Mutation Repair Mechanism and Related Disorders

Mutations, Repair mechanism and related disorders Overview  DNA damage  Sources of DNA damage  Mutations Types and their consequence Repair Repair mechanism Mutation vs. DNA damage  Related disorders of Repair mechanism 2 DNA DAMAGE  External to the cell  Ionizing radiation • gamma ray/ X rary cause ss/ ds break in sugar-phosphate backbone Ultraviolet radiation • UV-C(260nm) • UV-B (280-315nm) • Cross-link between adjacent pyrimidine Environmental chemicals • hydrocarbons (Cigarette smoke) • Aflatoxins in peanuts  internal cell chemistry. 3 Thymine dimers 4 Undesired effects of internal cell chemistry  Depurination:  about 5000 A or G are lost every day by spontaneous hydrolysis of sugar-base Deamination 100 C are deaminated to produce U Attack by reactive oxygen species  superoxide onion are generated as by-product of oxidative metabolism in mitochondria Non-enzymatic methylation 5 Common forms of DNA damage Common forms of DNA damage. (A) Depurination. (B) Deamination of cytosine to uracil. (C) Deamination of 5-methyl cytosine to thymine. 6 Nonenzymatic methylation  non-enzymatic DNA methylation by S-adenosyl methionine produce 3-methyl adenine and 7-methyl guanine  This is quite distinct from the enzymatic methylation of cytosine to produce 5-methyl cytosine, which cells use as a major method of controlling gene expression.  The methylated adenine or guanine bases distort the double helix and interfere with vital DNA-protein interactions. 7 8 Other Sources of DNA Damage  Errors arise during normal DNA metabolism. – A certain error rate (incorporation of the wrong nucleotide) is inevitable during DNA replication.  Proofreading mechanisms correct the great majority of the resulting mismatches, but a few may persist, producing sequence variants. – Failure of the proofreading mechanism in somatic cells is one cause of cancer.  In addition, occasional errors in replication or recombination leave strand breaks in the DNA, which must be repaired if the cell is to survive. 9 Mutation definitions, Classification  Any change in the nucleotide sequence or arrangement of DNA is called mutation  Why are these mutations important? They can affect mRNA and protein production, eventually determining the phenotype  Classification • classified by the size of the altered DNA sequence • functional effect of the mutation on gene expression. 10 Mutation types  Mutations that leave chromosomes intact but change the number of chromosomes in a cell (chromosome mutations)  Mutations that change : a portion of a chromosome  a change in the copy number of a sub chromosomal segment or a structural rearrangement involving parts of one or more chromosomes (regional or subchromosomal mutations)  substitution, deletion, or insert ion of DNA, ranging from a single nucleotide up to 100 kb (gene mutations) 11 The Origin and Frequency of Different Types of Mutations  Chromosome Mutations:  chromosome missegregation are among the most common mutations  one mutation per 25 to 50 meiotic cell divisions  Regional Mutations  Duplications, deletions, and inversions of a segment of a single chromosome are predominantly result of homologous recombination  spontaneous double- stranded DNA breaks  Gene Mutations  errors introduced during DNA replication or mutations arising from a failure to properly repair DNA after damage. 12 Types of mutations 13 14 Effect of a nonsense mutation on translation. 15 Some examples 16 Estimates of Mutation Rates for Human Disease Genes Based on data in V ogel F, M otulsky A G: Human genetics, ed 3, Berl in, 1997, Springer-V erlag 17 Nonsense mutations – change in codon to UGA, UAA, or UAG. This results in premature stopping of protein synthesis. They can be lethal or severe in phenotype 18 The 2nd most common type of mutations are insertion/deletions of base pairs. This can cause frameshifts. Deletions or insertions of one or two bases usually results in dramatic differences in protein production. 19  Silent mutations – produce no change in amino acid sequence (due to degeneracy of the genetic code.) CUU codes for leucine, but so does CUC, CUA, CUG, UUA, and UUG. 20 A base pair addition/deletion (frameshift) mutation results in multiple amino acid changes downstream from the point of mutation 21 DNA Repair  refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that consist its genome.  Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene(s) that the affected DNA encodes. 22 A cell that has accumulated a large amount of DNA damage , no longer effectively repairs damage to its DNA, can enter one of three possible states: – An irreversible state of dormancy, known as senescence. – Cell suicide (apoptosis or programmed cell death). – Unregulated cell division (cancerous tumor) 23 Repair mechanism At least 6 DNA repair mechanisms. 3 used to correct abnormal modified bases when are present in just one of the two strands of the DNA( they are paired with a normal base) The damaged base can be repaired, or removed and replaced 24 Base excision repair (BER)  Corrects much the commonest type of DNA damage (20,000 altered bases in each nucleated cell in our body each day). • BER glycosylase enzymes remove abnormal bases by breaking the sugar-base bond.  Humans have at least 8 genes encoding different DNA glycosylases, each responsible for identifying and removing a specific kind of base damage.  After removal of the damaged base, an endonuclease & a phosphodiesterase cut the sugar-phosphate backbone at the position of the missing base and remove the sugar-phosphate residue.  