Lec 53A DNA Replication, Mutation & Repair TTh .pdf

Introduction: Mutation Definition: Any change in the nucleotide sequence of the genetic material. A change may be a simple substitution of 1 nucleotide or the insertion or deletion of 1 or more nucleotides within a DNA sequence. Genetic code provides the basis for understanding the nature of mutatio...

Introduction: Mutation Definition: Any change in the nucleotide sequence of the genetic material. A change may be a simple substitution of 1 nucleotide or the insertion or deletion of 1 or more nucleotides within a DNA sequence. Genetic code provides the basis for understanding the nature of mutations. 22 NOV 2023 MIBS BATCH 29 42 Mutagen A mutagen is any substance/agent that can cause mutation. Mutagen can cause changes to the DNA, that can in turn affect transcription and replication of the DNA, which in severe cases can cause/lead to cell death. Strong mutagens can result in chromosomal instability by causing rearrangements of the chromosome such as translocation, deletion or inversion. These mutagens are called clastogens. Some mutagens can even cause aneuploidy, i.e. change the no. of chromosome in the cell. Accumulation of some mutations can lead to cancer. 22 NOV 2023 MIBS BATCH 29 43 Mutagen 3 types: Chemical Physical Biological Mutagens 22 NOV 2023 MIBS BATCH 29 44 Physical mutagens Radiation is the first mutagenic agent known. There are 2 types: 1) Ionizing radiation (Gamma & X-rays) 2) Non-ionizing radiation (UV) 22 NOV 2023 MIBS BATCH 29 45 Chemical mutagens 4 major types: 1. Base analogs - For example, 5-bromouracil (has the same base-pairing property as thymine). The mutagenic effect arises during the next round of replication as there is a relatively high chance of the polymerase encountering the enol form of 5bU (due to tautomerization), which (like enol-thymine) pairs with G rather than A. This results in a point mutation. 2. Deaminating agents. - Presence of nitrous acid can cause removal of amino group (NH2) in A, G & C. Deamination of A gives hypoxanthine, which pairs wt C, while deamination of C gives U. 22 NOV 2023 MIBS BATCH 29 46 Chemical mutagens 3. Alkylating agents - Ethylmethane sulfonate (EMS) add alkyl groups (CH3) to nucleotides. Cause point mutations. 4. Intercalating agents - Interact with bases of DNA & insert between them . This causes a “stretching” of the dsDNA & the DNA polymerase is ‘fooled’ into inserting an extra base opposite an intercalated molecule. Cause insertion mutation. - Eg. Ethidium bromide 22 NOV 2023 MIBS BATCH 29 47 Biological mutagens Transposon/Transposable elements - A section of DNA that undergoes autonomous fragment relocation or multiplication. Its insertion into chromosomal DNA disrupt functional elements of the genes. Virus - Virus DNA may be inserted into the genome and disrupt genetic function. - A risk factor for cancer. Eg. Human papilloma virus (HPV) is implicated in cervical cancer. Bacteria - some bacteria such as Helicobacter pylori cause inflammation during which oxidative species are produced. This causes DNA damage and increase mutation rate. 22 NOV 2023 MIBS BATCH 29 48 Mutation 2 major types of mutation: 1) Gene mutation 2) Chromosome mutation 22 NOV 2023 MIBS BATCH 29 49 Gene mutation 22 NOV 2023 MIBS BATCH 29 50 Gene mutation Change in the nucleotide sequence of a gene May be due to copying errors, chemicals, viruses, etc. Gene mutation can be subdivided to: 1) Point mutation/base pair substitution mutation/Single nucleotide polymorphism (SNP) 2) 22 NOV 2023 Frame shift mutation MIBS BATCH 29 51 Point mutation 22 NOV 2023 MIBS BATCH 29 52 Point mutation Involves substitution of 1 (ONE) nucleotide in a gene. 2 main types: 1) Transition mutation base substitution that changes a purine nucleotide to another purine (A <-> G) or a pyrimidine nucleotide to another pyrimidine (C <-> T). 