DNA Replication, Telomeres, and Mutations

DNA Replication and Mutation Very few errors are introduced during replication RNA polymerase error rate: 1 in 104 3’-->5’ Exonucleolytic Proofreading A cytosine is accidentally incorporated across from an adenine. The C does not base pair...

DNA Replication and Mutation Very few errors are introduced during replication RNA polymerase error rate: 1 in 104 3’-->5’ Exonucleolytic Proofreading A cytosine is accidentally incorporated across from an adenine. The C does not base pair properly, leaving the 3’ end unpaired and blocking further polymerization. 3’-->5’ Exonucleolytic Proofreading The exonuclease activity of DNA polymerase chews off the C. The correct nucleotide is inserted and polymerization continues.  Strand-directed mismatch repair catches mistakes It is best to fix mismatches during DNA synthesis, when the enzyme can clearly tell which strand is the template (and therefore “correct”) and which strand is currently being synthesized (which has the incorrect base). The newly synthesized base is always excised and replaced. What about mismatches that escape the exo? A second repair process catches them. However, it has a challenge: how to tell which strand is correct and which is wrong. If it can’t distinguish old from new strands, it will guess wrong half the time (that’s still better than leaving the mismatch in the DNA). In E. coli, the mismatch repair system uses the methylation state to tell old from new strands (old is methylated, new not). In eukaryotes, the mismatch repair system looks for nicks, which are more common on the new strand. Strand-directed mismatch repair catches mistakes The repair system does not just remove the mismatched base. Rather, all the new strand in the region is removed and resynthesized. Replication of Eukaryotic Chromosome: The End Replication Problem  The DNA At Telomeres Is Composed Of A Simple Repeated Sequence Telomeric repeat sequences Vertebrates: TTAGGG 3′ T T T T T T T T T T T T T T T T AG G G A G G G A G G G A G G G A G G G A G G G A G G G A G G G A A A A A A A A A A A A T C C C T C C C T C C C T C C C T C C C T Overhang 5′  The ends of our chromosomes have special structures called telomeres 5’ TTGGGGTTGGGGTTGGGGTTGGGG 3’ AACCCCAACCCC Replication at the chromosome end is problematic: – Degradation of the 5’ ends of the lagging strands – Undesirable recognition of ends as double strand breaks  Solution to these problems: Telomerase: An enzyme that adds repeats to the end of a chromosome. It is a Ribonucleoprotein: it contains an RNA molecule that serves as an internal template for repeat addition It adds telomeric repeats (TTGGGGTTGGGGTTGGGG) to maintain telomere length. (T2G4 Tetrahymena; T2AG3 mammals) Telomere capping mechanisms – G-quadruplexes and capping proteins – t-loops and capping proteins Telomerase adds telomeric repeats Telomerase adds telomeric repeats Telomerase is a DNA polymerase of the reverse transcriptase family. It carries its own RNA template that templates for 1.5 repeat units.  Telomere Capping: T-loops Telomeres are packaged into specialized structures that protect the ends of chromosomes  Telomere capping mechanisms T-loops and the shelterin complex Shelterin complex: From Effrossyni Boutou et al (2013). DNA Repair and Telomeres — An Intriguing Relationship, New Research Directions in DNA Repair, Prof. Clark Chen (Ed.). Available from: http://www.intechopen.com/books/new-research-directions-in-dna-repair/dna-repair- and-telomeres-an-intriguing-relationship Telomeres Are Protected by The Shelterin Complex Shelterin: a complex of 6 proteins TRF 1, TRF 2-Bind dsDNA POT1- Binds ssDNA TIN2- Links TRF1 and TRF2s TPP1 –Links POT1 with the TRF complexes know all roles offD  Telomere Length Is Regulated by Cells and Organisms Artificial chromosome with Telomere length is readjusted to wt after multiple rounds of cell division  Why Telomere Replication is important? In humans, the mutation of telomere maintenance genes results in dyskeratosis congenita (DC) DC is an inherited disease characterized by: Abnormal Skin Pigmentation Nail Dystrophy Mucosal Leukoplakia Pulmonary & Liver Disease Gastrointestinal Tract Abnormalities Predisposition to Cancer Bone marrow failure, or aplastic anemia, is the principal cause of premature mortality in patients Dyskeratosis Congenita (DC) Correlates With Extreme Telomere Shortening De Telo shortening Hayflick Limit: The number of times a normal cell can divide In normal cells, telomeres shorten with each cell division.  