The gap is filled by resynthesis with a DNA polymerase, and the remaining nick is sealed by DNA ligase Ill 25 Base excision repair (BER) base excision repair(BER), which repairs damage to a single nucleotide caused by oxidation, alkylation, hydrolysis, or deamination. The base is removed with glycosylase and ultimately replaced by repair synthesis with DNA ligase. 26 Nucleotide Excision Repair (NER)  Removes thymine dimers and large chemical adducts  NER removes & resynthesizes a large patch around the damage  The sugar–phosphate backbone is cleaved at the site of the damage, & exonucleases remove a large stretch  the gap is filled by re synthesis and sealed by DNA ligase 27 Direct reversal of the DNA damage  An infrequently used mechanism of DNA repair in humans.  Three human genes have been implicated in this mechanism, of which the best-characterized encodes: the O-6-methylguanine-DNA methyltransferase, which is able to remove methyl groups from guanines that have been incorrectly methylated.  In this repair process (also BER & NER), the second, undamaged DNA strand acts as a template for accurate reconstruction of the damaged strand.  Damage that affects both DNA strands, requires different mechanisms: HR and NHEJ. 28 Recombination repair  Recombinational repair requires the presence of an identical or nearly identical sequence to be used as a template for repair of the break.  The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover during meiosis 29 homologous recombination,  Human genes involved in this pathway include NBS (Nijmegen breakage syndrome), BLM (Bloom syndrome), and the breast cancer susceptibility genes BRCA2 and BRCAI 30 nonhomologous end joining  In NHEJ, DNA Ligase IV, directly joins the two ends regardless of their sequence .  NHEJ can cause mutations during repair. Loss of damaged nucleotides at the break site can lead to deletions, and joining of nonmatching termini forms translocations  NHEJ is especially important before the cell has replicated its DNA, since there is no template available for repair by homologous recombination * Recombinational repair and NHEJ correct doublestrand breaks. 31 Mismatch Repair (MMR)  MMR corrects errors of DNA replication and recombination that result in mispaired (but non- damaged) nucleotides following DNA replication. • Examples of mismatched bases include a G/T or A/C pairing. • Mismatch repair is strand-specific. During DNA synthesis the newly synthesised (daughter) strand will include errors commonly. • In order to do this the mismatch repair machinery distinguishes the newly synthesised strand from the template (parental). 32 Mismatch Repair (MMR) -2  The damage is repaired by recognition of the deformity caused by the mismatch, determining the template and non-template strand, and excising the wrongly incorporated base and replacing it with the correct nucleotide.  Cells deficient in mismatch repair have mutation rates 100-1000-fold normal.  In humans, the MMR mechanism involves at least 5 proteins, and defects cause Lynch syndrome (AD, Hereditary nonpolyposis colorectal cancer).  BER, NER and MMR correct single strand damages. 33 Translesion Synthesis  A DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites. – AP site (apurinic/apyrimidinic site), also known as an abasic site, is a location in DNA that has neither a purine nor a pyrimidine base, usually due to DNA damage. • Translesion synthesis polymerases often (but not always) have low fidelity (high propensity to insert wrong bases) relative to regular polymerases. • Pol η mediates error-free bypass of lesions induced by UV irradiation, whereas Pol ζ introduces mutations at these sites. 34 DNA Damage vs. Mutation  DNA damages and mutation are fundamentally different.  Damages are physical abnormalities in the DNA, such as single and double strand breaks.  DNA damages can be recognized by enzymes, and thus they can be correctly repaired if redundant information, such as the undamaged sequence in the complementary DNA strand or in a homologous chromosome, is available for copying.  If a cell retains DNA damage, transcription of a gene can be prevented and thus translation into a protein will also be blocked. Replication may also be blocked and the cell may die. 35  In contrast to DNA damage, a mutation is a change in the base sequence of the DNA.  A mutation cannot be recognized by enzymes, once the base change is present in both DNA strands, and thus a mutation cannot be repaired.  At the cellular level, mutations can cause alterations in protein function and regulation. 36  Mutations are replicated when the cell replicates.  In a population of cells, mutant cells will increase or decrease in frequency according to the effects of the mutation on the ability of the cell to survive and reproduce.  Although distinctly different from each other, DNA damages and mutations are related because DNA damages often cause errors of DNA synthesis during replication or repair and these errors are a major source of mutation. 37 DNA Repair Defects and Human Diseases  A variety of human genetic diseases show partly overlapping phenotypes that suggest defects in some aspect of DNA repair.  These include xeroderma pigmentosum, Cockayne syndrome, trichothiodystrophy, Fanconi anemia, ataxia-telangiectasia, Nijmegen breakage syndrome, and Bloom syndrome.  Patients may suffer symptoms including hypersensitivity to sunlight, neurological and/or skeletal problems, anemia, and especially a high incidence of various cancers.  In the laboratory, cells from patients with these disorders show hypersensitivity to various DNA-damaging agents. 38 39