2) Transversion mutation base substitution that changes a purine nucleotide to a pyrimidine nucleotide or vice versa. 22 NOV 2023 MIBS BATCH 29 53 Effects of point mutation Point mutations can result in : Genetic code 1) Silent mutation (same sense) - Changes codon but specifies the same amino acid. - Eg. UUG codes for Leu. If the G is substituted with A, it still codes Leu. 2) Missense mutation - Changes codon and specifies different amino acid. - Eg. In the codon UUG, if the G is substituted with U, it codes for Phe. 3) Nonsense mutation - Substitution specifies a termination codon. - Eg. In the codon UUG, if the second base, U is substituted with A, it codes for a stop codon. 22 NOV 2023 MIBS BATCH 29 54 Effects of point mutation (a) Wild type/ Original sequence DNA (template) DNA (template) (b) Missense mutation DNA (template) G DNA (template) C 22 NOV 2023 (c) Silent mutation MIBS BATCH 29 (d) Nonsense mutation 55 Missense mutation Missense mutation depending upon its location in the specific protein might be acceptable, partially acceptable or unacceptable in terms of its effect on the protein function. Thus, missense mutation can have: 1) Acceptable Missense effect 2) Partially Acceptable Missense effect 3) Unacceptable Missense effect Illustration of all 3 effects using hemoglobin (Hb) molecule, the protein which carries oxygen in the red blood cell, as an example. Acceptable missense 61 lys asn HbA, β Hb Hikari, β 22 NOV Hb Hikari - Normal physiological properties, only electrophoretic altered. 2 0 2mobility 3 Partially acceptable missense 6 glu HbA, β val HbS, β Hb S – Binds oxygen, but precipitates when deMIBS BATCH 29 oxygenated. Unacceptable missense 58 his tyr HbA, α HbM (Boston), α Hb Boston – Permits oxidation of the heme ferrous iron to ferric state, and therefore cannot bind oxygen at all. 56 Point mutation & sickle cell anemia Sickle Cell disease is the result of missense mutation with partially acceptable effect. The sequence of GAG (codes for glutamic acid) in the gene coding for β globin of Hb is changed to GTG (codes for valine). HbA, β is mutated to form HbS, β. This change causes profound structural distortion of Hb. 22 NOV 2023 MIBS BATCH 29 57 Frameshift mutation 22 NOV 2023 MIBS BATCH 29 58 Frameshift mutation Caused by the deletion or insertion of nucleotides that generate altered mRNA. This changes the reading frame for the gene & results in altered amino acid sequence. Insertion Wild type CUG ACG UAU UUU AAU GUC AUG Leu Thr Tyr Phe Asn Val Met Mutant CUG AAC GUA UUU UAA UGU CAU G (A added) Leu Asn Val Phe STOP Deletion Wild type G removed CUG ACG CAG GUA AAG GUG AGA Leu Thr Gln Val Lys Mutant Val Thr CUG ACG CAG UAA 22 NOV 2023 MIBS BATCH 29 Leu Thr Gln Stop 59 Frameshift mutation Frameshift mutation can cause : 1) shift in the reading frame 2) extensive missense 3) immediate nonsense Another important form of frameshift mutation is the insertion of 3 bases which codes for an amino acid resulting in a protein molecule containing an extra amino acid. Eg. Huntington disease, a neurodegenerative disorder. Also known as trinucleotide repeat disorder. 