It has been suggested thatchanges in the way we preparefood can reduce the amountsof HCAs produced. Ovenroasting,marinading, andcoating food with breadcrumbsbefore frying are modificationsthat may reduce the formationof HCAs.
The repairing process begins with the protein MutS which binds to mismatched base pairs. Then, MutL is recruited to the complex and activates MutH which binds to GATC sequences. Activation of MutH cleaves the unmethylated strand at the GATC site. Subsequently, the segment from the cleavage site to the mismatch is removed by exonuclease (with assistance from helicase II and SSB proteins). If the cleavage occurs on the 3' side of the mismatch, this step is carried out by exonuclease I (which degrades a single strand only in the 3' to 5' direction). If the cleavage occurs on the 5' side of the mismatch, exonuclease VII or RecJ is used to degrade the single stranded DNA. The gap is filled by DNA polymerase III and DNA ligase. The distance between the GATC site and the mismatch could be as long as 1,000 base pairs. Therefore, mismatch repair is very expensive and inefficient.
Mutation and dna repair mechanisms
MUTATIONS AND DNAREPAIR MECHANISMS
Mutations can occur in a number of ways:1. Errors can occur during DNA replication, DNA repair, or DNA recombination which can lead to base-pair substitutions, insertions, or deletions, as well as mutations affecting longer stretches of DNA.2. Mutagens are chemical or physical agents that interact with DNA to cause mutations.3. Physical agents include high-energy radiation like X-rays and ultraviolet light.4. Some errors can be corrected by direct repair, while others are repaired by more complex mechanisms.
MUTATIONS are changes in the genetic material of a cell (or virus). Some are large-scale mutations in which long segments of DNA are affected (example: translocations, duplications, and inversions). A chemical change in just one base pair of a gene causes a spontaneous or point mutation. A base-pair substitution is a point mutation that results in replacement of a pair of complimentary nucleotides with another nucleotide pair. Some base-pair substitutions have little or no impact on protein function. If these occur in gametes or gamete-producing cells, they may be transmitted to future generations and cause novel traits or defects.
Silent /synonymous mutations changes a codon but does not alter the amino acid encoded. Alterations of nucleotides still indicate the same amino acids because of redundancy in the genetic code. Such mutations may still have effects on mRNA stability. Nonsynonymous mutations result in an altered sequence in a polypeptide or functional RNA: one or more components of the sequence are altered or eliminated, or an additional sequence is inserted into the product. Transversions (blue): replacement of a purine by a pyrimidine or that of a pyrimidine by a purine. Transitions – (black ): replacement of one purine by the other or that of one pyrimidine by the other.
Missense mutations are those that still code for an amino acid but change the indicated amino acid. Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein.
Insertions and deletions are additions or losses of nucleotide pairs in a gene. These have a disastrous effect on the resulting protein more often than substitutions do. Unless these mutations occur in multiples of three, they cause a frameshift mutation. All the nucleotides downstream of the deletion or insertion will be improperly grouped into codons. The result will be extensive missense, ending sooner or later in nonsense - premature termination.
Mutation class Type of mutation Incidence Base Comparatively common type of mutation in coding All typessubstitutions DNA but also common in noncoding DNA Transitions and Transitions are more common than transversions, transversions especially in mitochondrial DNA Synonymous substitutions are more common than Synonymous and nonsynonymous substitutions in coding DNA; nonsynonymous conservative substitutions are more common than substitutions non-conservative Gene conversion-like Rare except at certain tandemly repeated loci or events (multiple base clustered repeats substitution) One or a few Very common in noncoding DNA but rare in coding Insertions nucleotides DNA where they produce frameshifts Triplet repeat Rare but can contribute to several disorders, expansions especially neurological disorders Rare; can occasionally get large-scale tandem Other large insertions duplications, and also insertions of transposable elements One or a few Very common in noncoding DNA but rare in coding Deletions nucleotides DNA where they produce frameshifts Rare, but often occur at regions containing tandem Larger deletions repeats or between interspersed repeats Rare as constitutional mutations, but can often beChromosomal Numerical and pathogenic. Much more common as somaticabnormalities structural mutations and often found in tumor cells
1.Purine bases are lost by spontaneous fission of the base- sugar link.2.Cytosines, and occasionally adenines, spontaneously deaminate to produce uracil and hypoxanthine respectively.3.Many chemicals, for example alkylating agents, form adducts with DNA bases.4.Ultraviolet light causes adjacent thymines to form a stable chemical dimer.5.Ionizing radiation causes single or double-strand breaks.6.Reactive oxygen species in the cell attack purine and pyrimidine rings.7.Mistakes in DNA replication result in incorporation of a mismatched base.8.Mistakes in replication or recombination leave strand breaks in DNA.