Cells senesce when their telomeres become too short Telomerase expression is lost in most cells of our body. Replicative senescence occurs when telomeres become too short. Replicative senescence is associated with aging. Mice that are engineered to lack telomerase age rapidly. Adding back telomerase (by controlled expression) reverses the aging process. Proliferating cells senescent cells Blue stain: b-galactosidase Marker of senescence Leontieva and Blagosklonny, “DNA damaging agents and p53 do not cause senescence in quiescent cells, while consecutive re-activation of mTOR is associated with conversion to senescence ” Aging, 2, 924-935 (2010) Hayflick Limit and Telomere length  Hayflick Limit and Telomere length A transformed cell has bypassed the Hayflick Limit (Senescence) and an immortalized cell has survived Crisis, by reactivating telomerase activity. By definition, all human cancer cells are transformed and immortal. The malignant ones are also tumorigenic. DNA Mutation  DNA Mutation DNA damage is the primary cause of cancers that kill ~600,000 Americans every year. $200 billion a year is currently spent on cancer treatment in the US. For example, one in four people in this room will at some point in their lives be diagnosed with a basal cell carcinoma (skin cancer) due to the mutation in a single cell of both copies of the tumor suppressor gene hptc. DNA can be damaged in several ways: A. Spontaneous: Occur through multiple mechanisms: oxidative damage, uncontrolled methylation hydrolytic attack (depurination and deamination) B. Radiation C. Chemical D. Errors in DNA replication  DNA Damage In one day a single human cell (x ~ 1013 cells/human body) is thought to suffer: Friedberg, et al, 2006. DNA repair and mutagenesis  Types of mutations: Silent mutations Because only 1.5% of our DNA comprise codons, changes in the other 98.5% of our DNA sequence are largely inconsequential. Even within the DNA that encodes amino acid residues of proteins (codons), some changes do not alter the primary sequence of the protein because the code is degenerate.  Mutations Any change in the DNA sequence of an organism is a mutation. Mutations are the source of the altered versions of genes that provide the raw material for evolution. Most mutations have no effect on eukaryotes, because a large portion of the DNA is not in genes and thus does not affect the organism’s phenotype. Mutations in essential genes lead to lethality Only a small percentage of mutations cause a visible but non-lethal change in the phenotype.  Types of Mutation Mutations can be classified according to their effects on the protein produced by the mutated gene. 1. Silent Mutations. Since the genetic code is degenerate, several codons code for the same amino acid. Especially, third base changes often have no effect on the amino acid sequence of the protein. These mutations affect the DNA but not the protein. Therefore they have no effect on the phenotype. 2. Missense mutations. Missense mutations substitute one amino acid for another. Some missense mutations have very large effects, while others have minimal or no effect. It depends on where the mutation occurs in the protein’s structure, and how big a change in the type of amino acid it is.  Types of Mutation 3. Nonsense mutations convert an amino acid into a stop codon. The effect is to shorten the resulting protein. These are often devastating mutations that result in completely non-functional proteins. However, sometimes nonsense mutations have only a little effect if they occur at the carboxy terminal end of proteins 4. Sense mutations are the opposite of nonsense mutations. Here, a stop codon is converted into an amino acid codon. Since DNA outside of protein-coding regions contains an average of 3 stop codons per 64, the translation process usually stops after producing a slightly longer protein.  