Genetics - BIO310 Gene Mutation and DNA Repair PDF

Genetics– BIO310 Gene Mutation and DNA Repair  Mutations Now and then cells make mistakes in copying their own DNA, inserting the wrong base or even skipping a base as a strand is put together. These variations are called mutations, from the Latin word mutare, meaning “to change....

Genetics– BIO310 Gene Mutation and DNA Repair  Mutations Now and then cells make mistakes in copying their own DNA, inserting the wrong base or even skipping a base as a strand is put together. These variations are called mutations, from the Latin word mutare, meaning “to change.”  Mutations Mutations are a change in the DNA nucleotide sequence (which can cause heritable changes in genetic information.) Since mutations can be quite harmful, organisms have developed ways to repair damaged DNA Types of Mutations All mutations fall into two basic categories: 1. Those that produce changes in a single gene are known as gene mutations. 2. Those that produce changes in whole chromosomes are known as chromosomal mutations.  Mutations can occur at the chromosomal or gene level Chromosomal changes in structure or number ◦Generally, affect more than one gene Gene mutation ◦ Usually affects one gene ◦ Change from one nucleotide to another ◦ Delete nucleotides ◦ Insert nucleotides 8  Gene Mutations – Point Mutations Mutations that involve changes in one or a few nucleotides are known as point mutations because they occur at a single point in the DNA sequence. They generally occur during replication. If a gene in one cell is altered, the alteration can be passed on to every cell that develops from the original one. Gene Mutations Change the DNA Sequence A point mutation is a change in a single base pair ◦ It involves a base substitution ◦ A transition is a change of a pyrimidine (C, T) to another pyrimidine or a purine (A, G) to another purine ◦ A transversion is a change of a pyrimidine to a purine or vice versa ◦ Transitions are more common than transversions 10 Deletions or Additions Mutations may also involve the addition or deletion of short sequences of DNA Deletion Addition 11  Mutations in the Coding Sequence Silent mutations do not alter the amino acid sequence ◦ Due to the degeneracy of the genetic code Missense mutations do alter the amino acid sequence ◦ Example:Sickle-cell anemia ◦Some may not affect function – neutral mutation Nonsense mutations change a codon to a stop codon ◦ Produces a truncated polypeptide 12 Access the text alternative for slide images. 13  Mutations in the Coding Sequence Frameshift mutations involve the addition or deletion of nucleotides in multiples of one or two but not three (the size of one codon) This shifts the reading frame so that a completely different amino acid sequence occurs downstream from the mutation Except for silent mutations, new mutations are more likely to produce polypeptides with reduced function than enhanced function Amutation can occasionally produce a polypeptide with an enhanced ability to function; may result in an organism with greater likelihood of surviving and reproducing 14 16  Gene Mutations in Noncoding Sequences These mutations can still affect gene expression ◦ Promoter Up promoter mutations increase transcription Down promoter mutations decrease transcription ◦ Splice junctions in eukaryotes ◦ 5’ and 3’ UTR – alter stability of RNA, translation ◦ Regulatory element/operator site – disrupt proper regulation of gene expression 17 Table: Possible Consequences of Gene Mutations Outside of a Coding Sequence Sequence Effect of Mutation Promoter May increase or decrease the rate of transcription Regulatory element/operator site May disrupt the ability of the gene to be properly regulated 5′-UTR/3′-UTR May alter the ability of mRNA to be translated; may alter mRNA stability Splice recognition sequence May alter the ability of pre-mRNA to be properly spliced  Gene Mutations and Their Effects on Genotype and Phenotype A neutral mutation does not alter protein function A deleterious mutation lowers the chance of survival and reproduction; Extreme deleterious mutation is a lethal mutation; results in death of the cell or organism A beneficial mutation enhances the survival or reproductive success 20 G e n e M u t a t i o n s a n d Their Ef f e ct s o n G e n o t y p e a n d Phenotype Occasionally, whether a mutation is beneficial or deleterious will depend on environmental conditions Example: Sickle cell allele Heterozygotes have increased survival in the presence of malaria A conditional mutation is one that affects the phenotype only under specific conditions Example: Temperature-sensitive (ts) mutants Used by geneticists to study gene function Ex: E. coli with a ts mutation may grow in the range 33-38°C but not in the range 40-42°C  Chromosomal mutations involve changes in the number or structure of chromosomes. These mutations can change the location of genes on chromosomes and can even change the number of copies of some genes. There are four types of chromosomal mutations: deletion, duplication, inversion, and translocation. Deletion involves the loss of all or part of a chromosome. This genetic information is lost and no longer available to be inherited. C h a n g e s in Chromosome Structure C a n Affect G e n e Expression A chromosomal rearrangement may affect a gene because the breakpoint occurred within the gene itself Or, a gene may be left intact, but its expression may be altered because of its new location ◦ This is called position effect Position Effects There are two common reasons