Types of Damages and Effects:
Living systems have perfected genetics of inheritance. This is achieved
by variety of natural process that leads to natural selection and speciation,
thus the genetic system of an organisms is inherited and perpetuated for
many thousands or even million years, occasionally make mistakes
because variety of causes; such mistakes can happen at gene level
or at chromosomal level, even at cytoplasmic level, means organelle DNA
can suffer damages. Often in course of time these changes if remain
and survived they lead to variation in plants and animals.
Carol Bernstein, A. R. Prasad et al; www.intechopen.com
Carol Bernstei, A. R.Prasad etal; www.intechopen.com
Chromosomal aberrations, as the above diagrams depict, can cause very serious
effect but also lead variations and speciation. http://www.tutorhelpdesk.com/
Chromosomal changes can be at numerical level or structural level:
Numerical level can be detected as aneuploidy and polyploidy.
Structural abnormalities can be deletions, duplications,
inversions and translocations. These changes have enormous
effects on organisms, some survive and others perish in the
struggle for existence, those survive inherit their characters differently.
Here is simple chromosomal diagram showing the broken nature of chromosomes. http://en.wikipedia.org/
Tautomerization involves nitrogenous bases; it is one of the common causes
of genetic errors. Bases under certain conditions can be in a state of enolic
form or immino form instead of their natural ketonic and amino forms.
Such tuatomeric forms can base pair with different bases, which leads to
change in the base pair sequences and generate mutations such as frame
shift in reading frame, silent mutation and nonsense and missense mutations.
Distribution of Transitions and Transversions among SNPs; A to G i.e one purine or pyrimidines to another purine or pyrimidines; change of
one purine to pyrimidine or pyrimidines to purine (rare), called transversions and transitions respectively. http://openi.nlm.nih.gov/
Look at the original triplet sequences, look at mutations- Point mutations can create silent mutations, missense mutations, nonsense mutations as shown above. http://utminers.utep.edu/
Point Mutations; http://www.studyblue.com/
Effect of tautomerization, where a normal base with either ketonic
or amino form can change into enolic or imminoform; these changes lead to different
base pairing; structural biochemistry; http://en.wikibooks.org/
AP sites are mainly generated by DNA glycosylases and depurination. Certain major alkylation products (e.g. 7-methylguanine and 3-methyladenine) increase rates of hydrolytic depurination by several orders of magnitude. Essentially error-free repair of AP sites is carried out by BER, NER and recombination repair, whereas error-prone DNA polymerases may synthesize DNA over the baseless site. This type of complexity in handling of damage may be more common than thought previously. ;https://www.researchgate.net/
· Transitions are due to substitution of purine with another purine and pyrimidine with another pyrimidine and substitution of one purine with a pyrimidine and vice versa invariably due to base substitutions and Tautomerisation. Cellular environment can easily cause such transitions. Even certain type of base modifications can cause transition type changes.
Very rarely Purines are replaced with Pyrimidines or Pyrimidines are replaced with Purines and such changes are called as Transversions.
Such Transversions type incorporations are possible due to mistakes during replication or bases are deleted and new base is added.
Enol form of guanines can easily methylated, which further cause more malformation in terms of information flow. The O-6methyl guanine can base pair with Thymidine,
When guanine gets methylated it can base pair with thymine; http://fbio.uh.cu/
Such changes can be due changes during replication are corrected; 3' to 5' Exonuclease Action If synthesis occurred in the opposite direction, the terminal end of a growing chain would contain a triphosphate group instead of an -OH group. This triphosphate would become the target of the proof-reading exonuclease and its removal would halt DNA replication. http://www.sparknotes.com/biology
In fact replication mechanism and the enzymes involved have inbuilt safety system in the form of proof reading, yet occasionally, perhaps one in one million nucleotides incorporation, there is a possibility, plausibility or probability for wrong nucleotide incorporation, which is enough to create an error. If the error happens in repetitive DNA or transposons does not result in any meaningful changes. If that error in the coding sequences is not repaired or corrected immediately and if the incorporated nucleotide in the codon codes for a wrong amino acid, which if it is important for protein function, such as binding site or catalytic site or both in enzymes or in structural proteins, then the error is manifested in biochemical dysfunction.
