DNA Damage and Repair Lecture Notes 2023/2024 PDF

DNA damage and repair Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology, 2023/2024 Lecture 9 Book Reference Essential Cell Biology by Bruce Alberts, 6th Edition. Focus on what is discussed in the lectures ⚫ Ch...

DNA damage and repair Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology, 2023/2024 Lecture 9 Book Reference Essential Cell Biology by Bruce Alberts, 6th Edition. Focus on what is discussed in the lectures ⚫ Chapter 6: Control of Gene Expression o Section starting page 215 ⚫ Chapter 9: How Genes and Genomes Evolve o Section starting page 298  Replication accuracy Replication cannot be error-free because infinite accuracy requires infinite energy Variability of genetic information is inevitable and is the basis of the evolutionary process.  Mutation Mutation: ❑ a sudden, unintended change in a nucleotide sequence of DNA that occurs under influence of internal or external mutagenic factors ❑ a change in information that is contained in the DNA of somatic and germ cells that is passed-on to daughter cells (change in genetic plan) ht tp: // lovv y.f iles.wordpress. com  Mutation Somatic - occur in cells of body not inherited by offspring may initiate the formation of cancer Germline - arise in the process of oogenesis, spermatogenesis or embryo development may be inherited may lead to genetic diseases  De novo mutations De novo mutations occur in an organism but not its parents. They can: ❑ arise spontaneously (e.g., with age) ❑ be induced by physical, chemical or biological factors De novo mutations that arise in cells can occur in: ❑ the body (somatic), which are not inherited by offspring ❑ the germline (reproductive cells / gametes), which can be inherited by offspring ❑ the developing embryo, which can be inherited by offspring  Spontaneous mutations Spontaneous mutations can result from errors during: ❑ DNA replication ❑ DNA transcription Replication errors are associated with: ❑ modification or loss of nitrogenous bases in DNA ❑ DNA polymerase slippage Transcription errors are associated with changes in: ❑ promoter or regulatory sequences ❑ alternative splicing  Base modifications Spontaneous base modifications in DNA can result from: ❑ tautomerization ❑ oxidative deamination ❑ oxidation ❑ methylation ❑ depurination and depyrimidination  Base tautomerization Nitrogen bases occur in tautomeric forms Tautomeric compounds are structural isomers that typically differ by the position of a proton(s) and double bond(s): H–A–B=C ⇌ A=B–C–H Some tautomers can transform into one another as the result of a spontaneous intramolecular reaction without the participation of other molecules  Base tautomerization Nitrogenous bases occur in two tautomeric forms Thymine, uracil and guanine may occur in the form of: ❑ a ketone (R-C=O) ❑ an enol (R=C-OH) Adenine and cytosine may occur in the form of an: ❑ amine (R-NH2) ❑ imine (R=NH) The ketone and amine forms are the dominant forms and confer proper pairing Base tautomerization Base tautomerization  Base tautomerization Enol and imino forms of nitrogenous bases in DNA occur rarely (1:10,000 nt) These forms can create bonds with other nontraditional bases besides the typical base pairing. Examples include, the: ❑ enol form of thymine binding with guanine (mutation) ❑ enol form of guanine binding with thymine (mutation) ❑ imine form of adenine binding with cytosine (mutation) ❑ imine form of cytosine binding with guanine (correct!)  Oxidative deamination Oxidative deamination involves the exchange of an amino - NH2 group for an oxygen atom =O, which, in turn, leads to the change of: ❑ adenine into hypoxanthine ❑ guanine into xanthine ❑ cytosine into uracil Oxidative deamination  Oxidative deamination Oxidative deamination of cytosine and adenine can lead to mutations because: ❑ uracil pairs with adenine, so one of the daughter strands will have a U:A pair instead of the correct C:G pair (mutation) ❑ hypoxanthine pairs with cytosine, so one of the daughter strands will have H:C pair instead of the correct A:T pair (mutation) Oxidative deamination of guanine to xanthine - does not lead to mutations because it pairs correctly with cytosine  Oxidation The reaction of guanine with reactive oxygen species (ROS) produces: 8-oxoguanine (8-oxG) Since 8-oxG (*G) pairs with adenine, one of the daughter strands will contain an 8-oxG:A pair, instead of the correct G:C pair (mutation) Formation 8-oxoguanine (8-oxG) is one of the most common type of spontaneous DNA damage Oxidation  Depurination and depyrimidination Depurination and depyrimidination involve the removal of a purine or pyrimidine base from a nucleotide in DNA, respectively A hydroxyl group (-OH) remains in place of a base Depurination is