22 NOV 2023 MIBS BATCH 29 60 Frameshift mutation can cause Extensive missense Change in 2 amino acids & absence of stop codon Immediate nonsense Frameshift caused premature termination of protein sequence Extensive frameshift Removal of 1 amino acid 22 NOV 2023 MIBS BATCH 29 61 Chromosome mutation 22 NOV 2023 MIBS BATCH 29 62 Chromosome mutation May Involve: – Changing the structure of a chromosome – The loss or gain of part of a chromosome 22 NOV 2023 MIBS BATCH 29 63 Chromosome mutation 5 types: +Deletion +Inversion +Duplication +Translocation +Nondisjunction 22 NOV 2023 MIBS BATCH 29 64 Chromosome mutation: Deletion Due to breakage A piece of a chromosome is lost 22 NOV 2023 MIBS BATCH 29 65 Chromosome mutation: Inversion Involves 3 steps: 1) Chromosome segment breaks off 2) Segment flips around backwards 3) Segment reattaches 22 NOV 2023 MIBS BATCH 29 66 Chromosome mutation: Duplication Occurs when a gene sequence is repeated 22 NOV 2023 MIBS BATCH 29 67 Chromosome mutation: Translocation Involves 2 chromosomes that aren’t homologous. Part of one chromosome is transferred to another chromosome. 22 NOV 2023 MIBS BATCH 29 68 Chromosome mutation: Nondisjunction Failure of chromosomes to separate during meiosis Causes gamete to have too many or too few chromosomes. Eg. of diorders: Turner syndrome (loss of an X chromosome), Down syndrome (extra copy of chromosome 21) 22 NOV 2023 MIBS BATCH 29 69 Chromosome mutation: Examples 22 NOV 2023 MIBS BATCH 29 70 Review +Topic Outcome 53.5 – Describe the general features and various types of DNA mutation. 22 NOV 2023 MIBS BATCH 29 71 DNA repair Mechanism 22 NOV 2023 MIBS BATCH 29 72 Introduction On a daily basis, DNA undergoes various forms of damage. What is DNA damage & what is the difference between DNA damage & DNA mutation???? Vs. DNA damage - Physical abnormalities or abnormal chemical changes - Cannot be inherited. -Effects: Prevents replication and transcription or may cause mutation if damaged DNA is replicated. 22 NOV 2023 MIBS BATCH 29 DNA mutation - Change in normal nucleotide sequence in DNA. - Can be inherited. 73 Agents that cause DNA damage • Highly reactive oxygen radicals Radiations • Ionizing (X & ϒ rays) • Non-ionizing (UV) • Aromatic hydrocarbon • Plant & microbial products (eg. Chemicals aflatoxin) • Chemotherapy agents Replication errors 22 NOV 2023 MIBS BATCH 29 74 Types of DNA damage DNA damage can be categorized into 4 types. • • • • • Depurination Deamination Alkylation Insertion/deletion 1) Single Base-analog base addition alteration 3) Chain breaks • UV light-induced thymine-thymine (pyrimidine) dimer 2) 2 base alteration s 4) Cross linkage • Ionizing radiation • Oxidative free radical formation 22 NOV 2023 MIBS BATCH 29 • Bifunctional alkylating agent 75 1) Single base alteration: depurination Depurination: Removal of A or G from deoxyribose through hydrolysis of the β-glycosylic bond. Spontaneously occurs due to heat & acid. Guanine Depurinated sugar Guanine 22 NOV 2023 MIBS BATCH 29 76 1) Single base alteration: deamination Deamination: Removal of an amino group (NH2) from a nucleotide by spontaneous hydrolysis. Cytosine Uracil Tautomeri c shift 22 NOV 2023 MIBS BATCH 29 77 2) 2 base alteration: thymine-thymine dimer formation UV irradiation causes covalent bond formation between adjacent thymines on the same strand of DNA. Covalent linkage may result in the dimer being replicated as a single base, which results in a frameshift mutation. 22 NOV 2023 MIBS BATCH 29 78 3) Chain break: Ionizing radiation When DNA interacts with ionizing radiation, the energy transferred from the radiation can break chemical bonds present in the DNA. This can result in base substitution, DNA single-strand breaks (SSBs) & double strand breaks (DSBs). 22 NOV 2023 Single-strand break MIBS BATCH 29 Double-strand break 79 3) Chain break: Oxidative free radical formation Free radical is any atom or molecule containing unpaired electrons. When oxygen is metabolised during regular cellular metabolism, it creates 'free radicals' which steal electrons from other molecules, causing damage. This can result in oxidation of bases & large deletions through single and double strand breaks. 