Chemical Modification Depurination Photodamage thymine dimer Chemical Modification by O2 free Deamination radicals
(A) depurination (loss of purine bases)resulting from cleavage of the bond between the purine bases and deoxyribose, leaving an apurinic (AP) site in DNA and (B)deamination (converts cytosine to uracil; adenine to hypoxanthine)
is the addition of methyl or ethyl groups tovarious positions on the DNA bases. Example: alkylation of guanine by ethylmethane sulfonate (EMS). At the left is anormal G-C base pair. Note the free O6 oxygen (red) on the guanine. EMS donates an ethyl group (blue) to the O6oxygen, creating O6-ethylguanine (right), which base-pairs with thymine instead of cytosine. Mustard gas (sulfur mustard) is the most well-known example because of itsuse and consequences observed during World War I. It has two reactive groups that form intra-chain and inter-chain cross-links on DNA directly.
This lesion can be repaired by an enzyme (O6- methylguanine methyltransferase) that transfers themethyl group from O6- methylguanine to acysteine residue in its active site, and the original guanine is restored. This reaction is widespread in both prokaryotes andeukaryotes, including humans.
from the sun is carcinogenic andis a principal cause of skin cancer. 3 types of ultraviolet radiation (UV) from the sun: UVA (wavelength 320–380 nm), UVB (wavelength 290–320 nm), and UVC (wavelength 200–290 nm). UVC penetrates into the superficial layer of the skin, UVB penetrates into the basal level of the epidermis, and UVA penetrates into the more acellular dermis level. UVB is the most effective carcinogen because it causes UV photoproducts. Cyclobutane pyrimidine dimers are responsible for at least 80% of UVB-induced mutations. The precise class of mutations resulting from pyrimidine dimers is a unique molecular signature of skin cancer. UVA indirectly damages DNA via free radical-mediated damage. Water is fragmented by UVA, generating electron-seeking ROS that cause DNA damage (transversions are characteristic of UVA damage).
most common type of DNA damage caused by UV irradiation. (a) UV light cross-links thetwo thymine bases on the top strand. This distorts theDNA so that these two bases no longer pair with their adenine partners. (b) The two bonds joining the two thymines form a 4-membered cyclobutane ring (red). http://highered.mcgraw-hill.com/olc/dl/120082/micro18.swf
UV-induced thymine dimers can be repaired byphotoreactivation. The enzyme (photolyase) absorbs visible light and binds to damaged DNA. The enzyme breaks the dimer, and finally dissociates from the repaired DNA. Repair of pyrimidine dimers byphotoreactivation is common to prokaryotic and eukaryotic cells, including E. coli, yeasts,and some species of plants and animals. Photoreactivation is not universal; many species (including humans) lack this mechanism of DNA repair. 15
UV-damaged skin cells are eliminated by initiating apoptosis (peeling of the skin after a sunburn). Mutations in the p53 gene (tumor suppressor p53 protein is an important regulator of apoptosis). These mutations yield 9 hot spots which are sites where removal of cyclobutane pyrimidine dimers is particularly slow, and consequently allows the proliferation of mutated p53 cells. UV radiation thus induces the formation of tumor cells by blocking apoptosis, and clonal expansion of the p53 mutants. Sunscreens work on the basis of including UV-absorbing organic chemicals (e.g. cinnamates), inorganic zinc- containing pigments, or titanium oxides in their ingredients to minimize UV absorption by the skin. Sunscreens must be used with care since some compounds may be photosensitized carcinogens, (chemicals that can be activated by UV to become carcinogenic), e.g. 5-methoxy psoralen, and fluoroquinolone antibiotics (stay out of the sun during their administration)!
