DNA Damage & Repair Flashcards
What are the two forms of double strand break (DSB) repair?
- Homologous Recombination (HR)- meiotic or mitotic
- End Joining (NHEJ)- non homologous
What is AICDA?
- Activation-induced cytidine deaminase, catalyzes the deamination of cytidines in DNA to uracil.
- Clusters of deaminated cytidines are seen as damage by BER and MMR, chromosome breaks
- Repaired by NHEJ
- Triggers class switching and affinity maturation in B cells
What are the 2 main radiation forms of DNA damage?
- UV Radiation- photoactivates nucleotides and induces covalent bonds (pyrimidines), causes kinks in DNA
- Ionizing Radiation- usually medical sources (X-ray or gamma) through radiolysis of water (ROS), causes strand breaks
What are the general steps of the maturation of the Humoral (B cell) response?
- Ig antigen and activation
- Proliferation (clonal expansion)
- Maturation into 4 types:
- Antibody secretion (plasma cell)
- Isotype Switch
- Affinity Maturation
- Memory Cell
What are the characteristics of Nucleotide Excision Repair (NER)?
- Recognizes general distortions in DNA (more flexible than BER)
- Removes most UV photoproducts, adducts
- Multi-protein machine removes ssDNA about 28 NT long
- Gap is repaird by fill-in synthesis and ligation (similar to BER)
- Usually coupled to transcription (more efficient in “non-silent” regions and template strand)
What are the characteristics of Mismatch Repair (MMR)?
- Reverse errors made during replication (misincorporation and slippage)
- Not obvious which strand is damaged (template not obvious)
- Looks for discontinuity of okazaki fragments, needs to occur before gaps/nicks are sealed (coupled to replication fork)
- Removes large chunk of ssDNA (300-500 NT)
What is the role of genes BRCA1 and BRCA2 in recombination?
- BRCA1 promotes homologous recombination and supresses end joining
- BRCA2 is required for early step in homologous recombination (helps broken chromosome find sister or homolog)
What diseases are associated with defects in VDJ recombination?
- Severe Combined Immunodeficiency (SCID)- both B and T cell arms of adaptive immune response. Due to loss of RAG1 and RAG2
- Radiosensitive+SCID (RS-SCID)- combined B/T cell immunodeficiency, cellular sensitivity to DSB (radiation). Hypomorphic mutations in NHEJ proteins.
- Omenn’s Syndrome- autoimmune disorder (hypomorphic mutations in RAG1 and RAG2)
What is VDJ recombination and how does it generate receptor diversity?
- Multiple versions of a coding segment (V, D, and J) are distributed at a specific locus in the chromosome.
- One each segment (V, D, and J) is assembled into code for the variable domain of mature receptor
- The variable domain provides specificity for different foreign agnets
- The constant domains mediate effector functions
At which two points in the cell cycle does the cell check for DNA damage?
Before S-phase (G1) and before mitosis (G2). Will enter cause a cell cycle arrest (repaid DNA) or apoptosis (programmed cell death)
Which antibodies are expressed by naive B cells?
IgM and IgD
What is affinity maturation and how does it occur?
- Increases the affinity or anitbody for a specific antigen (1000 fold) by altering the variable domains.
- Involves somatic hypermutation- AICDA deaminates cytidines specifically within variable domain exons and causes base changes.
- Cells with improved affinity survive during clonal expansion while “other” mutations die
What are some of the problems with AICDA function and deficiency?
- The targeting of AICDA is not perfect and can cause cancer (off-target hypermutation, off-target DSB and translocations)
- Absence of AICDA can cause immunodeficiency “hyper IgM syndrome” (failure to class switch)
What is adduct formation and what causes it?
Chemicals can promote covalen attachment to nucleotides and can interefere with replication or transcription of DNA. Often caused by carcinogens
What disorders are associated with defects in MMR?
- Hereditary non-polyposis colon cancer (HNPCC, Lynch Syndrome)- mutation in MMR gene, usually the TGF receptor (epithilia growth)
- Microsatellite instability can lead to other cancers and genetic diseases
What are 3 disorders associated with NER defects?
- Xeroderma pigmentosa- UV sensitive, prone to skin cancer
- Cockayne Syndrome and Trichothiodystrophy- developmental defects, “brittle hair syndrome”
What are the two main types of replication errors?
