BMB 462 1st Edition Lecture 21 Outline of Last Lecture I. Strand and Base PropertiesII. Base ConformationIII. Summary of DNA StructureIV. Comparing Types of DNAV. DenaturationVI. Techniques Utilizing DenaturationVII. Other Secondary StructuresVIII. Mutations in DNAOutline of Current Lecture I. Mutations in DNAII. Beneficial Methylation- Begin Unit on Chromosome Structure -III. Composition of the GenomeIV. Supercoiling in ChromosomesV. Function of TopoisomerasesVI. Nucleosome Formation and other Chromosome StructuresVII. Maintenance of Chromosomal StructureCurrent LectureConcepts to remembers from previous courses/lectures:- TautemerizationI. Mutations in DNAa. Review of Depurinationi. The glycosidic bond breaks so the sugar loses its base.b. Pyrimidine Dimersi. This occurs when there are adjacent thymines in the DNA sequence.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.1. The thymines are converted by UV light into cyclobutane thymine dimersii. Formation of the dimer creates a kink in the DNA, which can be repaired by the cell.1. A single kink in E. coli DNA is enough to kill the bacterium, which iswhy UV light is used for sterilizationc. Methylation of Guaninei. Another mutation is caused by alkylation of bases; i.e. methylation of guanine.1. Tautomerization occurs, moving the H from N5 to create a hydroxyl group on C6. In this form, the molecule can be methylated. 2. The methylguanine formed can no longer base pair with cytosine.ii. The methylation is done by alkylating groups like dimethylsulfate, a very toxic environmental agent. II. Beneficial Methylationa. Some methylation can be beneficial to the cell; SAM does beneficial methylation.b. 2 important typesi. Cytidine methylation (done in mammals)ii. Adenosine methylation (only done in bacteria)1. Bacteria use the N6 adenosine methylation to distinguish their DNA from foreign DNA; i.e. viral DNAc. Beneficial methylation is also used as a specific repair mechanism in the cell.d. Cytidine methylation in mammals is quite common; CPGs are common residues in the mammalian cells and so are frequently methylated (~5% of CPGs in eukaryotic cells)i. CPG methylation plays a role in gene silencing.III. Composition of the Genomea. Genomei. The genome is comprised of all the DNA in a cell (or virus)ii. This includes mitochondrial or (in plants) chloroplast DNAiii. The genome is partitioned into individual chromosomes.b. Chromosomei. In bacteria, chromosomes are circular with one origin of replication and several termination sites.ii. Eukaryotic cells have linear chromosomes.1. The centromeres are the site of attachment for the mitotic spindles.2. Telomeres are located at the ends, and protect the ends of the chromosome.3. Multiple origins of replication.iii. Each chromosome has many genesc. Genesi. A gene is a sequence of DNA that encodes a protein or RNA.1. The promoters/regulatory sequences control gene expression.ii. Eukaryotic genes have introns and exons (bacteria do not)1. Exons have the coding DNA that codes for protein 2. Introns are intervening sequences of DNA that are removed prior to translation of mRNA into protein.d. Composition of the Human Genomei. Only about 1.5% of DNA is comprised of exons to code for proteinsii. There are a lot of regulatory genes controlling gene expressioniii. A large portion of LINEs and SINEs are transposons, which are kind of like viruses. These are self-promoting sequences that can hop around the genome.iv. Miscellaneous sequences whose purpose is not known; these may play a role in regulation.e. Comparing Genome Sizesi. Saccharomyces cerevisiae (common bakers' yeast) has a genome of 12 million base pairs. When comparing that to Drosophila melanogaster (fruit fly), there is a 10-fold increase in genome size. 1. There is only a 4-5-fold increase in the number of genes, though.ii. Going from the genome of the fruit fly to humans, there is a 30-fold increase in the total number of base pairs, but the number of genes did not increase much, only about 1.5-fold (from 20,000 genes to 29,000 genes).1. What has increased is the regulatory sequences; there is much more regulation in higher organisms.IV. Supercoiling in Chromosomesa. The DNA in cells is under-wound, which helps facilitate processes like conscription and replicationb. Variablesi. Twist (Tw) - how often single strands cross over each other.ii. Writhe (Wr) - how often double strands cross over each other.iii. Linking Number (Lk) - in a circular piece of DNA, the Linking Number is the twists + the writhe.1. If the DNA is relaxed, (like in the first block), there is no writhe so Lk = Tw.2. Lk also = (for a relaxed pieces of DNA) the length of base pairs/# ofbase pairs per turn3. If you do not break the circular DNA while coiling/uncoiling, the Lkdoes not change.iv. ΔLk = ΔWr + ΔTwc. Positive vs. Negative Supercoilingi. A right-handed helix has a positive twist ii. Right-handed supercoiling has a negative writhe, and left-handed supercoils have a positive writhe.d. Superhelical Density (σ)i. This is the degree of supercoiling over relaxed DNA. It is defined as σ = ΔLk/Lko1. Lko = the Lk of relaxed DNAe. Alternate forms of Writhe - Instead of the double strands simply crossing over each other, there are other types of writhe.i. i.e. plectonemic and solenoidalV. Function of Topoisomerasesa. Overviewi. Topoisomerases are enzymes that can alter DNA topologyii. Type I and Type II topoisomerases function slightly differently, but both control supercoiling, changing the linking number1. They actually break the backbone2. Usually they relax supercoiling.b. Type I Topoisomerasei. Type I topoisomerases change the Lk by 1.ii. They do not require ATP1. Use transesterification to break and reform the backbone.iii. Mechanism: A tyrosine residue in the active site of the topoisomerase carries out a nucleophilic attack on the backbone of DNA and breaks the bond. Later, a 3' OH in the backbone carries out a nucleophilic attack on a phosphodiester bond, attacking the phosphate currently attached to the topoisomerase, reforming the bond and sealing the backbone.1. While the backbone is broken, the supercoiling is changed by threading one strand through the other.2. Lk is changed by 1, so 1 strand can pass through during each break.c. Type II Topoisomerasei. Change the linking number by 2 and require 2 ATPs per cycle. 1. They thread both double
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