DNA Structure and Replication PPT PDF

THE STRUCTURE OF NUCLEIC ACIDS DNA and RNA  TERMINOLOGY Genetics The scientific study of how living organisms determine and inherit physical characteristics. DNA Deoxyribonucleic acid = the material used to determine inheritance & expr...

THE STRUCTURE OF NUCLEIC ACIDS DNA and RNA  TERMINOLOGY Genetics The scientific study of how living organisms determine and inherit physical characteristics. DNA Deoxyribonucleic acid = the material used to determine inheritance & expression of physical characteristics in in living organisms. Nucleotide A monomer (building block, basic structural unit) within a DNA polymer Gene A segment of DNA that directs the production of one polypeptide. Genome The complete set of genetic material in one living organism. Genetic Code The association between nucleotides and corresponding Amino Acids of peptides.  TIMELINE OF DISCOVERIES MOLECULAR STRUCTURE OF DNA 1903 - Mendel’s Laws were accepted. 1903 - Chromosome Theory of Inheritance proposed. Very little detailed knowledge about the molecules involved in the above was known at that time. 1952 - DNA determined to be the universal inheritance material. 1953 – Molecular structure of DNA proposed by Watson & Crick. 2003 – Human Genome sequenced.  1928 – GRIFFITHS TRANSFORMING PRINCIPLE Research with smooth & rough strains of Streptococcus pneumoniae  1944 -AVERY, MCCART Y & MACCLEOD TRANSFORMING AGENT IDENTIFICATION Streptococcus pneumoniae separated into macromolecule fractions. Conclusion: DNA is the inheritance material. 1952 -HERSHEY & CHASE – PHAGE LABELING  DNA STRUCTURE: EARLY (PRE-1950) CONCEPTS Microscopy + Chemistry DNA specific stains to visualize chromosomes. to generate basic information about nucleic acid composition Biochemistry sugar + base = nucleoside nucleoside + phosphate = nucleotide Nucleotides are the monomers in Nucleic Acids. Note: Science is interdisciplinary. Life is interdisciplinary. How do these nucleotides look? How do these nucleotides assemble into polymers of DNA? DRAW A NUCLEOTIDE DRAW A NUCLEOTIDE  NITROGENOUS BASES Adenine & Guanine are Purines double ring Cytosine & Thymine & are pyrimidines. single ring Uracil (RNA) Chargaff ’s Rule: In dsDNA - # purines = # pyrimidines Adenine forms double hydrogen bond with Thymine. Cytosine forms triple hydrogen bond with Guanine  THE BIRTH OF MOLECULAR GENETICS DNA STRUCTURE DISCOVERIES IN 1950’S Three Dimensional models were proposed by many groups – all had technical errors. X-Ray Crystallography Performed by Rosalind Franklin, Maurice Wilkins The overall shape of DNA is helical. The individual nucleotides are perpendicular to the main axis of the molecule. Watson & Crick connected the pieces of information together to propose the accurate model.  DRAW A SEGMENT OF DNA The figure should include a total of four nucleotides. The DNA segment should be double stranded. Include one molecule of each nucleotide. DESCRIBE THE MOLECULAR STRUCTURE OF DNA  DESCRIBE THE MOLECULAR STRUCTURE OF DNA DNA structure is described as a “double helix”. Two linear polynucleotide strands are created by phosphodiester bonds. These two strands are held to each other by forming hydrogen bonds between their nitrogenous bases. The orientation of the two strands occurs is “anti- parallel”. The structure coils upon itself @ ten nucleotides (34 angstrom) per revolution.  THINK AND APPLY Define the word transformation for non-scientific purposes. How does this generic definition apply to the concept of transformation in genetics (e.g., Griffith’s experiment). What structural feature of DNA enables it to store specific information and provide direction concerning phenotypes? The nucleic acid content of a virus was analyzed. It contained: 14.1% A, 35.7% C, 36.2% G, 14.0% U. How much Thymine does it contain? DNA REPLICATION CENTRAL DOGMA OF GENETICS  THE CENTRAL DOGMA OF GENETICS : DNA is the genetic material. Its significance to cells is manifested in two ways. First, DNA undergoes replication to produce additional DNA --- this process enables generational continuity (i.e., enables the DNA to control what the next generation is going to be able to do = the future). Second, DNA undergoes transcription to synthesize RNA and the RNA will be translated to produce polypeptides --- these events determine what a cell/organism will be able to do in its current existence/‘lifetime’ (i.e., directs the present). REPLICATION OF DNA  When DNA structure was accurately described, the three ideas about how the replication process might occur: Conservative Dispersive Semi-Conservative Semi-Conservative Replication was proved to be the accurate mechanism by Meselsohn & Stahl’s - Density Gradient Centrifugation Experiment  REPLICATION: THREE CONSIDERATIONS What is the sequence of steps? Which enzymes are required? What are the physical/geometrical considerations? An over-simplified sequence of steps: A helix unwinds. New nucleotides are brought in to the unwound template helix. Phosphodiester bonds are catalyzed between the newly added nucleotides. The new nucleotide strands form hydrogen bonds with the template strands.  