Frameshifts and Reversions 5. Insertion or deletion of a nucleotide causes a frameshift. Frameshift mutations result in all amino acids downstream from the mutation site being completely different from wild type. These proteins are generally non-functional. A “reversion” is a second mutation that reverse the effects of an initial mutation, bringing the phenotype back to wild type (or almost). Frameshift mutations sometimes have “second site reversions”, where a second frameshift downstream from the first frameshift reverses the effect. npulau polarAA  11 11 Effect of Point Mutations Within a Coding Region Sickle Cell Anemia: Hbb E6V (147 aa) ; Cystic Fibrosis CFTR DF508 (1480 aa) Does frameshifting ever occur on purpose? Many retroviruses, including HIV, use frameshifting to generate different proteins from the same mRNA. For example, the gag pol mRNA encodes 2 proteins that overlap by 200 nucleotides and are in different reading frames. To produce the pol proteins, frameshifting must occur. A gag-pol fusion protein is produced, which is later proteolytically processed to release the two proteins in their active forms. The frameshift occurs at the ‘Ribosome’ (translation) level, and not through alteration of DNA sequence. This mechanism is known as ‘Ribosomal frameshifting’.  Mutations that alter the amino acid sequence of a protein Two ways mutations alter the behavior of a protein. Recessive: A mutation that does not manifest a phenotype in the presence of a normal copy. e.g. Mutations that destroy the activity of a protein are often tolerated because we have two copies of most genes, and we can get by with half the amount of most proteins. (Sickle cell anemia, Cystic Fibrosis) Dominant: A mutation that manifests itself to change the outcome for that protein’s role in the system, even in the presence of a normal copy (Huntington disease)  Polymorphisms You have two copies of each chromosome, but they are not the same. About 1 – 2% of the nucleotides are different, most of the these are inconsequential and are called polymorphisms. Many of these differences are a single base pair difference and are called SNP (snips) for single nucleotide polymorphism. The term SNP is sometimes replaced by SNV for single nucleotide variation. Numerous differences in longer DNA sequences have been found in our genome, meaning that some chromosomes are duplicated for some genes whereas other chromosomes may be deleted for another set of genes. These are called CNV for copy number variation.  1. Spontaneous Damage to DNA : Depurination and Deamination These two reactions are the most frequent spontaneous reactions known to create serious DNA damage in cells. During the time it will take you to read this sentence, a total of about six trillion (1012) purine bases will be depurinated in your body. Depurination affects both purines and deamination can occur on other nucleotides as well as cytosine.  Spontaneous Damage to DNA Sites that are known to be modified by spontaneous oxidative damage, hydrolytic attack, and uncontrolled methylation are shown, with the width of each arrow indicating the relative frequency of each event.  Spontaneous Damage to DNA: Depurination and Deamination Depurination is the process whereby purine bases (adenine and guanine) are lost because their N-glycosyl linkages to deoxyribose are spontaneously hydrolyzed About 5,000 purine bases per cell per day are lost this way! Deamination occurs on all bases but the deamination of cytosine to uracil is the most common, with a rate of about 100 bases per cell per day. Depurination breaks the glycosidic bond between the sugar and the A or G base In deamination, an extracyclic amino group is lost from the base Deamination of cytosine creates uracil: what do you think will happen when this damaged DNA is replicated? Depuration Can Result In A Deletion Mutation Deamination can occur on any base except T. What problem does this cause? Changes base Pt mutation Deamination Of Cytosine Can Result in a CG > TA Mutation  Deamination of 5-Methyl Cytosine Eukaryotic cells have a lot of CpG sequences that are methylated 5-Me 5-Me CH3 CH3 5 –Me Cytosine > 5 –Me Uracil = Thymine ??? Thymine is a base in DNA. The repair system does