DNA damage ,satuanarayan.pdf

mutations the Some of the diseases caused by his due to insertion of transpons into a genes. me Sa WO he DAMAGE AND REPAIR OF DNA the re Being the carrier of ge...

mutations the Some of the diseases caused by his due to insertion of transpons into a genes. me Sa WO he DAMAGE AND REPAIR OF DNA the re Being the carrier of genetic information, A. cellular DNA must be replicated (duplicated), on maintained, and passed down to the daughter cells ne accurately. In general, the accuracy of replication is ed extremely high. However, there do OCCur replication errors. It is estimated that approximately ct one error is introduced per billion base pairs during on each cycle of replication. The cells do posses the capability to repair damages done to DNA to a large extent. Consequences of DNA damage Despite an efficient repair system for the damaged DNA, replication errors do accumulate that ultimately result in mutations. The human body possesses 10 nucleated cells, each with 3 x 10 base pairs of DNA. It is estimated that about 10!6 cell divisions occur in a lifetime. If 10-10 mutations per base pair per cell generation escape repair, this results in about one mutation per 106 base pairs in genome.  34 BIOTECHNOLOGY Besides the possible errors in replication, the TABLE 3.1 Major types of DNA damagesS by both DNA is constantly subjected to attack include Types physical and chemical agents. These which also Category radiation, free radicals, chemicals etc., Single-base alteration Deamination result in mutations. (C’U; A’hypoxanthine) majority of the Depurination It is fortunate that a great that does not mutations probably occur in the DNA Base alkylation not have encode proteins, and consequently will Insertion or deletion of organism. This is not, nucleotides any serious impact on the mutations do however, all the time true, since Incorporation of base analogue There are occur in the coding regions of DNA also. Two-base alteration UV light induced pyrimidine situations in which the change in a single base pair dimer alteration (T-T) serious disease in the human genome can cause a Oxidative free radical formation e.g. sickle-cell anemia. Chain breaks lonizing radiation TYPES OF DNA DAMAGES Cross-linkage Between bases in the same or opposite strands The damage done to DNA by physical, chemical Between the DNA and protein and environmental agents may be broadly classified molecules into four categories with different types (Table 3.1). The DNA damage may occur due to single-base MUTATIONS alterations (e.g. depurination, deamination), two base alterations (e.g. pyrimidine diamer) chain The genetic macromolecule DNA is highly breaks (e.g. ionizing radiation) and cross-linkages stable with regard to its base composition and (e.g. between bases). Some selected DNA damages sequence. However, DNA is not totally exempt are briefly described. from gradual change. A general picture of DNA damage and its repair is also described in this The occurrence of spontaneous deanmination bases in aqueous solution at 37°C is well known. chapter. Cytosine gets deaminated to form uracil while Mutation refers to a change in the DNA structure adenine forms hypoxanthine. of agene. The substances (chemicals) which can Spontaneous depurination, due to cleavage of induce mutations are collectively known as mutagens. glycosyl bonds (that connect purines to the The changes that occur in DNA on mutation backbone) also occurs. It is estimated that are reflected in replication, transcription and translation. 2000-10,000 purines may be lost per mammalian cell in 24 hours. The depurinated sites are called as abasic sites. Originally, they were detected in Types of mutations purines, and called apurinic sites (AP sites) which Mutations are mainly of two major types-point represent lack of purine. Now, the term AP sites is mutations, frameshift mutations (Fig. 3.14). generally used to represent any base lacking in 1. Point mutations : The replacement of one DNA. base pair by another results in point mutation.They The production of reactive oxygen species is are of two sub-types. often associated with alteration of bases e.g. (a) Transitions : In this case, a purine (or a formation of 8-hydroxy guanine. Free radical pyrimidine) is replaced by another. formation and oxidative damage to DNA increases (b) Transversions : These are characterized by with advancement of age. replacement of a purine by a pyrimidine or Ultraviolet radiations result in the formation of vice versa. covalent links between adjacent pyrimidines along 2. Frameshift mutations : These occur when the DNA strand to form pyrimidine dimers. DNA chain breaks can be caused by ionizing radiations one or more base pairs are inserted in or deleted (e.g. X-rays). trom the DNA, respectively, causing insertion or deletion mutations.  35 Chapter 3 : DNA REPLICATION, RECOMBINATION, AND REPAIR no detectable (A) genetic code. Therefore, there are effects in silent mutation. -C-GA-G Transition -C-GG-G changed 2. Missense mutation : In this case, the acid. For -G-CT-C -GCCC base may code for a different amino codes example, UCA codes for serine while ACA C GA-& Transverson -C--G-T-G for threonine. The mistaken (or missense) amino acid may be acceptable, partially acceptable or -G-CT-C -G-C-A-C protein unacceptable with regard to the function of molecule. Sickle-cell anemia is a classical example (B) of missense mutation. -C-G-A-T-G codon Insertion 3. Nonsense mutation : Sometimes, the a termination (or -G-CT-A-C with the altered base may become CG-A-G change in the nonsense) codon. For instance, result in second base of serine codon (CA) may -G-CT-C UAA, The altered codon acts as a stop signal and -CG-C Deltion -GCG causes termination of proteinsynthesis, at that Consequences of frameshift mutations point. of a base in a The insertion or deletion Fig. 3.14 : An illustration of mutations (A)-Point of the mutations; (B)-Frameshift mutations. gene results in an altered reading frame mRNA(hence the name frameshift). The machinery of mRNA (Containing codons) does not recognize Consequences of point mutations that a base was missing or a new base was added. Since there are no punctuations in the reading of The change in a single base sequence in codons, translation continues. The result is that the point mutation may cause one of the following protein synthesized will have several altered amino (Fig. 3.15). acids and/or prematurely terminated protein. 