Whether it is a prokaryote or a eukaryote, they have inbuilt enzymes with 3’->5’ exonuclease activities, such as DNA pol-I in PK and DNA pol-delta, even DNA pol-gamma have 3’->5’ exonuclease activity, so they perform proof reading, which is called co-replication repair or proof reading mechanism; may have post replication repair mechanism.
Prokaryotes are more amenable for creating and detecting single gene mutations than eukaryotes, it is easy to identify and isolate such mutants and genes involved in repair system.
In higher organisms, the DNA damage repair systems are more or less on the same pattern as one finds in E.coli.
Though both PK and EK systems have foolproof mechanism of replication, occasional incorporation of non-complementary nucleotides is a possibility, but the enzymes or enzyme complexes that operate have specific subunits with 3’->5’ exonuclease activity. In addition, they have gap filling functions by polymerase functions. In spite of these functions, incorporation of wrong nucleotides does happen, but at a very low frequency, and they escape from the molecular proof reading enzymes called molecular vigilante.
Slippage error during replication; The mutation caused by replication slippage. In this figure, mispairing involves only one repeat. In fact, the slippage could cause several repeats to become unpaired. (a) Normal replication.(b) Backward slippage, resulting in the insertion mutation. (c) Forward slippage, resulting in the deletion mutation. http://www.web-books.com/MoBio
To combat these mismatch errors, cells are endowed with enzymes and the required factors that remove or correct the errors post replication stages and restore the normalcy. Replication can also produce what is called slippage error leading to duplication or deletion of some segments DNA. This can affect the information in terms of reading frame.
During recombination, whether it is homologous or site-specific process, occasionally, recombination leads to errors such as loss of certain regions of the DNA or gain in some segments of DNA, which leads to the change in the sequence of the reading frame. This can also produce substitutions, single base deletions and single base additions; all of them generate wrong reading frame, which may work or may not. Such recombination errors are also made during somatic recombination events. Recombination at repetitive DNA often creates mutations.
DNA break and repair; http://www.bioch.ox.ac.uk
. DSBs are sensed by the ring-shape heterodimer Ku70/Ku80 which then stabilizes the two DNA ends and recruits DNA-PK. Next, DNA-PK phosphorylates and activates the NHEJ effector complex (ligase IV/XRC44/XLF) that finally religates the broken DNA. . The ATM kinase is recruited to DSB via an interaction with the MRN (Mre11-Rad50-Nbs1) complex. Once at the break, ATM that becomes activated, phosphorylating multiple substrates. In a reaction that depends in multiple endo and exonuclease activities (including Mre11, Exo1 and CtIP) DSBs are resected forming ssDNA strands. These ssDNA regions attract Rad51 and other associated proteins. The Rad51-coated nucleoprotein filaments then invade the undamaged sister strands forming HJ structures. HR is completed by new DNA synthesis and still to be identified HJ “resolvase” enzymes.;https://www.intechopen.com/
Radiations like Gamma and X-rays can also cause ionizations or ring opening in bases, or they can open sugar rings or break strands and even they can cause chromosomal breaks. The radiations that cause ionization of bases can lead to wrong base pairing. This is one of the methods used to generate large number of mutations in plant breeding to generate new strains or varieties.
A daily dosage of daylight UV exposure, skin cells can accumulate 1000 nucleotides of Thymidine dimers per day. Dimrization prevents normal base pairing and disrupts continuous new strand formation during replication. This leads to skin cancer, which is called Xeroderma pigmentosa. Some people who work daily in very hot sun light are prone to such diseases than others.