high-energy radiation capable ofproducing ionization in substances through which itpasses, e.g. x-rays, alpha and beta rays, and neutrons froma nuclear reaction. It can directly ionize atoms comprising DNA, or indirectly by the interaction with water molecules (radiolysis) that generate dangerous reactive oxygen species (ROS): the hydroxyl radical (–OH), hydrogen peroxide (H2O2), and the superoxide radical (O–2). A free radical reacts very strongly with other molecules as it seeks to restore a stable configuration of electrons. A free radical may drift about up to 1010 longer than the time needed for the initial ionization, increasing the chance of it disrupting DNA and cause mutations.
Oxidation of DNA is one of the main causes of mutation, and explains why free radicals produced by radiation exposure as well as endogeneous cellular reactions (e.g., oxidative respiration and lipid peroxidation) are such potent carcinogens. Oxidation can produce oxidized bases, e.g., adenine mispairs with 8-oxoguanine during replication leading to a G→T transversion mutation. The -OH radical removes electrons from any molecule in its path, turning that molecule into a free radical and so propagating a chain reaction. H2O2 is more dangerous to DNA than the -OH radical. Its slower reactivity gives it time to travel into the nucleus of a cell, where it is free to wreak havoc upon DNA. The superoxide radical is not very reactive but acts more as a catalyst for the generation of the other ROS intermediates. Double-strand DNA breaks cause ionizing radiation-induced carcinogenesis.
: The common mechanism of action is that an electrophilic (electron-deficient) form reacts with nucleophilic sites (sites that can donate electrons) in the purine and pyrimidine rings of nucleic acids. Some chemicals are base analogues that may be substituted into DNA, and pairs incorrectly during DNA replication. Other mutagens interfere with DNA replication by inserting into DNA and distorting the double helix. Still others cause chemical changes in bases (DNA adducts) that change their pairing properties.Carcinogens can be segregated into 10 groups: polycyclic aromatic hydrocarbons carbamates halogenated compounds aromatic amines nitrosamines and nitrosamides azo dyes hyrazo and azoxy compounds natural products inorganic carcinogens miscellaneous compounds (alkylating agents, aldehydes, phenolics)
) are carcinogens produced by cookingmeat, formed from heating amino acids and proteins. About 20 HCAs havebeen identified. Three examples, Phe- P-1, IQ, and Mel Q, are shown.These are examples of carcinogens towhich we are exposed daily and which are produced in our own kitchens!Oven roasting, marinading, and coatingfood with breadcrumbs before fryingare modifications that may reduce the formation of HCAs.
are found in tobacco or are formed when preservative nitrites react with amines in fish and meats during smoking. Their principal carcinogenic product is alkylated O6 guanine derivatives. (a) An example of nitrosamines: alkylnitrosoureas.(b) A potential carcinogenic product of nitrosamines: O6 adduct of guanine. Guanine is shown for comparison.
treatment of DNA results in the conversion of adenine into hypoxanthine, which pairs with cytosine, inducing a transition from A-T to G-C. induce frameshift mutations byintercalating into the DNA, leading to the incorporation of an additional base on the opposite strand.
. The compound, produced by molds that grow on peanuts, is activated by cytochromeP450 to form a highly reactive species that modifiesbases such as guanine in DNA, leading to mutations.
Asbestos is a group of fibrous silicate minerals that was used extensively in building materials because of its insulating properties but is now prohibited due to association with several diseases of the lung, including lung cancer and mesothelioma. Erionite is a fibrous zeolite mineral formed from volcanic rock. Mechanisms of carcinogenesis include generation of ROS and induction of a chronic inflammatory response. Genetics may predispose some people to the carcinogenic effects of fibrous materials.