1. Silent mutation : The codon (of mRNA) Mutations and cancer containing the changed base may code for the Mutations are permanent alterations in DNA same amino acid. For instance, UCA codes for serine and change in the third base (UCU) still structure, which have been implicated in the codes for serine. This is due to degeneracy of the etiopathogenesis of cancer. REPAIR OF DNA UCU As alreay stated, damage to DNA caused by (codon for Ser) replication errors or mutations may have serious consequences. The cell possesses an inbuilt system Silent to repair the damaged DNA. This may be achieved mutation by four distinct mechanisms (Table 3.2). UCA 1. Base excision-repair (codon for Ser) Missense 2. Nucleotide excision-repair mutaiNonse 3. Mismatch repair m u t a t i o n 4. Double-strand break repair. ACA K UAA (codon for Thr) (termination codon) Base excision-repair Fig. 3.15 : An illustration of point mutations The bases cytosine, adenine and guanine can (represented by acodon of mRNA). undergo spontaneous depurination to respectively form uracil, hypoxanthine and xanthine. These  36 BIOTECHNOLOGY repair Malor mechanlsms of DNA TABLE 3.2 DNA repair Damage toDNA Mechanism base due to Removal of the base by N-glycosylase; Damage to a single abasic sugar removal, replacement. Base excision-repair spontaneous alteratlon or by means. chemical or radiatlon DNA by Removal of the DNA fragment (- 30 nt Nucleotide excision-repair Damage toa segment of radiation length) and replacement. spontaneous, chemical or means. Removal of the strand (by exonuclease Damage due to copying errors digestion) and replacement. Mismatch repair (1-5 base unpaired loops). Unwinding, alignment and ligation. radiations, Double-strand break repair Damage caused by ionizing free radicals, chemotherapy etc. TCCT DNA, and altered bases do not exist in the normal out need to be removed. This is carried AGGA therefore Normal DNA by base excision repair (Fig. 3.16). A defective DNA in which cytosine is deaminated to uracil is acted upon by the enzyme uracil DNA glycosylase. This results in the removal of the defective base uracil. An endonuclease cuts TCUT the backbone of DNA strand near the defect and removes a few bases. The gap so created is filled AGGA Defective DNA up by the action of repair DNA polymerase and DNA ligase. Uracil DNA Nucleotide excision-repair Uglycosylase The DNA damage due to ultraviolet light, TCXT ionizing radiation and other environmental factors often results in the modification of AGGA certain bases, strand breaks, cross-linkages etc. Nucleotide excision-repair is ideally suited for such large-scale defects in DNA. After the identification Endonucleases of the defective piece of the DNA, the DNA double helix is unwound to expose the damaged part. An excision nuclease (exinuclease) cuts the DNA on either side (upstream and downstream) of the AGGA damaged DNA. This defective piece is degraded. The gap created by the nucleotide excision is filled up by DNA polymerase which gets ligated by DNA DNA polymerase ligase (Fig. 3.17). DNA ligase Xeroderma pigmentosum (XP) is rare TCCT autosomal recessive disease. The affected patients are photosensitive and susceptible to skin It is now recognized that XP is due to a cancers. AGGA defect in the nucleotide excision repair of the Fig. 3.16 : A diagrammatic DNA. damaged representation of base excision-repair of DNA.  Chapter 3 : DNA REPLICATION, RECOMBINATION, AND REPAIR 37 3 5 3 CH3 CH3 5 3 5 3 Defect recognition and unwinding Single strand cut by GATC endonuclease CHg CH, Exonuclease Cutting at two sites to remove defective oligonucleotide DNA polymerase CH3 CH3 Degradation of defective DNA Resynthesis and religation Ligase 5' 3 CHg 3 3 3' 5 5 Fig. 3.18 : Adiagrammatic representation of Fig. 3.17 : A diagrammatic representation of mismatch repair of DNA. nucleotide excision-repair of DNA. Double-strand break repair Mismatch repair replication, defects do Double-strand breaks (DSBs) in DNA are Despite high accuracy in recombination is copied. For instance, cytosine dangerous. They result in genetic Occur when the DNA translocation, be incorporated opposite which may lead to chromosomal (instead of thymine) could cell death. DSBs single broken chromosomes, and finally recombination or Mismatch repair corrects a to adenine. instead of T to A. can be repaired by homologous mismatch base pair e.g. C to A, non-homologous end joining. Homologous DNA exists in a in yeasts while in mammals, The template strand of the synthesized strand recombination occurs methylated form, while the newly non-homologous and joining dominates. This difference allows the is not methylated. enzyme GATC AND CANCER recognition of the new. strands. The an adjacent DEFECTS IN DNA REPAIR endonuclease cuts the strand at certain genes This is develops when methylated GATC sequence (Fig. 3.18). defective Cancer division fail or are by an exonuclease digestion of the that regulate normal cell followed encoding proteins new DNA strand is altered. Defects in the genes strand, and thus its removal. A mismatch synthesized to replace the damaged one. involved in nucleotide-excision repair, linked to now are cancer (HNPCC) repair and recombinational repairalready referred colon Hereditary nonpolyposis inherited cancers. This human cancers. For instance, as is one of the mnost common above, HNPCC is due to a defect in mismatch faulty mismatch repair cancer is now linked with repair. of defective DNA.