General Biology 2 BZE: DNA Damage & Repair Class Notes PDF

General Biology 2 BZE: Mistakes happen! Class 15: DNA Damage & Repair  TODAY’S MENU: 1) DNA Proofreading and Repair  Proofreading  Repair 2) DNA Packaging 3) The fundamentals of transcription  Discovery of the model  Transcription and Translation  Overview of the st...

General Biology 2 BZE: Mistakes happen! Class 15: DNA Damage & Repair  TODAY’S MENU: 1) DNA Proofreading and Repair  Proofreading  Repair 2) DNA Packaging 3) The fundamentals of transcription  Discovery of the model  Transcription and Translation  Overview of the steps Campbell: Chapter 16  1) DNA PROOFREADING & REPAIR replication of an enormous amount of genetic information is achieved with very few errors: only about one per 10 billion nucleotides The copying of DNA is remarkable in its speed and accuracy. This is due to:  the high specificity of base pairing during replication  Proofreading and error-checking mechanisms ensure near perfect matching in base pairs during DNA replication.  Other enzymes can further fix errors left behind by DNA polymerase or created after DNA synthesis  PROOFREADING ABILITY During DNA replication, DNA polymerase proofreads each nucleotide against its template as soon as it is covalently bonded to the growing strand. It immediately replaces the ‘wrong’ nucleotide by the ‘right’ one. In this example, a mismatched “C” is replaced with the correct “T”. This is possible because DNA polymerase has exonuclease abilities (can cleave off nucleotides).  REPAIR: NUCLEOTIDE EXCISION REPAIR (NER) 1. Mismatched base What can damage DNA? pairs are detected by teams of enzymes.  Reactive chemicals,  radioactivity,  X-rays,  ultraviolet light, 2. Nuclease enzyme cuts  other harmful chemicals (ie. the damaged strand mycotoxins, asbestos, at two points releasing the nanoparticles) damaged section. Mismatched nucleotides sometimes evade DNA polymerase proofreading. In mismatch repair, other enzymes 3. DNA polymerase remove and replace incorrectly paired places corrects nucleotides in gap. nucleotides that have resulted from replication errors. Nuclease enzyme 4. Ligase seals the DNA polymerase nucleotides together. DNA ligase  DNA MISMATCH REPAIR E. coli mechanism: How to know which strand has the right sequence? Parental DNA is methylated and daughter DNA is not Repairing these errors is essential to the survival of an organism Specific enzymes exist to catalyze repair: about 100 DNA repair enzymes have been found in E. coli & over 130 in humans UV-RELATED DNA DAMAGE IN SKIN CELLS  UV covalently crosslinks two adjacent pyrimidines  Ex: Thymine dimer  Distorts the DNA molecule  Repair machinery detects the distortion in the DNA  REPAIR: UV DAMAGE Inherited disease: xeroderma pigmentosum (XP) Defect in nucleotide excision repair mechanisms. Hypersensitivity to sunlight. Mutations in skin cells caused by ultraviolet light are left uncorrected, often resulting in skin cancer. Without sun protection, children who have XP can develop skin cancer by age 10.  2) DNA PACKAGING Stretched out, the DNA of an E. coli cell would measure about a millimeter in length, which is 500 times longer than the cell. However, certain proteins cause the chromosome to coil and “supercoil,” densely packing it so that it fills only part of the cell. Unlike the nucleus of a eukaryotic cell, this dense region of DNA in a bacterium, called the nucleoid, is not bound by membrane.  DNA PACKAGING: EUKARYOTES Each eukaryotic chromosome contains a single linear DNA double helix in humans, that averages about 1.5 × 108 nucleotide pairs (per chromosome) If completely stretched out, a chromosome would be ~4 cm long (x 46 chromosomes) Remember from one of your Bio 1 labs, the diameter of a human cell nucleus is ~ 10 mm Eukaryotic DNA is precisely combined with a large amount of protein. This complex of DNA and proteins is called chromatin.  DNA PACKAGING: CHROMATIN DNA double Histones: Nucleosome: Fibers: helix: (Proteins with Basic unit of More complex (DNA) high content DNA packaging. arrangement and Looped domains: of positively Unfolded folding of Folding of the DNA arranges charged chromatin. nucleosomes. fibers. itself in a amino acids) (Beads on a Results from the These also fold double-helix. Responsible string, interactions during for the first separated by between histone replication. level of DNA linker DNA). tails and linker Prophase: 300 packaging Present during DNA further nm fibres form. interphase away. Present Metaphase: 700 during interphase nm fibres form.  DNA PACKAGING: CHROMATIN PHASES Chromatin undergoes striking changes in its degree of packing during the cell cycle.  Interphase: Highly extended  Prophase: Chromatin coils/folds (condenses)  Metaphase: Short and thick chromosomes Though interphase chromatin is generally much less condensed than the chromatin of mitotic chromosomes, it shows several of the same levels of higher-order packing.  CHROMATIN: SPATIAL ORGANIZATION Looped domains of chromatin seem to be attached to the nuclear envelop during interphase. These attachments may help organize regions of chromatin where genes are active. (More of this in transcription and gene expression class notes) The chromatin of each chromosome occupies a specific restricted area within the nucleus. The chromatin fibers of different chromosomes do not appear to be entangled.  