one of most common spontaneous DNA damages Depyrimidination occurs in cells twenty times less frequently than depurination Depurination  Replication slippage DNA polymerase can make mistakes due to "slippage errors” referred to as as "slipped strand mispairing" This can result in can duplicatation of a fragment of DNA coined expansion/contratction usually with length of two (trinucleotide expansion), three (trinucleotide expansion), four, or [less often] five extra nucleotides in the DNA sequence This mainly concerns lagging strand Replication slippage  Transcriptional mutations Occur in regions associated with transcriptional regulation: ❑ promoter sequences (e.g., TATA) ❑ enhancer or silencer sequences Can cause a decrease in the level of gene transcription with a decrease in mRNA production and as a result a decrease in protein concentration Can cause an increase in the level of gene transcription with an increase in mRNA production and as result an increase in protein concentration  Splicing mutations During post-transcriptional processing, the following may occur: ❑ exon skipping ❑ exon shuffling ❑ intron retention This causes changes in nucleotide sequence in mature mRNA and thus changes in amino acid sequence in proteins Mutagenic factors  Cell response to mutation Cell response: ❑ The cell repairs the damage restoring the DNA molecule to its original state ❑ The cell dies (apoptosis) ❑ The cell replicates small damages during replication and passes them on to daughter cells (mutation) ❑ The cell partially repairs DNA damage and passes it on to daughter cells (mutation)  Influence of mutations Influence of mutations on the phenotype: ❑ beneficial ❑ inert ❑ adverse ❑ lethal Leopard gecko  Mutation types Mutation types: ❑ point (genetic) ❑ chromosome - structure ❑ genome – number of chromosomes ❑ beyond chromosomal: ❑ mitochondrial ❑ chloroplast  Point mutations Point mutations are changes at the molecular level of DNA, consisting of: ❑ substitution of one nucleotide in place of a proper one ❑ insertion of one or more nucleotides ❑ deletion (i.e., loss of one or more nucleotides) ❑ inversion (i.e., change in the order of nucleotides) ❑ duplication (i.e., doubling the number of nucleotides)  Point mutations Point mutations cause the nucleotides located where the mutation site occured to be read as completely different codons resulting in an incorrect and/or dysfunctional protein to be produced Types of point mutations Types of substitution  Effects of point mutations Point mutations are divided based on their effects: ❑ silent mutations ❑ nonsense mutations ❑ missense mutations ❑ frameshift mutations  Silent mutations Silent mutations The change in the nucleotide concerns the third position of the codon and does not cause a change in the encoded amino acid Occur as result of substitution, both transversion and transition Occuring in >1% of a population is termed as a polymorphism rather than a mutation Single nucleotide polymorphism (SNP)  Nonsense mutations Nonsense mutations Changing one or several nucleotides causes the premature creation of the STOP termination codon, and thus creating a shortened protein without its function  Missense mutations Missense mutations Change in nucleotide affects first or second position in codon and causes change in the encoded amino acid They are divided into: ❑ conservative - when changing the amino acid to another, it does not change function of the protein ❑ non-conservative - when changing the amino acid to another, it causes a change in function of the protein  Frameshift mutations Frameshift mutations Insertion or deletion of one or more nucleotides in the coding region of DNA, that are not divisible by 3, causes a shift in reading frame All codons are changed and thus the amino acid sequence in the protein A protein with changed function is created Point mutations  Chromosomal aberrations Chromosomal aberrations are mutations in structure or the number of chromosomes This mutation is large enough to be visible under a light microscope The smallest aberrations that are visible under a microscope are 2 - 4 x 106 base pairs in length Chromosomal aberrations can affect both: ❑ autosomes (somatic chromosomes) ❑ allosomes (sex chromosomes)  Chromosomal aberrations Structural chromosomal aberrations occur as a result of breakage of one or more chromosomes and incorrect connection of chromosomal fragments during: ❑ cell division (mitosis, meiosis) ❑ fertilization ❑ embryo development Balanced structural aberrations do not cause loss or an increase in the amount of genetic material Unbalanced structural aberrations lead to a loss or an increase of genetic material  Structural Aberrations Types of structural aberrations: ❑ deletion ❑ insertion ❑ inversion ❑ duplication ❑ translocation ❑ Robertsonian translocation (ROB, centric fusion) ❑ centric division ❑ isochromosomes ❑ ring chromosome ❑ dicentric chromosomes  Deletion Terminal (distal) deletion is the loss of an end of a chromosome due to a break in one place Interstitial deletion is a loss of a section within a chromosome due to a break in two places Terminal Interstitial  Insertion Insertion involves inserting a chromosomal fragment into another location of: ❑ the same chromosome ❑ another non-homologous chromosome It results in 3 breaks of two chromosomes Insertions are always associated with translocation Insertion  Inversion An inversion is a break in chromosome in two places and a 180° inversion of the respective chromosome fragment Types of inversion: ❑ pericentric inversion - breaks occurred in both arms of chromosome, and the inverted fragment contains centromere ❑ paracentric inversion - both breaks occurred in one arm and inverted fragment does not contain centromere  Inversion Pericentric inversion Paracentric inversion centromere centromere  Duplication A duplication is the doubling of a specific fragment of a chromosome Types of duplication: ❑ tandem duplications - duplicated fragments occur next to each other and have the same gene order ❑ inverted duplications - duplicated fragments are reversed with respect to each other, so genes are arranged in the reverse order  Duplication Normal chromosome Duplication Tandem Inverted tandem  Translocation Translocation involves the exchange of genetic material between two or three (less common) chromosomes Types of translocation: ❑ reciprocal translocation involves breakage of two non- homologous chromosomes and mutual exchange of fragments between these chromosomes ❑ non-reciprocal translocation involves breakage of two non-homologous chromosomes and attachment of fragment of one chromosome to another chromosome  Types of translocation Reciprocal translocation Non-reciprocal translocation  Robertsonian translocation Robertsonian translocation (ROB, centric fusion) occurs as a result of: ❑ breaks in the centromeric region of two acrocentric chromosomes ❑ fusion of long arms of both chromosomes ❑ loss of short arms of both chromosomes Robertsonian translocations in humans most often occur between chromosomes 13 and 14, and 14 and 21 Robertsonian translocation  The Philadelphia chromosome The Philadelphia chromosome is formed by translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)] resulting in the formation of the Bcr-Abl fusion gene The Abl gene is a proto-oncogene, and when combined with the Bcr gene it becomes an oncogene  The Philadelphia chromosome The new protein, BCR-ABL, is produced, which: ❑ blocks DNA repair ❑ impairs cells' ability to undergo apoptosis ❑ is created de novo in somatic cells The Philadelphia chromosome occurs: ❑ in 95% of chronic myeloid leukaemias ❑ in 25 - 30% in adults and about 6% in children with acute lymphoblastic leukaemias ❑ In less than 1% of acute myeloid leukaemias  Centric division As result of centromere breaking, centric division occurs, which leads to a separation of chromosome arms It leads to the formation of two telocentric chromosomes The number of chromosomes in the cell increases  Isochromosomes Isochromosomes are formed by the transverse division of a centromere within chromosome Isochromosomes consist of two identical, long or short arms  Other aberrations Ring chromosomes Formed when terminal parts of the chromosome are lost and the remaining part of chromosome forms ring Dicentric chromosomes Formed by fusion of two damaged chromosomes and contain two centromeres Acentric chromosomes These are fragments of chromosomes without a centromere that are removed during the subsequent mitosis Other aberrations  Numerical aberrations Autosome aneuploidy - set of chromosomes (typical for given species and sex) in all somatic cells is enriched or depleted by one or more chromosomes Allosome aneuploidy - abnormal number of sex chromosomes Polyploidy - presence of more than two complete haploid sets of chromosomes  Causes of aneuploidy Cause of aneuploidy is a process called, nondisjunction, which is a lack of separation of homologous chromosomes or sister chromatids during: ❑ meiosis I ❑ meiosis II ❑ mitosis in embryogenesis Such processes are a result of damage to the mitotic spindle during the anaphase process Nondisjunction Nondisjunction  DNA repair Genetic information contained in DNA is exposed to constant damage as result of: ❑ spontaneous errors during replication ❑ spontaneous errors during transcription ❑ the action of mutagenic factors Damage is a source of mutations that lead to: ❑ cancer ❑ genetic diseases Cells have special repair mechanisms that protect the genome from change or loss