22 NOV 2023 MIBS BATCH 29 80 4) Cross linkage: bifunctional alkylating agent Similar to monofunctional alkylating agent, bifunctional alkylating agent also adds an alkyl group to the DNA. However, bifunctional agent can cause interstrand (between) and intrastrand (within) DNA-DNA cross links and these cross links are responsible for inhibition of DNA synthesis. Sugar-phosphate backbone Intrastrand crosslinking Interstrand crosslinking Usually used as anticancer drug. 22 NOV 2023 MIBS BATCH 29 81 DNA repair 22 NOV 2023 MIBS BATCH 29 82 Important terms Interstrand: Between different DNA strands Intrastrand: Within the same DNA strands Endonuclease: Enzyme which cleaves within DNA strands. Exonuclease: Enzyme which cleaves at the ends of DNA strands. Nick: discontinuity in dsDNA due to absence of phosphodiester bond 22 NOV 2023 MIBS BATCH 29 83 DNA repair Estimated rates of DNA damage per human cell per day: 22 NOV 2023 Form of damage No. Single strand breaks 50,000 Double-strand breaks 10 Depurination 10,000 Deamination 600 Oxidative free radical damage 2,000 Alkylated base 5,000 Intrastrand cross links 10 Total DNA damaging events per cell per day: 60,000 Total DNA damaging events per cell per hour: 2,500 Since there are 1013 – 1014 cells in human body, ~ 3 × 1017 DNA damaging events per hour. MIBS BATCH 29 84 DNA repair Since DNA undergoes many damaging events, it is essential that cells possess efficient repair system. Without proper repair systems, key genes will lose its functions and the cell will not be able to maintain important cellular functions. Most cells possess 4 categories of DNA repair mechanism: 1) Direct repair 2) Excision repair: Base & nucleotide excision repair 3) Mismatch repair 4) Double-stranded break repair 22 NOV 2023 MIBS BATCH 29 85 Direct repair This system act directly on damaged nucleotides, converting each one back to its original structure. Is the simplest repair mechanism available. Nick Involved in repairs of: 1) Nicks DNA ligase 2) Some forms of alkylation damage Nick is repaired 22 NOV 2023 MIBS BATCH 29 86 Excision repair Unlike the direct repair mechanism, a longer stretch of polynucleotide is excised. 2 major forms of excision repair: a) Base excision repair (BER) b) Nucleotide excision repair (NER) 22 NOV 2023 MIBS BATCH 29 87 Base excision repair (BER) Essential to repair minor damages to the base resulting from oxidation, methylation, deamination & depurination. These damages, although simple in nature, are highly mutagenic and therefore represent a significant threat to genome fidelity (accuracy) and stability. Also used to repair single-stranded break (SSB). involves removal of 1/> damaged nucleotide base, excision of a short piece of the polynucleotide around the AP (apurinic/apyrimidinic) site created and resynthesis with a DNA polymerase. 22 NOV 2023 MIBS BATCH 29 88 Base excision repair (BER) BER pathway (a) A DNA glycosylase recognizes a damaged base and cleaves between the base & deoxyribose in the backbone. (b) An AP endonuclease cleaves the phosphodiester backbone near the AP site. (c) DNA polymerase I initiates repair synthesis from the free 3’ OH at the nick, removing a portion of the damaged strand (with its 5’ to 3’ exonuclease activity) & replacing it with undamaged DNA. (d) The nick remaining after DNA polymerase I has disassociated is sealed by DNA ligase. AP = apurinic or apyrimidinic (a = without) 22 NOV 2023 MIBS BATCH 29 89 Nucleotide excision repair (NER) most flexible of the DNA repair pathways. similar to BER but is not preceded by removal of a damaged base and can act on more substantially damaged areas of DNA such as intrastrand