L3 dna damage, repair, mutations

• hereditary/germline mutations are inherited. while acquired/somatic mutations cannot be passed on to the next generation.
• mutagenesis is a relative weak force for changing allele frequencies because typical mutation rates are too low
• there are mutations at 2 different levels. 1. chromosome and 2. dna
• Mutations at DNA levels (point or frameshift mutation): changes in a single base such as base pair exchange or addition, deletion, shift of a base
• chromosomal aberrations: changes in chromosomes, such as chromosome gaps, breaks, translocations, and changes in number of chromosomes
• A mutagen is a chemical or physical agent capable of inducing mutations
• there are two types of mutagens: chemical and physical
• examples of chemical agents: alkylating agents, dimethylnitrosoamine, arsenic, nickel, chromium, base analogs
• examples of physical agents: ionizing radiations (x rays, gamma rays alpha particles) and non ionizing radiations (uv)
• chemical mutagens: base analogs, hydroxylating agents, alkylating agents, deaminating agents, intercalating agents
• base analogs: mimics the structure of one of the four bases found in DNA. ex. T:A --> C:G
• hydroxylating agents: add an OH group to a carbon atom on the sugar-phosphate backbone of DNA. ex. Hydroxy-C pairs with A.

  
  

  insert between bases: intercalating agents. roughly the same size and shape as a base pair of the double helix. insertions and deletions.
• alkylating agents: attach an alkyl group to a nitrogenous base. ex. G:C --> ethyl-G pairs with T
• deaminating agents: agents remove amine (-NH2) groups; nitrous acid modifies cytosine to uracil, pairs with A instead of G.
• 5 types of DNA Damage

  
  

  1. Depuration and Depyrimidination

  • The hydrolysis of A or G bases leaves a DNA strand w/ an unspecified base
  • Lose purine/pyrimidine

  2. Deamination

  • REMOVAL OF AN AMINE GROUP

  3. DNA breaks

  • x rays break the DNA backbone
  • break the sugar phosphate backbone and split DNA into smaller pieces
  • high E x rays

  4. Formation of Thymine Dimers

  • UV light produces thymine dimers
  • T-T --> T ----T. bridge forms , distorts the DNA structure
  • not as strong as x ray tho

  5. Oxidation

  • irradiation causes formation of free radicals that can damage individual bases.
  • ex. changes G-C to T-A
• DNA repair: cells possess the ability to correct or repair many of the mutations. If not corrected, the incorrect info may be transcribed into RNA. And the mutation is then expressed as an altered protein.
• Repair process:

  1. recognize the damage

  2. remove it

  3. repair DNA synthesis

  4. dna ligation

  DNA repair enzymes:

  • endonuclease, DNA polymerase, dna ligase, methyl transferase

  repair mechanisms

  • base excision repair, nucleotide excision repair, double strand break repair, mismatch repair, MGMT (meghylguanine Dna methyltransferase repair)
• DNA DAMAGE AND REPAIR MECHANISMS

  Base Excision Repair: caused by ROS, X RAY, alkylating agents, spontaneous rxns. a pathway of excision repair that recognizes damage to single bases, such as deamination or alkylation, and either repairs the base alone (short patch) or replaces 2-10 nucleotides (long patch)

  
  

  Nucleotide Excision repair: caused by UV light. bulky adducts. bigger size

  
  

  Mismatch Repair: caused by replication errors. A-G and T-C; insertion, deletion. small scale

  
  

  Double Strand break repair: caused by X-ray, ionizing radiation, anti-tumor agents. types: non homo and homo
• double strand break repair

  nonhomologous end joining: occurs during all cell cycle, often deletion made. no reference, harder to repair.

  homologous recombination: occurs during S/G2 period, more accurate DNA repair. easier to repair because information from sister chromatid
• humans have 46 chromosomes in the nucleus of each body cell. each chromosome carries hundreds to thousands of genes
• karyotype: the chromosomes are arranged in a standard presentation format of longer to shortest. arrangement of chromosomes in descending order of size
• clastogenesis: process resulting additions, deletions, or rearrangements of parts of the chromosomes detectable by light microscopy. same as mutagenesis but not DNA, rather chromosome.
• gaps: achromatic lesions in a chromosome, may vary in length and are thought to be due to loss of DNA
• breaks: broken ends of chromatids that are dislocated but still contained within metaphase.
• chromosomal rearrangements (typically end in -ion): deletions, duplications, inversion, translocations, transpositions

  changes in chromosome number: euploidy, polyploidy, aneuploidy
• translocations have 2 types: reciprocal and non reciprocal.

  non: unequal exchanges (only one trades, the other disappears)

  reciprocal: equal (trade places)
• euploidy: complete set number of chromosomes. ex. diploidy (2x), monoploid (1x)

  polyploidy: more than diploid. 3, 4x (triploidy, tetraploidy)

  aneuploidy: loss or gain of one or more chromosomes producing a chromosome number that's not an exact multiple of the haploid number.. ex. monosomy (2n-1), trisomy (2n+1), tetrasomy (2n+2)
• Lipitor: not mutagenic or clastogenic. was negative in the in vivo mouse micronucleus test
• Ames Test: rapid assay/test to screen potential mutagenic and carcinogenic compounds. based on a strain of salmonella that cant synthesize histidine. doesn't have enzymes to repair DNA . leaky cell walls.
• metaphase: stage of mitosis at which condensed chromosomes are aligned equidistant between poles at the mitotic spindle
• chromosome

  p: petit

  q: long
• micronuclei assay: micronuclei are formed when a chromosome or fragment of a chromosome is not incorporated into one of the daughter nuclei during cell division
• metacentric vs Afrocentric homologous chromosomes

  metacentric: centromere is in middle

  afro: centromere is higher up
• BrdU (bromodeoxyuridine) - a nucleoside analog of thymine. marker for if there is sister chromatid exchange u will know.
• G bands: series of striations...stained
• Bar code: each chromosome displays a unique banding pattern . allows it to be readily differentiated from other chromosomes of same size and centromeric position
• in situ hybridization (ISH): powerful technique for localizing specific nucleic acid targets within fixed tissues and cells, allowing you to obtain temporal and spatial information about gene expression and genetic loci. can tell u location of specific gene of interest

  
  

  • treating samples with single stranded RNA or DNA probes that hybridize to sequence of interest
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  FISH (fluorescent in situ hybridization)

  • colors; alternative to radioactive labeling
  • attached to probe , use fluorescent microscope
  • drop cells onto glass slide, denature dna , add probes labeled with fluorescent dye and wash away unhybridized probe, expose to UV light
  • can provide gene mapping/location, detect large segment of chromosome painting, be used for interphase and metaphase chromosomes, identify chromosomal abnormalities
• chromogenic assay (chromosome painting)

  • diagnosis of aneuploidy is improved using fish
  • normal vs cancer cells u can easily tell the difference ...translocation,...missing pieces ..hyperploidy
  • similar to FISH, but use permanent peroxide reaction instead of the fluorescent dye. use reg light microscope