In some cases the linking can be inter strands, which has devastating effects.
If radiations cause ionization in bases, that can lead to wrong base pairing in subsequent replication.
DNA interstrand crosslink repair and cancer Interstrand cross linking between G and G
Thymidine dimers C5-C5 and C6-C6 linkage; http://mb207.blogspot.com
Thymine dimerization induced by UV light ;
Highly reactive free radical induced damage:
Hyperbaric oxygen, gamma and UV radiations, ozone, peroxides and recycling drugs can cause serious damage to tissues; all of them adversely affect the tissues. Reactive oxygen radicals can cause severe damages via H2O2 production. However the activation superoxide dismutase (SOD) can take care of it, but if free radicals are produced in large amounts and for longer duration the effect will be devastating
Cells contain a variety of metal ions and many a times they generate free radicals such as iron and other metal ions. These ions are highly reactive and they can cause damage to DNA in the form of strand break or modifying bases or opening of rings. Free radicals are energy rich ions and they can easily attack deoxy ribose sugars and break the ring structure to generate opened rings with 3’phosphate groups. These free radicals also cause tissue damage. Such radicals generate during blood transfusion and also during certain biochemical reactions, where hydrogen peroxide generating enzymes are involved in the reactions. The damage to DNA by oxygen radicals is mediated by metal ions especially Fe^2+ ions, which leads to Fenton reactions.
Cells do have enzyme to combat intracellular free radical damage. One such enzyme is super oxide dismutases (SOD), which acts like scavengers of free radicals and remove them from circulation. Among many free radicals, H2O2^- very reactive and they can act on deoxyribose sugar rings and break them to generate 3’P-open sugar rings. Even iron free radicals can severely damage DNA.
Heat and Acid induced damages:
Within the body of an organism, most of the tissues are subjected to changes in pH and body temperature. Eating and drinking of hot food or liquid can cause damages to gut, stomach and intestinal tracks; they are the most abused systems. This can cause the loss of bases, because glycoside bonds between the base and the sugar residues for they are very week. When glycosidic bonds break, the bases can easily break, thus create apurinic sites. In the region of apurinic sites the sugar-phosphate-sugar backbone remains intact. In most of the cases guanine is removed and infrequently adenine is also removed. However it is not surprising to observe depyriminidation. In all these cases holes are generated. A single genome can suffer about 10000 base losses per day. If these depurination and depyriminidation are not corrected, cells don’t work even for a single day.
Chemical induced damages:
Ames test is one of the standard test to identify a chemical or an agent that can induce mutations which can be identified easily. Chemical damages to DNA can have devastating effect on environment and organisms. Chemical induced mutation can be detected by a famous test called Ames test.
The Ames Test for mammalian environmental mutagenicity; http://www.mun.ca/
Ames devised a simple but an effective test to detect mutagenic effect of chemicals on Salmonella typhimurium strains that are defective in Histidine pathway. When cells are grown on a medium without histidine as a nutrient or metabolite supplement the cell fail to grow. When such cells treated with chemical mutagens to be tested and then plated on to minimal medium, only those mutants that repaired the gene to normal Histidine synthesis grow, others won’t. This gives an identity that the said chemical is capable of causing mutation. Then this strain can be used for further investigation, where one can study, what kind of mutation has occurred.
Ames test and detection of mutation; http://www.mun.ca/
Studies have shown that a whole lot of chemicals cause damage to DNA in one form or the other. The list of chemicals that can cause damage is a long one; here only some important ones are quoted.
Nitrous acid causes Deamination of cytosine, adenine, guanine and 5’methyl cytosine.
If 5- methylated cytosines are deaminated, they produce thymidine and the glycosylase enzyme does not act on Thymidines; this results in a change in the sequence and cause mutations (transition mutations). Such 5-methylated cytosines sites act as mutation hot spots. Lac-I genes in bacteria one finds at least 80 mutational hot spots and this is due to the presence of 5-methylated cytosine sites.