of information contained in it  DNA repair Stage I Damaged DNA is recognized and removed in a variety of ways, which requires the presence of nucleases Nucleases are enzymes that cut phosphodiester bonds that connect damaged nucleotides to the rest of DNA strand A smaller or larger break occurs on one or both strands of the DNA helix  DNA repair Stage II DNA repair A replication polymerase: ❑ binds to the -OH group on the released 3’ end of the strand cut site ❑ Synthesizes a copy complementary to the respective sequence present in the undamaged strand ❑ catalyzes DNA strand synthesis in the 5’ to 3’ direction and has proofreading activity  DNA repair Stage III Breaks in the sugar-phosphate backbone of the repaired strand are joined together by DNA ligase This is the same enzyme that joins Okazaki fragments during replication in the lagging strand of DNA DNA repair stages  DNA repair systems Direct repair Repair of mismatched nucleotides Excision repair involves damage to single strands of DNA: ❑ base excision repair ❑ nucleotide excision repair Repair of double-stranded DNA breaks: ❑ non-homologous end bonding ❑ homologous recombination  Direct repair Direct repair involves the removal of alkyl groups from certain positions in DNA bases The MGMT enzyme catalyzes the transfer of a methyl group from O6-methylguanine to the thiol group (SH) in the cysteine ​residue of the enzyme Repair occurs without breaking the continuity of the DNA strand  Direct repair methyltransferase  MMR-type repair Stages of repair of mismatched nucleotides: ❑ recognition of the damage by MutS, MutL and MutH proteins ❑ unwinding of double-stranded DNA around damaged nucleotide by helicase ❑ excision of the DNA segment containing the damaged nucleotide by endonuclease ❑ filling in gap of the DNA strand by polymerase δ ❑ joining of the new fragment of DNA strand by ligase Repair of mismatched nucleotides  Base excision repair Stages of base excision repair: ❑ DNA glycosylase removes a damaged nitrogenous base (via breakage of the N-glycosidic bond) ❑ An apurinic/apyrimidinic site (AP site) is created ❑ An AP endonuclease recognizes this site and cuts DNA in the 5’ direction from the AP site creating a free 3’-OH end ❑ polymerase β inserts the correct nucleotide ❑ nucleotides are joined together by DNA ligase Base excision repair damaged base DNA glycosylase AP endonuclease β polymerase DNA ligase repaired DNA  Nucleotide excision repair Stages of nucleotide excision repair: ❑ recognition of damage by XPA and XPC proteins ❑ unwinding of double-stranded DNA around the damaged nucleotide by helicases ❑ cutting of DNA strand on both sides of the damage by XPG and XPF endonucleases ❑ removal of the damaged DNA fragment (approx. 25 nucleotides long) ❑ synthesis of a new DNA strand fragment by polymerase β ❑ joining of the new DNA strand fragment by ligase Nucleotide excision repair removal of damaged section polymerase and ligase rebuild the removed fragment repaired DNA Repair of double-stranded DNA breaks DNA double-strand breaks (DSBs) are particularly difficult to repair A break in both DNA strands within in a chromosome causes: ❑ broken DNA strands to separate ❑ a lack of a copy that could be used to recreate missing information Repair of double-stranded DNA breaks Eukaryotic cells have developed two basic strategies for repairing double-stranded DNA breaks: ❑ non-homologous end joining (NHEJ) ❑ homologous recombination (HR) In mammalian cells, double-stranded DNA breaks: ❑ are primarily repaired by non-homologous end joining ❑ homologous recombination occurs primarily during a crossing-over event in meiosis Repair of double-stranded DNA breaks  NHEJ type repair Stages of NHEJ: ❑ recognition of ends of broken DNA strands by Ku70 and Ku80 proteins ❑ removal of damaged part of DNA by Artemis endonuclease ❑ joining of DNA strands by XRCC4-ligase complex ❑ formation of multi-nucleotide deletion in the damaged region NHEJ type repair Ku 70 Ku 80 Artemis endonuclease XRCC4-ligase complex,  HR-type repair The main function of HR is to repair double-strand breaks, which occurs shortly after DNA replication in a cell but before it divides (splits sister chromatids) The stages of HR: ❑ marking the break (via ATM kinase) and notifying of the cell of DNA damage to allow the cell to stop the cell cycle ❑ formation of single-stranded ends by the action of 3'exonuclease  HR-type repair ❑ the single-stranded DNA fragment searches for the homologous, undamaged fragment of the corresponding sister chromatid ❑ polymerase δ or ε builds the missing DNA fragment ❑ the new DNA strand detaches from sister chromatid ❑ ligase joins the DNA ends ❑ The previously broken DNA is recreated without errors Homologous recombination Homologous recombination