HETEROCHROMATIN vs EUCHROMATIN Some regions of chromatin are more or less condensed than other regions even during interphase. Regions of highly condensed chromatin (heterochromatin) are characterized by DNA which is largely inaccessible to transcriptional machinery. Regions of less tightly-packed chromatin (euchromatin) render the DNA in those regions more easily transcribed. These regions are usually associated with more actively- transcribed genes. Chemical modifications of histones affect the state of chromatin condensation and have effects on gene activity.  3) TRANSCRIPTION: GENE  PROTEIN The DNA inherited by an organism dictates the synthesis of proteins and of RNA molecules involved in protein synthesis. This produces specific traits of an organism Proteins link genotype and phenotype. Gene expression is the process by which DNA directs the synthesis of proteins (or, in some cases, just RNAs). The expression of genes that code for proteins includes two stages: transcription and translation.  INDIVIDUAL GENES SPECIFY INDIVIDUAL ENZYMES Neurospora has modest food requirements (it’s a fungus) It can grow in the laboratory on a minimal nutrients solution incorporated into agar, a support medium. Neurospora cells use this minimal medium (MM) and their own metabolic pathways to produce all the other molecules they need to grow. Beadle and Tatum performed experiments that supported a one gene – one enzyme model, wherein each enzyme is encoded by a specific gene… ONE GENE – ONE ENZYME Beadle and Tatum bombarded Neurospora cells with X rays to generate mutants that require arginine specifically to grow. Testing revealed 3 classes of genetic mutants They then isolated the mutants that were unable to grow in MM without the addition of arginine into 3 separate subclasses.  ONE GENE – ONE ENZYME 3 classes of genetic mutants that needed arginine to grow Each mutant class had a defective gene  ONE GENE – ONE ENZYME Since each mutant was defective each in one gene and each were unable to produce one specific enzyme, Beadle & Tatum developed the hypothesis: “one gene – one enzyme” They demonstrated that the enzymes were coded for by genes by showing that these mutations were heritable mutations  ONE GENE – ONE ENZYME: FLAWS & REVISIONS The issue with the one gene – one enzyme model is:  not all genes encode enzymes.  Many genes encode proteins without enzymatic properties. Also, many proteins are constructed from two or more different polypeptide chains, and each polypeptide is specified by its own gene. As such, we turned to a one gene – one polypeptide model. Unfortunately, this is also inaccurate:  Many eukaryotic genes can each code for a set of closely related polypeptides via a process called alternative splicing  Quite a few genes code for RNA molecules that have important functions in cells even though they are never translated into protein.  TRANSCRIPTION & TRANSLATION Genes are typically hundreds or thousands of nucleotides long, each gene having a specific sequence of nucleotides. Each polypeptide of a protein also has monomers arranged in a specific linear order (primary structure) but made of amino acids. Transcription:  The synthesis of RNA using information in the DNA.  Different types of RNA can be produced through transcription Translation:  The synthesis of a polypeptide using the information in the mRNA.  Change in language: nucleic acids to amino acids  TRANSCRIPTION & TRANSLATION Although the basic principles of transcription and translation are similar between bacteria and eukaryotes, there exists some notable differences: Bacteria:  Don’t have nuclear membranes, therefore they can initiate translation before transcription is finished. Eukaryotes:  Transcription and translation occur in different compartments  Transcription produces a pre-mRNA which must undergo post-transcriptional modifications to be allowed out of the nucleus.  Primary transcript: All types of initial RNAs transcribed.  TRANSCRIPTION DNA RNA RNA nucleotides are matched with A:T A:U the sequence found on the DNA template strand. C:G C:G Polymerization of the RNA deoxyribose ribose polynucleotide strand occurs in the 5’ to 3’ direction.  GENE COMPONENTS Prokaryotic Genes:  Regulatory Sequences (includes promoter)  Coding Region (Exon) Eukaryotic Genes:  Regulatory Sequences (includes promoter)  Coding Regions (Exons)  Noncoding Regions (Introns) The stretch of DNA downstream from the promoter that is transcribed into an RNA molecule is called a transcription unit.  STEPS OF TRANSCRIPTION Transcription involves the enzyme RNA polymerase.  Only synthesizes in the 5’  3’ direction  Does not require a primer to start transcribing! In prokaryotes there is one type of RNA polymerase, while in Eukaryotes there are three known variants. Steps of transcription: Initiation Elongation Termination Now, you should be able to: Describe DNA mismatch repair and nucleotide excision repair Relate the different levels of DNA packaging to histone-DNA interactions and the cell cycle Explain the one gene / one enzyme hypothesis using the experiment of Beadle and Tatum and how that definition has changed until now. Describe the two main stages of gene expression Identify the broad differences between gene expression in prokaryotes and eukaryotes