cross-linked DNA (thymine-thymine dimers). The NER process requires the action of several proteins in a stepwise manner that includes: (a) damage recognition (b) local unwinding of the DNA duplex around the lesion (c) dual incision of the damaged DNA strand (d) gap repair synthesis and (e) strand ligation 22 NOV 2023 MIBS BATCH 29 90 Nucleotide excision repair (NER) NER pathway (a) 2 excinuclease (excision endonucleases) bind DNA at the site of damage. (b) 1 cleaves the 5’ side & the other cleaves 3’ side of the lesion. (c) The DNA segment is removed by a helicase (d) DNA polymerases fills in the gap & (e) DNA ligase seals the nick. 22 NOV 2023 MIBS BATCH 29 91 Mismatch repair (MMR) This system is involved only in correcting errors of replication, i.e. a mismatched nucleotide is removed. Eg. Instead of A, C is added to pair with T. The parent template strand is methylated because adenine is converted to 6-methyladenines in the sequence 5′-GATC-3′. The daughter strand is initially unmethylated. This difference is used as a clue by the GATC endonuclease to determine which strand to cleave (cut). 22 NOV 2023 MIBS BATCH 29 92 Mismatch repair (MMR) MMR mechanism (a) Specific proteins MutS which complexes with Mut L & MutH scan daughter strand and pass the mismatched information to specific GATC endonucleases. (b) The GATC endonuclease cuts the strand bearing the mutation. (c) An exonuclease then digests this strand from the GATC. (d) This is followed by resynthesis and religation. 22 NOV 2023 MIBS BATCH 29 93 Double-stranded break repair (DSB) it is used to mend double-strand breaks. Unlike BER, NER & MMR, DSB affects both strands of the DNA duplex. This prevents use of the complementary strand as a template for repair. 2 basic mechanisms are available: 1) homologous recombination (HR) 2) non-homologous end-joining (NHEJ) 22 NOV 2023 MIBS BATCH 29 94 Homologous recombination (HR) HR corrects defects in an error-free manner using a mechanism that retrieves genetic information from a homologous, undamaged DNA molecule. The majority of HR-based repair takes place in late S - and G2-phases of the cell cycle when an undamaged sister chromatid is available for use as repair template. 22 NOV 2023 MIBS BATCH 29 95 Non-homologous end-joining (NHEJ) NHEJ mechanism (a) Ku proteins and DNA-PK bind to the free ends of DNA. (b) This allows approximation of the separated ends. (c) DNA-PK phosphorylates Ku, which activates helicase & helps in unwinding. (d) The unwound DNA forms base pairs, the extra nucleotide tails are removed by exonuclease and the gaps are filled by ligase. DNA-PK: DNA protein kinase 22 NOV 2023 MIBS BATCH 29 96 Summary of DNA repair mechanism (A) Direct repair (B) Excision repair (C) Mismatch Repair (D) Double-stranded break repair 22 NOV 2023 MIBS BATCH 29 97 Disorders due to defects in DNA repair mechanism The importance of DNA repair is emphasized by the number and severity of inherited human diseases that have been linked with defects in one of the repair processes. One of the best characterized of these is Xeroderma pigmentosum, which is caused by a mutation in the enzymes involved in NER. This results in defect in the ability to repair damage caused by UV light. 22 NOV 2023 MIBS BATCH 29 98 Disorders due to defects in DNA repair mechanism Xeroderma pigmentosum: Defective NER mechanism; sensitive to UV light; skin cancer. Bloom’s Syndrome: Defective DSB (DNA ligase or Helicase); sensitive to alkylating agents; lymphoma & leukemias. Ataxia telangiectasia: Defective NER mechanism; sensitive to damage by UV and X-rays. Hereditary Nonpolyposis Colon Cancer: Defective MMR; sensitive to UV radiation; colon & ovary cancer 22 NOV 2023 MIBS BATCH 29 99