How deamination reactions change the character of bases. http://www.sivabio.50webs.com/
Cytosine deamination reactions
Bases in DNA can change other forms, which change the codon information
Cytosine + HNO2 + (O) -----> Uracil,
In G=C base pairing if C-->converted to Uracil*, in the next round of replication U* base pairs with A (U*= A), in the next round of replication, in the place of U, Thymidine will be incorporated,
U = A----> T = A,
Here the C = G base pair is converted to T =A.
Hypoxanthin base pairs with cytosine,
A = T------> Hypoxanthine = T -----> HPX = C ------> G = C.
A=T base pairs are changed to G=C base pairing.
G* = C à Xanthine*, ----> X = C, ------> C =G.
In this G=C is changed to C=G.
5’CH3-C = G-----> T = G, ---T = A, C=G is converted to T = A. The bases thus produced are not removed and remain so. Thus these regions containing 5’methyl cytosines considered as mutational hot spots.
Change of 5’CH3 C=G to A=T
C = G ----> C is converted to U, then U pairs with A, the consequence is C=G is converted to U = A and U=A is converted T=A
· Spontaneous deamination of cytosine is also a process by means of
which 100 or more deamination reactions take place per genome per day?
Hydroxylamine treatment causes cytosine to be aminated at 6th position to produce 6- Hydroxylamine cytosine. This hydroxylamine group is a heavy adduct, which is not desirable.
There are many base analogues, which can be incorporated into DNA as substitutes. When such analogues get incorporated, they change base pairing subsequently, thus cause change in the information.
BURD pairs with Guanine, A=T is changed to A=BuRD, BuRD=G,
So A=T is changed to G=C
Similarly 2’aminopurine and 2, 6’ di aminopurine can act as substitute and provide base pairing. The 2’ aminopurine when incorporated pairs with Thymidine. But if this is ionized, it can easily base pair with cytosine.
Such base analogues are used in vitro mutational studies; they are 8’Azaguanine, 6’Azauracil, 5’ Fluorouracil and Thioguanine. Azaguanine is used in place of Guanine for sequencing DNA, which is rich in GC contents. Ionosine is another base, which can be incorporated as a nucleotide, and this nucleotide can pair with Adenine, Uracil and Cytosine.
Ionized Bases and Oxidative Bases:
As mentioned above ionized bases substitute base pair differently. Ionized Thymidine, Adenine and Cytosine, instead of base pairing with Adenine, Thymidine and Guanine, they base pair with Guanine, Guanine and Guanine (the last one with Hoogsteen base pairing) respectively.
T = A, ionized T instead base pairs with guanine; T*= G; -> C=G
A = T, ionized A base pairs with Guanine; A* = G;-> C=G
C = G, ionized C pairs with guanine by Hoogsteen mode, C* = G.
Guanine can be oxidized to 8-oxoguanine; https://en.wikipedia.org
O-6 methylation of Guanine base pairs; http://www.atdbio.com
There are several Alkylating chemicals, which transfer either ethyl or methyl groups to reacting positions of the bases, which can lead to hydrolysis of glycoside bond between the base and the sugar. This causes Apurinic or Apyrimidinic sites, leaving phosphate-sugar-phosphate backbone intact.
DNA + Dimethyl sulphoxide--> 7’CH3-G DNA.
7’CH3-G.DNA + Piperidine -> 5’ (p-G-DNA) n (more frequently) + 5’ (p-A-DNA) rarely.
7’CH3-G-DNA + Formic acid -> 5’ (P-G-DNA) n.
If the DNA is treated with Hydrazine followed by piperidine, the cleavage is before Cytidine and Thymidine, but if the DNA is treated with hydrazine in the presence of 1.5mM NaCl, cleavage is preferred only at Cytidine.
C=Gà C= CH3-G, leads to CH3G pairs with Thymidine, and then Thymidine pairs with Adenine.
