DA: The Genetic Material Chapter 14 Griffith s experiment with Streptococcus pneumoniae Live S strain cells killed the mice Live R strain cells did not kill the mice eat-killed S strain cells did not kill the mice eat-killed S strain + live R strain cells killed the mice 2 Avery, MacLeod, & McCarty 1944 Transformation Information specifying virulence passed from the dead S strain cells into the live R strain cells ur modern interpretation is that genetic material was actually transferred between the cells Repeated Griffith s experiment using purified cell extracts Removal of all protein from the transforming material did not destroy its ability to transform R strain cells DA-digesting enzymes destroyed all transforming ability Supported DA as the genetic material 3 4 ershey & Chase 1952 Investigated bacteriophages Viruses that infect bacteria Bacteriophage was composed of only DA and protein Wanted to determine which of these molecules is the genetic material that is injected into the bacteria Bacteriophage DA was labeled with radioactive phosphorus ( 32 P) Bacteriophage protein was labeled with radioactive sulfur ( 35 S) Radioactive molecules were tracked nly the bacteriophage DA (as indicated by the 32 P) entered the bacteria and was used to produce more bacteriophage Conclusion: DA is the genetic material 5 6 1
Chargaff s Rules Erwin Chargaff determined that Amount of adenine = amount of thymine Amount of cytosine = amount of guanine Always an equal proportion of purines (A and G) and pyrimidines (C and T) Rosalind Franklin Performed X-ray diffraction studies to identify the 3-D structure Discovered that DA is helical Using Maurice Wilkins DA fibers, discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm 7 8 James Watson and Francis Crick 1953 Deduced the structure of DA using evidence from Chargaff, Franklin, and others Did not perform a single experiment themselves related to DA Proposed a double helix structure 9 DA Structure DA is a nucleic acid Composed of nucleotides 5-carbon sugar called deoxyribose Phosphate group (P 4 ) Attached to 5 carbon of sugar itrogenous base Adenine, thymine, cytosine, guanine Free hydroxyl group ( ) Attached at the 3 carbon of sugar itrogenous base 2 7 6 5 1 Phosphate group 8 2 4 9 3 P C 2 4 2 1 in RA in DA Purines Pyrimidines C 2 C C C C Adenine 2 C C C C Cytosine (both DA and RA) itrogenous Base C C C C C 2 Guanine 3C C C C C Thymine (DA only) C C C C Uracil (RA only) 10 Phosphodiester bond Bond between adjacent nucleotides Formed between the phosphate group on the 5 carbon of one nucleotide and the 3 of the next nucleotide Phosphodiester bond The chain of nucleotides has a 5 -to-3 orientation P 4 C 2 C P C 2 Base Base Double helix 2 strands are polymers of nucleotides Phosphodiester backbone repeating sugar and phosphate units joined by phosphodiester bonds Wrap around 1 axis Antiparallel 11 12 2
DA Replication Complementarity of bases A forms 2 hydrogen bonds with T G forms 3 hydrogen bonds with C Gives consistent diameter G ydrogen bond ydrogen bond C C 3 3 possible models 1. Conservative model 2. Semiconservative model 3. Dispersive model A T 13 Conservative Semiconservative Dispersive 14 Meselson and Stahl 1958 Bacterial cells were grown in a heavy isotope of nitrogen, 15 All the DA incorporated 15 Cells were switched to media containing lighter 14 DA was extracted from the cells at various time intervals The semiconservative method was confirmed DA Replication Requires 3 things Something to copy Parental DA molecule Something to do the copying Enzymes Building blocks to make copy ucleotide triphosphates 15 16 DA replication includes Initiation replication begins Elongation new strands of DA are synthesized by DA polymerase Termination replication is terminated RA polymerase makes primer DA polymerase Matches existing DA bases with complementary nucleotides and links them All have several common features Add new bases to 3 end of existing strands Synthesize in 5 -to-3 direction Require a primer of RA DA polymerase extends primer 17 18 3
Prokaryotic Replication E. coli model Single circular molecule of DA Replication begins at one origin of replication Proceeds in both directions around the chromosome Replicon DA controlled by an origin E. coli has 3 DA polymerases DA polymerase I (pol I) Acts on lagging strand to remove primers and replace them with DA DA polymerase II (pol II) Involved in DA repair processes DA polymerase III (pol III) Main replication enzyme 19 20 Semidiscontinous elicase opens the double helix DA polymerase can synthesize only in 1 direction Leading strand synthesized continuously from an initial primer Lagging strand synthesized discontinuously with multiple priming events kazaki fragments Partial opening of helix forms replication fork DA primase RA polymerase that makes RA primer RA will be removed and replaced with DA 21 22 Leading-strand synthesis Single priming event Strand extended by DA pol III Processivity β subunit forms sliding clamp to keep it attached Lagging-strand synthesis Discontinuous synthesis DA pol III RA primer made by primase for each kazaki fragment All RA primers removed and replaced by DA DA pol I Backbone sealed DA ligase DA ligase Lagging strand (discontinuous) Termination occurs at specific site RA primer DA polymerase I DA gyrase unlinks 2 copies kazaki fragment made by DA polymerase III Leading strand (continuous) Primase 23 24 4
Replisome Enzymes involved in DA replication form a macromolecular assembly 2 main components Primosome Primase, helicase, accessory proteins Complex of 2 DA pol III ne for each strand Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the ormal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http:// get.adobe.com/flashplayer. 25 26 Eukaryotic Replication Complicated by Larger amount of DA in multiple chromosomes Linear structure Basic enzymology is similar Requires new enzymatic activity for dealing with ends only Multiple replicons multiple origins of replications for each chromosome ot sequence specific; can be adjusted Initiation phase of replication requires more factors to assemble both helicase and primase complexes onto the template, then load the polymerase with its sliding clamp unit Primase includes both DA and RA polymerase Main replication polymerase is a complex of DA polymerase epsilon (pol ε) and DA polymerase delta (pol δ) 27 28 Telomeres Specialized structures found on the ends of eukaryotic chromosomes Protect ends of chromosomes from nucleases and maintain the integrity of linear chromosomes Gradual shortening of chromosomes with each round of cell division Unable to replicate last section of lagging strand Telomeres composed of short repeated sequences of DA Telomerase enzyme makes telomere of lagging strand using and internal RA template (not the DA itself) Leading strand can be replicated to the end Telomerase developmentally regulated Relationship between senescence and telomere length Cancer cells generally show activation of telomerase 29 30 5
DA Repair Errors due to replication DA polymerases have proofreading ability Mutagens any agent that increases the number of mutations above background level Radiation and chemicals Importance of DA repair is indicated by the multiplicity of repair systems that have been discovered DA Repair Falls into 2 general categories 1. Specific repair Targets a single kind of lesion in DA and repairs only that damage 2. onspecific Use a single mechanism to repair multiple kinds of lesions in DA 31 32 Photorepair Specific repair mechanism For one particular form of damage caused by UV light Thymine dimers Covalent link of adjacent thymine bases in DA Photolyase Absorbs light in visible range Uses this energy to cleave thymine dimer 33 Excision repair onspecific repair Damaged region is removed and replaced by DA synthesis 3 steps 1. Recognition of damage 2. Removal of the damaged region 3. Resynthesis using the information on the undamaged strand as a template 34 6