- Substitutions- improved by proofreading and mismatch repair, made worse by imbalanced nucleotide pools
- Slippage- due to repetitive DNA and seconday structures (contracts and expands these)
What 3 things does the cell use for genomic surveillance?
- Damage sensors- see DNA damage and activate response
- Transducers- usually protein kinases, activated by sensors, phosphorylate mediators
- Mediators- proteins that directly or indirectly activate cell cycle check points (p53) or DNA repair
Why does the constant heavy chain domain change during isotype switching and not the light chain or variable heavy chain?
Because the variable domains are deteremined by VDJ and are essential for recognition. The constant domain, however, is essential for functions. So changing this domain changes the functino of the B cell, but not the type of antigen it recognizes.
What is RAGs involvement in VDJ recombination?
- RAG1 and RAG2 (only expressed in B/T cells) make a pair of DSB between signaling and coding segments.
- The break is repaired by NHEJ, with the excised DNA circularized and then lost in subsequent cell divisions.
What are the main excision repair systems called?
- Base Excision Repair (BER)- deaminations and depurinations
- Nucleotide Excision Repair (NER)- UV photoproducts, adducts, cross-links
- Mismatch Repair (MMR)- replication errors
What are the characteristics of Base Excision Repair (BER)?
- DNA damage is recognized and removed by specific glycosylase (removes base, leaving AP site)
- Single nucleotide gap is filled in by polymerase and sealed by DNA ligase
- Least “flexible” in responding to DNA damage (due to specific glycoyslases)
When and where does VDJ recombination occur?
Occurs at the DNA level and only in developing lymphocytes
What are the general steps of VDJ recombination?
- D to J recombination (DNA)
- V to DJ recombination (DNA)
- Transcription (RNA)
- Translation (protein)
What are the characteristics of non-homologous end joining (NHEJ)?
- Damage at ends of DNA is removed (nuclease) and endsare made compatible (polymerase), rejoin ends with ligase
- Usually involves a loss of squence, but it is easier and more flexible than HR
What are the two main types of adduct formation?
- Alkylating agents- chemicals with electrophilic groups bond with electron rich atoms in DNA and adds methyl, ethyl, or larger group. Can be activated by metabolic breakdown (BPDE and smoke)
- Cross-linking agents- bifunctinoal agents (two reactive groups) bond two positions on DNA. Can be intrastrand or interstrand (highly toxic)
What are the three main types of spontaneous DNA damage?
- Deamination- loss of amine group, changes A, G, or C (U, HX, or X). Results in transition mutation
- Base loss- rate of depurination (A/G) higher than depyrimidation (T/C), sugar/phosphate backbone left intact
- ROS- cleave deoxyribose sugar or phosphate backbone (strand break) or damage the base (replication block or mispair)
How is VDJ recombination related to cancer?
- Cancer can occur where translocations have happened between VDJ and cancer-causing gene, drives high expression of gene
- Many of the Non-Hodgkin Lymphomas and some Acute Leukemias (ALL)
Which protein induces DSB in meiosis?
Spo11
What are some examples of DNA damaging chemotherapy used against cancer?
- Alkylating agens (cytoxan)
- Crosslinking agents (cis-platin)
- Topoisomerase Inhibitors (etoposide)
What are the characteristics of homologous recombination (HR)?
- In meiosis, cells induce a DSB and recombination between homologous chromosomes generates genetic variability (gene conversion and cross-over)
- In mitosis, the intact sister chromatid is used as template for repair (not homologous because identical)
What is the difference in receptors for B cells vs. T cells?
B cells- immunoglobulins, Heavy chain (IgH) + light chaing (Igk or Ig-gamma)
T cells- T cell receptors, alpha-chain + beta-chain (alpha-beta or gamma-delta)
* Variable domains exist on each receptor so that different antigens can be seen*
What is transletion synthesis?
The ability of replication forks to progress through DNA damage, error-prone and uses specialized DNA polymerase (pols eta, zeta, and mu)
How are defects in genome surveillance involved in cancer?
Cancer is almost always associated with surveillance defects
- p53 can activate cell cycle arrest or apoptosis, lost in majority of all cancers (turmor supressor)
- BRCA1 is important for all cells, but mutated in 80% of all herediary breast cancer