CH3G=T à A=T
Most of the alkylating agents transfer their reactive methyl groups to suitable bases at specific positions. This kind of alkylation, generally leads to the break down of the glycosidic bonds and also breakdown of p-s-p backbone at depurinated sites.
Ethyl methane sulphonate transfers ethyl group to Guanine to its 6’O position creating 6’O-CH2 guanidine. This kind of ethylation can facilitate the O-ethylated Guanosine to pair with thymidine.
C=Gà C=CH2.Gà T=CH2.Gà T=A
If E.coli cells are exposed to higher levels of nitroso-guanine or to any other alkylating compounds, cells become resistant to mutagenic and toxic effects of mutagens. This is because, the drugs induce the synthesis of O-6-methyl guanine by methyl transferase, which is responsible for the removal of methyl group from O-6 th position, at the same time another enzyme that is induced is Glycosylase, which removes the modified bases and creates Apurinic or Apyrimidinic sites. The induction is 100 fold and thus cells become resistant to the methylating drugs. A group of genes called ‘Ada’ produce these enzymes, and these genes are positively regulated.
Effect of Bleomycin: It is an antibiotic and it can cause strand breaks.
Mitomycin effect: It is another antibiotic, which react with ds DNA strands and brings about covalent linkages between complementary bases. This distorts the structure of the DNA prevents the separation of ds DNA at the time of replication. It can also cause strand breaks.
Effect of Acridine, Proflavins and Ethidium dyes:
These dyes are fluorescent compounds, they intercalate or insert the dyes into spaces found in between flat base pairs and lengthen the DNA. Such lengthening of the DNA can cause either deletion or addition during next round of replication, these are manifested as bulges.
Effect of chemical carcinogens: Afflotoxin, Benzo (a) pyrene and Methyl cholanthrene:
Afflotoxin are the products of Fungus Amanita species, Benzo (a) pyrene is the product of burnt food or burnt gasoline found in emissions. They bind and covalently link to bases as bulky adducts. Afflotoxin by alkylation at N7th position can lead to the break of P-S-P bonds. Potato chips and French fries fried in oil produce acrylic compounds, which are carcinogenic.
Benzo(a)pyrene, a chemical produced by internal combusion engines and thus common in the environment, is not itself mutagenic. However, in the mammalian liver, benzo(a)pyrene is metabolized to diol epoxide, which binds covalently to guanine bases, preventing proper base pairing with cytosine bases. Bulky Addition Products such as diol epoxide or Aflatoxin B1 may result in depurination mutagenesis and are known to be carcinogens.;http://www.mun.ca
Carcinogen bound foodmaterials; https://aghealth.wordpress.com;https://www.daf.qld.gov.au;http://www.yourhealthremedy.com http://bioweb.uwlax.edu
Benzo(a)pyrene, a chemical produced by internal combusion engines and thus common in the environment, is not itself mutagenic. However, in the mammalian liver, benzo(a)pyrene is metabolized to diol epoxide, which binds covalently to guanine bases, preventing proper base pairing with cytosine bases. Bulky Addition Products such as diol epoxide or Aflatoxin B1 may result in depurination mutagenesis and are known to be carcinogens; inding of carcinogen leads to change in Guanine affinity to cytosine; http://www.mun.ca
Some carcinogens; http://www.biologydiscussion.com
From the above account it can be understood why DNA is damaged and how DNA can be damaged. The following are few important forms of damages:
Combination of X-rays and UV radiations is deadly.
In all the above said cases, cells are endowed with factors and enzymes to sense the kind of damage, respond to different types of damages and repair the DNA before it is perpetuated. In spite of this kind of molecular vigilance some damages escape the repairs system and cause heritable defect, which can be devastating, or cancer in a lifetime of an individual.
Types and frequency of DNA damage on daily basis:
Single strand break
Double strand braks
Interstrand cross link/dimerization