for position effects: 1. Movement to a position near regulatory sequences for a different gene 2. Movement to a heterochromatic region Example: position effect alters eye color in Drosophila ◦ Normal eyes are red ◦ Mutant flies can have a chromosomal rearrangement where the gene affecting eye color has been relocated to a heterochromatic chromosome; variegated eyes  Regulatory sequences are often bidirectional 25 26 Mutations C a n O c c u r in G e r m - Line or Somatic Cells Geneticists classify animal cells into two types 1. Germ-line cells Cells that give rise to gametes such as eggs and sperm 2. Somatic cells All other cells Ex: Muscle, nerve, or skin cells  The earlier the mutation, the larger the patch 29  Causes of Mutation Mutations can occur spontaneously or be induced Spontaneous mutations ◦ Result from abnormalities in cellular/biological processes ◦ Example: Errors in DNA replication Induced mutations ◦ Caused by environmental agents ◦ Agents known to alter DNA are called mutagens ◦ These can be chemical or physical agents  TABLE. Causes of Mutations Common Causes Description of Mutations Spontaneous Abnormal crossing over may cause deletions, duplications, translocations, and inversions (see Chapter 8). Aberrant recombination Abnormal chromosomal segregation may cause aneuploidy or polyploidy (see Chapter 8). Errors in DNA A mistake by DNA polymerase may cause a point mutation (see Chapter 13). replication Transposable elements Transposable elements can insert themselves into the sequence of a gene (see Chapter 12). Depurination On rare occasions, the linkage between a purine (i.e., adenine or guanine) and deoxyribose can spontaneously break. If not repaired, this can lead to mutation. Deamination Spontaneous changes in base structure can cause mutations if they occur immediately prior to DNA replication. Tautomeric shifts The products of normal metabolic processes, such as reactive oxygen species, may be chemically reactive agents that can alter the structure of DNA. Induced Chemical agents Chemical substances may cause changes in the structure of DNA. Physical agents Physical phenomena such as UV light and X-rays can damage DNA. 27  Causes of Spontaneous Mutations Spontaneous mutations can arise by three types of chemical changes ◦Depurination ◦Deamination ◦Tautomeric shift  Depurination Depurination ◦ The most common type of chemical change ◦ Removal of a purine (guanine or adenine) from the DNA ◦ Covalent bond between deoxyribose and a purine base is somewhat unstable Occasionally undergoes a spontaneous reaction with water that releases the base from the sugar ◦ This is then called an apurinic site ◦ Fortunately, apurinic sites can be repaired However, if the repair system fails, a mutation may result because there is no complementary base present to specify the base  A, T and G are incorrect. There’s a 75% chance of a mutation 30 D eamination Deamination ◦ Removal of an amino group from the cytosine base; produces uracil ◦ The other bases are not readily deaminated ◦ DNA repair enzymes can recognize uracil as an inappropriate base in DNA and remove it ◦ If repair system fails to correct the problem, a mutation could result during subsequent rounds of DNA replication Deamination of 5-methylcytosine Deamination of 5-methyl cytosine can also occur However deamination of 5-methyl cytosine does not result in uracil ◦ It results in thymine – a normal constituent of DNA ◦ This poses a problem for repair enzymes ◦ They cannot determine which of the two bases on the two DNA strands is the incorrect base ◦ For this reason, methylated cytosine bases tend to create hot spots for mutation Tautomeric Shifts Tautomeric shifts ◦ Involves a temporary change in base structure ◦ The common, stable form of thymine and guanine is the keto form At a low rate, T and G can interconvert to an enol form ◦ The common, stable form of adenine and cytosine is the amino form At a low rate, A and C can interconvert to an imino form ◦ These rare forms promote AC and GT base pairs ◦ To cause a mutation it must occur immediately prior to DNA replication  Temporary Shifted back to its tautomeric shift normal form 38  Induced Mutations An enormous array of agents can act as mutagens to permanently alter the structure of DNA Mutagens are often involved in the development of human cancers Mutagenic agents are usually classified as chemical or physical mutagens Examples of Mutagens Mutagen Effect(s) on DNA Structure Chemical Nitrous acid Deaminates bases Nitrogen mustard Alkylating agent Ethyl methanesulfonate Alkylating agent Proflavine Intercalates within DNA helix 5-Bromouracil Base analog 2-Aminopurine Base analog Physical X-rays Cause base deletions, single-strand breaks in the DNA backbone, crosslinking, and chromosomal breaks UV light Promotes formation of pyrimidine dimers, such as thymine dimers Mutagens Alter DNA Structure in Different Ways Chemical mutagens come in three main types ◦Base modifiers ◦Intercalating agents ◦Base analogs Base Modifiers Base modifiers covalently modify the structure of a nucleotide ◦ For example, nitrous acid, replaces amino groups with keto groups (–NH2 to = O) ◦ This can change cytosine to uracil and adenine to hypoxanthine These modified bases do not pair with the appropriate nucleotides in the daughter strand during DNA replication 57 58 Alkylating Agents Some chemical mutagens disrupt the appropriate pairing between nucleotides by alkylating bases within the DNA (also base modification) ◦ Methyl or ethyl groups are covalently attached to the bases ◦ Examples: Nitrogen mustard and ethyl methanesulfonate (EMS) 59 Intercalating Agents Intercalating agents contain flat planar structures that intercalate themselves into the double helix ◦ This distorts the helical structure ◦ When DNA containing these mutagens is replicated, the daughter strands may contain single-nucleotide additions and/or deletions resulting in frameshifts ◦ Examples: Acridine dyes Proflavine 60 Base Analogs Base analogs become incorporated into daughter strands during DNA replication ◦ For example, 5-bromouracil is a thymine analogue It can be incorporated into DNA instead of thymine ◦ 5BU undergoes a tautomeric shift and base pairs with G; When this occurs during DNA replication, TA base pair is changed to a 5BU-G base pair 61 62 63 Physical Mutagens Physical mutagens come into two main types 1. Ionizing radiation 2. Nonionizing radiation Ionizing Radiation Ionizing radiation ◦ Includes X-rays and gamma rays ◦ Has short wavelength and high energy ◦ Can penetrate deeply into biological materials ◦ Creates chemically reactive molecules termed free radicals ◦ Can cause ◦ Base deletions ◦ Single-strand and double-strand breaks in DNA backbone ◦ Cross-linking ◦ Oxidized bases Nonionizing Radiation Nonionizing radiation ◦ Includes UV light ◦ Has less energy ◦ Cannot penetrate deeply into biological molecules material ◦ Causes the formation of cross-linked thymine dimers ◦ Thymine dimers may cause mutations when that DNA strand is replicated ◦ Tanning greatly increases a person’s UV light exposure, raising the potential for thymine dimers and mutation 66 67 DNA Repair  DNA Repair Living cells contain several DNA repair systems that can fix different types of DNA alterations In most cases, DNA repair is a multi-step process 1. An irregularity in DNA structure is detected 2. The abnormal DNA is removed 3. Normal DNA is synthesized  TABLE : Common Types of DNA Repair Systems System Description Direct repair An enzyme recognizes an incorrect alteration in DNA structure and directly converts the structure back to the correct form. Base excision repair and An abnormal base or nucleotide is first nucleotide excision recognized and removed from the DNA, and a segment of DNA in this region repair is excised, and then the complementary DNA strand is used as a template to synthesize a normal DNA strand. Mismatch repair Similar to excision repair except that the DNA defect is a base pair mismatch in the DNA, not an abnormal nucleotide. The mismatch is recognized, and a segment of DNA in this region is removed. The parental strand is used as a template to synthesize a normal daughter strand of DNA. Homologous Occurs at double-strand breaks or when DNA damage causes a recombination gap in synthesis during DNA replication. The strands of a normal sister repair chromatid are used to repair a damaged sister chromatid. Nonhomologous end Occurs at double-strand breaks. The broken joining ends are recognized by proteins that keep the ends together; the broken ends are eventually rejoined. 68 Direct Repair In a few cases, the covalent modifications of nucleotides can be reversed by specific enzymes ◦ Photolyase can repair thymine dimers Splits the dimers restoring the DNA to original condition Uses light so called photoreactivation Nucleotide Excision Repair Can repair many types of DNA damage, including ◦ Thymine dimers and chemically modified bases ◦ Missing bases, some types of cross-links NER is found in all eukaryotes and prokaryotes ◦ Molecular mechanism best understood in prokaryotes Proteins in Nucleotide Excision Repair In E. coli, the NER system requires four key proteins ◦ UvrA, UvrB, UvrC and UvrD Named as such because they are involved in Ultraviolet light repair of thymine dimers ◦ They are also important in repairing chemically damaged DNA ◦ UvrA, B, C, and D recognize and remove a short segment of damaged DNA ◦ DNA polymerase and ligase finish the repair job 80 81 Diseases Involving NER Genes Several human diseases have been shown to involve inherited defects in genes involved in NER ◦ These include xeroderma pigmentosum (XP) and Cockayne syndrome (CS) ◦ A common characteristic in both syndromes is an increased sensitivity to sunlight xeroderma pigmentosu m Mismatch Repair The structure of the DNA double helix obeys the AT/GC rule of base pairing ◦ However, during DNA replication an incorrect base may be added to the growing strand by mistake ◦ Creating a base pair mismatch DNA polymerases have a 3’ to 5’ proofreading ability that can detect base mismatches and fix them  Mismatch Repair If proofreading fails, the mismatch repair system comes to the rescue Mismatch repair systems are found in all species In humans, mutations in the system are associated with particular types of cancer  Mismatch Repair in E. coli Mismatch repair has been studied extensively in E. coli ◦ MutL, MutH and MutS detect the mismatch and direct its removal from the newly made strand ◦ MutH can distinguish between the parental strand and the daughter strand Prior to replication, both strands are methylated Immediately after replication, the parental strand is methylated but the daughter strand is not 87 88 Recombination Repair DNA double-strand breaks are very dangerous ◦ Breakage of chromosomes into pieces ◦ Caused by ionizing radiation, chemical mutagens and free radicals ◦ 10 to 100 breaks occur each day in a typical human cell ◦ Breaks can cause chromosomal rearrangements and deletions They may be repaired by two systems: ◦ Homologous recombination repair (HRR) ◦ Nonhomologous end joining (NHEJ)