1. Areas of investigation on the molecular events behind the mechanism of bacteria-induced transformation include: the promotion of host cell proliferation, the generation of oxygen free radicals and subsequent DNA damage, and the activation of oncogenes.2. DNA tumor viruses encode viral proteins that block tumor suppressor genes, often by protein–protein interactions. Retroviruses may cause cancers in animals by encoding mutated forms of normal genes (i.e. oncogenes) that have a dominant effect in host cells. Examples: Human papillomavirus (HPV) - cervical cancer Kaposi’s sarcoma-associated herpes virus (KSHV) - Kaposi’s sarcoma Hepatitis B virus - liver cancer Epstein–Barr virus (EBV) - nasopharyngeal carcinoma Human T-cell leukemia virus type 1 (HTLV-1) – a retrovirus known to cause acute T-cell leukemia (ATL) Helicobacter pylori - a Gram-negative spiral bacterium that establishes chronic infection and ulcers in the stomach and one of the causative agents of gastric cancer. The typhoid pathogen, Salmonella enterica serovar Typhi (S. typhi), establishes chronic infection in the gallbladder and has been linked to hepatobiliary and gallbladder carcinoma.
The bases of DNA can exist in rare This base tautomeric forms. The imino analog of thymine has atautomer of adenine can pair with higher tendency to form an cytosine, eventually leading to a enol tautomer than does transition from A-T to G-C. thymine itself. The pairing of (Tautomerization is the the enol tautomer of 5-interconversion of two isomers that bromouracil with guanine willdiffer only in the position of protons lead to a transition from T-A and often, double bonds). to C-G.
(a)(b) Metabolic activation of BP (Benzopyrene)
Benzopyrene ( found in cigarette smoke) reacts with DNA bases, resulting in the addition of large bulky chemical groups to the DNA molecule and cause G→T transversions. Locations of these adducts matched the distribution of p53gene mutations in lung tumors from smokers (Science,1996). It is estimated that 104 to 106 mutations occur in a single human cell per day. Each day the DNA of a human cell loses about 5,000 purines, and about 100 cytosines spontaneously deaminate to uracil. Damage to DNA can block replication ortranscription, and can result in a high frequency of mutations.
Under normal circumstances, the immense error burden is successfully dealt with by the highly efficient cellular DNA repair mechanisms.Major DNA repairing mechanisms: base excision, nucleotide excision and mismatch repair.
A DNA glycosylase specific for G-T mismatches, usually formed by deamination of 5-methyl C residues, flips the thymine base out of the helix and then cuts it away from the sugar-phosphate DNA backbone (1), leaving just thedeoxyribose (black dot). An endonucleasespecific for the resultant baseless site then cuts the DNA backbone (2), and the deoxyribose phosphate is removed by an endonuclease associated with DNApolymerase (3). The gap is then filled in by DNA Pol ß and sealed by DNA ligase (4), restoring the original G-C base pair.
DNAs bases may be modified by deaminationor alkylation. The position of the modified (damaged) base is called the "abasic site" or "AP site". DNA glycosylase can recognize the AP site and remove its base. Then, the AP endonuclease removes the AP site and neighboring nucleotides. The gap isfilled by DNA polymerase I and DNA ligase.
Proteins UvrA, UvrB, and UvrC areinvolved in removing the damaged nucleotides(e.g., the dimer induced by UV light). The gap is then filled by DNA polymerase I and DNA ligase. In yeast, theproteins similar to Uvrs are named RADxx (radiation), such as RAD3, RAD10, etc.
