BMB 462 1st Edition Lecture 21 Outline of Last Lecture I Strand and Base Properties II Base Conformation III Summary of DNA Structure IV Comparing Types of DNA V Denaturation VI Techniques Utilizing Denaturation VII Other Secondary Structures VIII Mutations in DNA Outline of Current Lecture I Mutations in DNA II Beneficial Methylation Begin Unit on Chromosome Structure III IV V VI VII Composition of the Genome Supercoiling in Chromosomes Function of Topoisomerases Nucleosome Formation and other Chromosome Structures Maintenance of Chromosomal Structure Current Lecture Concepts to remembers from previous courses lectures Tautemerization I Mutations in DNA a Review of Depurination i The glycosidic bond breaks so the sugar loses its base b Pyrimidine Dimers i 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 dimers ii 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 is why UV light is used for sterilization c Methylation of Guanine i 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 Methylation a Some methylation can be beneficial to the cell SAM does beneficial methylation b 2 important types i 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 DNA c 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 Genome a Genome i The genome is comprised of all the DNA in a cell or virus ii This includes mitochondrial or in plants chloroplast DNA iii The genome is partitioned into individual chromosomes b Chromosome i 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 genes c Genes i 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 Genome i Only about 1 5 of DNA is comprised of exons to code for proteins ii There are a lot of regulatory genes controlling gene expression iii 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 Sizes i 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 Chromosomes a The DNA in cells is under wound which helps facilitate processes like conscription and replication b Variables i 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 of base pairs per turn 3 If you do not break the circular DNA while coiling uncoiling the Lk does not change iv Lk Wr Tw c Positive vs Negative Supercoiling i 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 i This is the degree of supercoiling over relaxed DNA It is defined as Lk Lko 1 Lko the Lk of relaxed DNA e 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 solenoidal V Function of Topoisomerases a Overview i Topoisomerases are enzymes that can alter DNA topology ii Type I and Type II topoisomerases function slightly differently but both control supercoiling changing the linking number 1 They actually break the backbone 2 Usually they relax supercoiling b Type I Topoisomerase i Type I topoisomerases change the Lk by 1 ii They do not require ATP 1 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 Topoisomerase i Change the linking number by 2 and require 2 ATPs per cycle 1 They thread both double strands through per break ii Mammalian cells can only relax supercoiling but can relax both positive and negative supercoils iii Bacteria E coli has a Type II
View Full Document
Unlocking...