme removes the sugar.
DNA polymerase synthesizes the correct base, and DNA ligase seals the nick.

Base Excision Repair: Thymine and Uracil

Deamination of cytosine produces uracil, which is removed by uracil DNA glycosylase.
Uracil is flipped out of the helix and cleaved, leaving an abasic site repaired by BER.

Nucleotide Excision Repair (NER)

Detects and corrects distortions in the DNA helix (e.g., thymine dimers, bulky adducts).
NER endonuclease (excinuclease) cuts the DNA backbone on both sides of the lesion.
Helicase unwinds the DNA between nicks, freeing the damaged segment.
DNA polymerase and ligase fill and seal the gap.

Mismatch Repair

Corrects errors remaining after DNA replication.
Bacteria use methylation to distinguish template from newly synthesized DNA.
Process involves MutS (detects mismatch), MutH (introduces nick), and exonuclease (removes incorrect nucleotides).

Error-Prone Repair: Translesion Synthesis

When damage is too severe, specialized bypass polymerases (e.g., Pol IV and Pol V in E. coli) replicate across lesions.
In eukaryotes, translesion synthesis can allow replication past thymine dimers, sometimes with errors.

Double-Strand Break Repair

Double-strand breaks are repaired by nonhomologous end-joining (NHEJ) or synthesis-dependent strand annealing (SDSA).
NHEJ: Proteins bind to broken ends and ligate them, often losing nucleotides and risking errors.
SDSA: Uses homologous sequences on sister chromatids for accurate repair via strand invasion and D-loop formation.

Comparison of NHEJ and SDSA

Feature	NHEJ	SDSA
Template Use	No template; direct ligation	Uses homologous sequence on sister chromatid
Error Rate	High (nucleotide loss possible)	Low (accurate repair)
Process	Ku proteins bind ends, nucleases trim, ligase seals	Rad51 mediates strand invasion, D-loop forms, repair synthesis, ligation

Summary Activity Questions

List mechanisms of DNA damage that lead to NER and BER.
Identify enzymes involved in NER and their functions.
Compare nonhomologous end joining and synthesis-dependent strand annealing.

Additional info:

G-quadruplex structures in telomeres may play roles in chromosome stability and regulation of telomerase activity.
DNA repair mechanisms are essential for maintaining genome integrity and preventing disease.