A DNA lesion that causes distortion of the double helix, such as a thymine dimer, is initially recognized by a complex of the XP-C (Xeroderma pigmentosum C protein) and 23B proteins (1). This complex thenrecruits transcription factor TFIIH,whose helicase subunits, poweredby ATP hydrolysis, partially unwind the double helix. XP-G and RPA proteins then bind to the complex and further unwind and stabilize the helix until a bubble of ≈25 bases is formed (2). Then XP-G (now acting as an endonuclease) and XP-F, a 2nd endonuclease, cut the damaged strand at points 24–32 bases apart on each side of the lesion (3).
This releases the DNA fragment with the damaged bases, which is degraded to mononucleotides. Finally the gap is filled by DNA polymerase exactly asin DNA replication, and the remaining nick is sealed by DNA ligase (4 )
The mismatch repair system detects and excises mismatched bases in newly replicated DNA, which is distinguished from theparental strand because it hasnot yet been methylated. MutSbinds to the mismatched base,followed by MutL. The bindingof MutL activates MutH, whichcleaves the unmodified strandopposite a site of methylation.MutS and MutL, together withhelicase II, SSB proteins, and an exonuclease, then excisethe portion of the unmodified strand that contains the mismatch. The gap is thenfilled by DNA polymerase and sealed by ligase.
Mismatch repair in eukaryotes may be similar to that in E. coli. Homologs of MutS and MutL have been identified in yeast, mammals, and other eukaryotes. MSH1 to MSH5 are homologous to MutS; MLH1, PMS1 and PMS2 are homologous to MutL. Germline mutations of MSH2, PMS1 and PMS2 are related to colon cancer. Loss of function of the protein products encoded by these genes is responsible for complete loss of mismatch repair. In eukaryotes, the mechanism to distinguish the template strand from the new strand is still unclear, but maybe related to the action of DNA methylases (the old DNA strand is methylated).
A complex of the MSH2 and MSH6 proteins binds to a mispaired segment of DNA such as to distinguish between the template and newly synthesized daughter strands (1). This triggers binding of the MLH1 endonuclease, as well as other proteins such as PMS2, which has been implicated inonco-genesis through mismatch-repair mutations. A DNA helicase unwinds the helix and the daughter strand is cut; an exonuclease then removes several nucleotides, includingthe mismatched base (2). Finally, as with base excision repair, the gap is then filled in by a DNA polymerase (Pol, in this case) and sealed by DNA ligase (3 ).
The presence of a thymine dimer blocks replication, but DNA polymerase can bypass the lesion and reinitiate replication at a new site downstream of the dimer. The result is a gap opposite thedimer in the newly synthesized DNA strand. In recombinational repair, this gap is filled by recombination with the undamaged parental strand. Although this leaves a gap in the previously intact parental strand, the gap can be filled by the actions of polymerase and ligase, usingthe intact daughter strand as a template. Two intact DNA molecules are thus formed, and the remaining thymine dimer eventually can be removed by excision repair.
If the replication fork encounters anunrepaired lesion or strand break, replication generally halts and the fork maycollapse. A lesion is left behind in an unreplicated, single-stranded segment ofthe DNA; a strand break becomes a double-strand break.There are two possible avenues for repair: recombinational DNArepair or, when lesions are unusuallynumerous, error-prone repair. The latter involves DNA polymerase V, encoded by the umuC and umuD genes that can inaccurately replicate over many types of lesions. The repair mechanism is referred to as error- prone becausemutations often result.
UV light activates the RecA co-protease, which stimulates theLexA protein (purple) to cleave itself, releasing it from the umuDC operon. This results in synthesis of UmuC and UmuD proteins, which somehow allow DNA synthesis acrossfrom a thymine dimer,even though mistakes (blue) will be made.
The black and red DNAs represent the homologous sequences on sister chromatids. (1) A double- strand DNA break forms in the chromatids. (2) The double- strand break activates the ATM kinase; this leads to activation of a set of exonucleases thatremove nucleotides at the break from the 3’ and 5’ ends of both broken strands, ultimatelycreating single stranded 3’ ends. In a process that is dependent on the BRCA1 and BRCA2 proteins, as well as others, the Rad51 protein (green ovals) polymerizes on single-strandedDNA with a free 3’ end to form a nucleoprotein filament.
