Cell Cycle and DNA Repair Mechanisms

Introduction

• Organisms face constant exposure to DNA damage from both internal (spontaneous mutations) and external sources (induced mutations).
• DNA repair processes, involving a multitude of proteins, are present in both prokaryotic and eukaryotic organisms.
• The regulation of DNA repair is intricately linked to the control of the cell cycle.
• Checkpoint mechanisms during the cell cycle ensure DNA integrity before and after replication, as well as before cell division. Failures in these checkpoints can lead to the accumulation of damage.

DNA Damage Response

• The cell responds to DNA damage through:
  • Repair mechanisms
  • Tolerance strategies
  • Activation of DNA damage checkpoints
  • Cell cycle arrest
  • Induction of apoptosis

DNA Damage Repair Mechanisms

• DNA damage can be caused by:
  • Crosslinking agents
  • Carcinogenic agents
  • Replication errors
  • UV light
  • Alkylation
  • Oxidation
  • Deamination
  • Ionizing radiation
• Types of DNA damage include:
  • T

Mismatch Repair (MMR)

• Repairs errors in DNA replication by removing base mismatches and small insertion/deletion loops.
• The mechanism is strand-specific, distinguishing between the newly synthesized strand and the template strand. This is crucial for maintaining the fidelity of DNA replication.
  • Mechanism Details: MMR systems often involve proteins that recognize distortions in the DNA helix caused by mismatches. These proteins then recruit other factors to excise the erroneous nucleotides specifically from the newly synthesized strand, which is identified through marks like nicks or methylation patterns.

Human Pathology and MMR

• Germline allelic variants in MMR genes (MLH1, MSH2, MSH6, or PMS2) predispose individuals to hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome (MIM #120435; #609310; #613244; #614331; #614337; #614350; #614385).
  • This significantly increases the risk of developing colorectal cancer.
  • Clinical Significance: Identifying these genetic predispositions through testing can inform screening and preventative strategies, such as more frequent colonoscopies, to detect and manage potential cancers early.

Nucleotide Excision Repair (NER)

• Repairs DNA damage induced by environmental factors such as radiation and various mutagens, including chemical mutagens.
• Essential for excising UV light-induced DNA damage, which commonly leads to the formation of pyrimidine dimers that distort the DNA structure.
• Two primary repair sub-pathways exist:
  • Transcription-coupled NER (TC-NER): targets transcriptionally active DNA, ensuring that actively transcribed genes are efficiently repaired to prevent transcription errors.
  • TC-NER Process: This pathway is initiated when RNA polymerase stalls at a site of DNA damage. This stalling recruits NER factors to the site, leading to the removal and repair of the damaged DNA segment.
  • Global genome NER (GG-NER): scans and repairs damage throughout the entire genome, including non-transcribed regions.
  • GG-NER Process: GG-NER involves proteins that patrol the genome, recognizing distortions caused by DNA damage. Once damage is detected, the NER machinery is recruited to carry out the repair.

Human Pathology and NER

• Xeroderma pigmentosum (XP) (MIM #278700):
  • Symptoms include extreme skin photosensitivity, pigmentary lesions, significantly elevated risk for skin and internal cancers, skin telangiectasia, ocular changes, and potential mental retardation.
  • All XP proteins play crucial roles in NER and transcription, often functioning as chromatin remodeling complexes to facilitate access to damaged DNA.
  • Allelic variants in germ cells lead to the disease, emphasizing the inherited nature of XP.

Human Pathology and NER Continued

• Cockayne Syndrome (CS) (#216400, #133540):
  • Incidence in Western Europe is approximately 2.7 per million live births.
  • Symptoms include mental and developmental retardation, pronounced photosensitivity, progressive sensorineural hearing loss, short stature, a typical bird-like face, deep-set eyes, loss of subcutaneous fat, premature aging, and progressive neurodegeneration.
  • Arises from defects in NER, particularly those connected to mitochondrial base excision repair (BER). These defects highlight the interplay between nuclear and mitochondrial DNA repair pathways.
  • Defective XP and CS proteins result in defective TCR and also play a critical role in overall transcription processes. This broader impact on transcription contributes to the pleiotropic effects observed in these syndromes.
  • Case Report: A male patient who died at age 31 \frac{1}{2} with a body weight of 11.3 kg provides a stark illustration of the severity of CS.
  • His developmental milestones included walking unsupported at 15 months but were followed by severe progressive symptoms:
    • Spasticity of joints began at 42 months.
    • Intention tremor and staggering gait developed by 9 years.
    • Inability to maintain trunk and head erect by age 30.
    • He was wheelchair-bound by 25 years.
    • Vision was limited to light perception by age 28 and complete blindness a year later.

The Bases for Cockayne Syndrome

• CS proteins are integral in signaling the TCR part of NER, which explains the characteristic photosensitivity seen in CS patients.
• CS proteins localize to mitochondria following oxidative stress, where they interact with mitochondrial proteins to protect the cell from oxidative stress-induced damage. These protective mechanisms are crucial, and their defects may significantly contribute to premature aging.
• NER defects alone cannot fully explain the diverse clinical features of CS. Mitochondrial changes, resulting from defective CS protein function, may be a significant underlying cause of the disease.

Base Excision Repair (BER)

• Removes damaged bases in the DNA sequence and is primarily responsible for correcting small, non-helix-distorting errors.
• BER is capable of repairing various types of damage:
  • Oxidized bases resulting from oxidative stress.
  • Alkylated bases caused by exposure to alkylating agents.
  • Deaminated bases, where amino groups are removed, leading to incorrect base pairing.
  • Inappropriately incorporated uracil, which should only be present in RNA.
  • Single-strand DNA breaks.

BER - Continued

• BER is initiated by a DNA glycosylase that recognizes and removes the damaged base, creating an abasic site (AP site).
• Two main pathways then proceed:
  • Short-patch BER: removes one-base lesions and involves replacing a single nucleotide.
  • Long-patch BER: removes 2-10 base lesions, replacing a longer stretch of nucleotides to ensure accurate repair.
• No human disease is currently known to be directly associated with a defect in BER, which may be due to the critical nature of this pathway, leading to embryonic lethality if severely disrupted.

Homologous Recombination Repair (HR) and Non-Homologous End Joining (NHEJ)

• Both HR and NHEJ are critical pathways for repairing double-strand breaks (DSB), which are particularly hazardous to the cell.
• HR uses a homologous DNA template to guide the repair, making it a highly accurate process.
• NHEJ rejoins broken ends without needing a template and is often accompanied by the loss of some nucleotides, making it a more error-prone mechanism.
• The choice between HR and NHEJ depends largely on the cell cycle stage:
  • NHEJ is more active in the G1 phase when a sister chromatid is not available as a template for HR.
  • HR dominates during the S and G2 phases when the sister chromatid can be used as a template for accurate repair.

Human Pathology Involving HR and NHEJ

• Defective repair of DSBs can lead to chromosomal instability, which is characterized by rearrangements and loss of entire chromosomes. This instability can drive