recognize the G-T mismatch but is not very efficient in fixing it leading to a mutation when the DNA is replicated Tautomeric Shifts Common Rare Tautomeric Shift Causes Mis-pairing Of Bases (rare form) (common form) (rare form) (common form)  Chemical Damage to DNA The Ames test is a famous test for determining the mutagenicity of chemicals and is the first test typically done when a new drug or chemical is synthesized that will be used in humans. Bacterial cells unable to make their own histidine due to a point mutation in a single gene are grown with and without the chemical of interest. Mutagenic compounds will cause mutations in the bacteria; among these will be rare mutations that revert the original mutations and allow the cells to grow on media lacking histindine. Often, those same chemicals will cause cancer in lab animal tests. Improvement to the test: treat the chemical with mammalian liver extract before putting it on the disc. Why? Ames Test: Panel B cells have been treated with a mutagen on the small white disc, which caused an increase in the number of colonies. Colonies on the plate in panel A are due to spontaneous mutations. Mutagens Chemical Modification Of Bases Leading To Mispairing Base Alkylation Deamination by Nitrous Acid Inter-strand cross-linking 5-bromo Uracil Causes Mutations Due To Frequent Tautomeric Shifts H N H Br O H N N N H N Sugar N N Sugar O H 5-bromouracil Adenine (keto form) N H Br O H O N N H N Sugar N N Sugar O H N H 5-bromouracil Guanine (enol form)  Chemical Damage to DNA DNA adducts are chemical groups that are added onto __ DNA as a consequence of high exposure to a mutagenic chemical. A common example is benzo[a]pyrene, which is a component of cigarette smoke. Benzo[a]pyrene is not carcinogenic until it is oxidized within cells to benzo[a]pyrene diol epoxide. Then, it can intercalate into DNA and covalently bind to guanine residues in DNA, interrupting hydrogen bonding in G-C base pairs. What’s wrong with this picture? Beno Pyrene Intercalacated  Why would the cell oxidize benzopyrene to the diol epoxide? It is oxidized by cytochrome P450 enzymes, probably in an attempt to detoxify it. Other tobacco compounds are made into more dangerous chemicals by detoxifying enzymes. Another bad thing about benzopyrene: Stribinski and Ramos (2006). Activation of Human Long Interspersed Nuclear Element 1 Retrotransposition by Benzo(a)pyrene, an Ubiquitous Environmental Carcinogen. Cancer Res 66: 2616-2620.  Chemotherapy Drugs Cisplatin Cross-links adjacent G residues- Intra- and Inter- molecular Doxorubicin Bleomycin Intercalator, blocks replication fork movement to cause strand breakage Binds to DNA, forms oxygen-radicals leading to strand breakage 2. Mutations caused by reactive oxygen species Consequences of oxidation: Different base pairing possible – Point mutations during replication Transversion Transition From Hsu et al, Nature 431 pp. 217-221 (2004) Reminder: Transitions vs Transversions  Radiation Damage to DNA Ultraviolet light can cause covalent linkages between adjacent pyrimidines, known as pyrimidine dimers or more commonly, thymine dimers. Ionizing radiation, for example X-rays and gamma rays, causes double stranded breaks in DNA.  The Thymine Dimer UV light can cleave the C=C double bond in the pyrimidine ring. The bond can reform between adjacent pyrimidines, particularly thymines, but CT dimers can also form. This causes problems for DNA replication as the dimers can not be recognized by DNA polymerase.  3. Mutations caused by UV radiation Causes covalent linkage between adjacent pyrimidines, called : thymine dimer Pyrimidine dimer Cyclobutane dimer  4. Mutations caused by X-rays Consequences of ionizing radiation: double strand break What happens if this damage is not corrected? These reactions cause mutations. Uncorrected depurination can lead to either the substitution or the loss of a nucleotide pair. When the replication machinery encounters a missing purine on the template strand, it may skip to the next complete nucleotide, producing a deletion. Uncorrected deamination of cytosine results in a substitution mutation. When the DNA is replicated, an adenine will be inserted instead of a guanine.