(3): Aided by yet other proteins, one Rad52 nucleoprotein filament searches for the homologous duplex DNA sequence on the sister chromatid, then invades the duplex to form a joint molecule in which the single stranded 3’ end is base-paired to thecomplementary strand on the homologous DNAstrand. (4) The replicative DNA polymeraseselongate this 3’ end of the damaged DNA (greenstrand), templated by the complementary sequences in the undamaged homologous DNA segment.
(5) Next this repaired3’ end of the damaged DNA pairs with thesingle stranded 3’ endof the other damaged strand. (6) Any remaining gaps are filled in by DNApolymerase and ligase (light green), regenerating a wild- type double helix in which an entire segment (dark and light green) has been regenerated from thehomologous segment of the sister chromatid.
A double-strand break activates the ataxia telangiectasia mutated (ATM) kinase. The RAD50/MRE11/NBS1 complex (a substrate of ATM) uses its 5′–3′ exonuclease activity to create single-stranded 3′ ends. BRCA1/2 aids in the nuclear transport of RAD51. RAD52 facilitates RAD51 binding to these exposed ends to form a nucleoprotein filament. RAD51 can exchange a homologous sequence from a single strand within a double-stranded molecule (e.g. a sister chromatid), with a single-stranded sequence. The sequences from the double- stranded molecule are then used as a template sequence for repair. Resolvases restore the junctions formed as a result of homologous recombination, called Holliday junctions. Two copies of intact DNA molecules are produced with rarely any errors.
In general, nucleotide sequences are butted together that were not apposed in the unbroken DNA. These DNA ends are usually from the same chromosome locus, and when linked together, several base pairs are lost. Occasionally, ends from different chromosomes are accidentally joined together. A complex of two proteins, Ku and DNA-dependent protein kinase,binds to the ends of a double-strand break (1). After formation of a synapse, the ends are further processed by nucleases, resulting in removal of a few bases (2), andthe two double-stranded moleculesare ligated together (3). As a result,the double-strand break is repaired, but several base pairs at the site of the break are removed.
Several conventional therapies aim to induce extensive DNAdamage in order to trigger apoptosis and paradoxically includeagents classified as carcinogens. Other conventional therapiesinhibit DNA metabolism in order to block DNA synthesis in therapidly dividing cancer cells. Still other drugs interfere with themechanics of cell division. The development of drug resistanceis a major problem for chemotherapy. and : have the ability to form DNA adducts by covalent bonds via an alkyl group or a platinum atom, e.g. clorambucil and cisplatin. The resulting DNA damage triggers apoptosis. Cisplatin had a major impact on ovarian cancer, but associated with irreversible kidney damage. Carboplatin is a less toxic platinum analog. : are compounds that are structurally similar to endogenous molecules (e.g. nitrogenous bases of DNA) and therefore can mimic their role and inhibit nucleic acid synthesis (e.g. 5-FU and methotrexate).
: Doxorubicin is a fungal anthracyclineantibiotic that inhibits topoisomerase II. The plantalkaloids vincristine and vinblastine (from the periwinkleplant) bind to tubulin and prevent microtubule assembly.Paclitaxel (taxol) binds to the β-tubulin subunit inpolymers and stabilizes the microtubules againstdepolymerization. Thus two opposing strategies can beused to disrupt the mitotic spindle. Ionizing radiation is delivered to thetumor by electron linear accelerators. Radiation-induceddamage can become permanent due to the generationof ROS if oxygen is present. More double-strand breaksoccur in cells irradiated in the presence of oxygen thanin cells irradiated in the absence of oxygen. Targeting ofthe tumor has been made more precise by moderntechniques such as magnetic resonance imaging (MRI)and computed tomography (CT) which produce 3-Dimages of the tumor within the body.
If DNA can repair itself, Go ahead, indulge yourself and enjoy life’s pleasures! After all, life is short … But DNA can only do so much for itself…Abusing its potentials can cause YOU and your future generations major, major problems!