PROKARYOTIC REPLICATION ENZYMES Gyrase (a member of the topoisomerase enzyme family) relaxes supercoils. This occurs by creating small temporary nicks/cuts in one of the strands. Helicase completes the untwisting process and denatures the DNA (separates the two strands from each other. SSBP/single strand binding proteins attach to the separated single strands, stabilizing them and holding them “open” The above three items create a “Replication Fork”.  PROKARYOTIC REPLICATION ENZYMES DNA primase (a form of RNA polymerase) attaches to helicase, creating a “primosome” complex. Primosome initiates the replication process by catalyzing the production (phosphodiester bonds) of a 10-11 base RNA fragment. The fragment is called a primer. None of the DNA polymerases are able to start the phosphodiester catalysis on their own. This is why primase is so important.  PROKARYOTIC REPLICATION ENZYMES DNA Polymerase I removes primers after replication of true DNA polymers is complete. It appears to have a degree of proofreading ability as well. (i.e., it can detect and excise an incorrectly added nucleotide. This is called exonuclease activity.) DNA Polymerase II is believed to be a part of the proofreading and repair process, because it has exonuclease activity. However the complete role of this enzyme is not well understood. DNA Polymerase III is responsible for catalyzing phosphodiester bonds between the DNA nucleotides that are destined to become a part of the growing strand. These are referred to as Kornberg enzymes, acknowledging the work of Arthur Kornberg who initially isolated them. PROKARYOTIC REPLICATION ENZYMES Ligase seals the Okasaki fragments created on the “lagging” strand. Due to the directionality of DNA strands, One strand of the template is read easily & continuously by the polymerase enzyme. The other strand is read discontinuously, and essentially synthesizes DNA fragments. The two template strands are given the terms “Leading” and “Lagging”.  DIRECTION OF REPLICATION The Template is Read from 3 -> 5 The new DNA is created DNA from 5 -> 3 The Template & Newly synthesized DNA have Base Complementarity with each other. INITIATION OF REPLICATION IN PROKARYOTES Where does a circle start? Where does replication begin in Prokaryotes?  PROKARYOTIC REPLICATION INITIATION There is a designated location called OriC (origin of copying). Replication proceeds in 2 directions around the circle. Ori C  PROKARYOTIC REPLICATION INITIATION Where does a circle start? Where does replication begin in Prokaryotes? Called Theta replication ORI C – A CLOSER LOOK DIRECTION OF SYNTHESIS  ASSIGNMENT Watch the video. Go to the discussion board on Brightspace, and write a 200- 300 word analysis of the implications of this video. https://futurism.com/scientists-capture-dna- replication-on-video-for-the-first-time-heres-what-it- revealed  EUKARYOTIC REPLICATION ENZYMES ALPHA functions on the lagging strand (nuclear). It requires the molecule Replication factor C as a cofactor. BETA is a repair molecule for nuclear DNA. GAMMA synthesizes mitochondrial DNA. DELTA functions on the leading strand (nuclear). It requires the molecule PCNA as a cofactor. EPSILON is a repair molecule for nuclear DNA. XI is needed for mitochondrial DNA synthesis.  EUKARYOTIC REPLICATION PROCESS Eukaryotic Chromosomes are linear. We tend to imagine that replication will begin at one end and proceed toward the other - however, it does not happen this way. To save time, many origins of replication are initiated (hundreds, or thousands). Replication occurs bi-directionally from each initiation point. Eventually, adjacent replication forks will fuse/overlap. The area in between an origin of replication and the point where its replication fork will fuse with the next replication fork is called a replicon. REPLICATION BUBBLES  TELOMERASE In eukaryotic Replication, the very ends of chromosomes are not copied during regular replication process. This could lead to chromosome length being shortened. Enzyme TELOMERASE – fills in nucleotides at the ends; creates the Telomeres of the chromosomes. Telomeres are composed of highly repetitive DNA (they do not include many essential genes). High telomerase activity correlates with high rates of cell division/mitosis. Cancer cells have extremely high usage of Telomerase.  APPLY CONCEPTS Isolate: brain neurons, liver cells, epidermal cells Measure Telomerase activity in all 3 cell types. How will telomerase activity compare? Highest rate of mitosis = Epidermal cells highest use of telomerase Lowest rate of mitosis = Neurons lowest rate of telomerase activity Replication Where in the cell? When during cell cycle? Product Template Substrates Initiation Sequences Initiation Enzymes Synthetic Enzymes Types of Bonds # Directions of Synthesis Termination Process Where is product used? Percentage of Template Copied Modifications to Product Replication Event Nucleus Where in the cell? S phase When during cell cycle? ds DNA Product ds DNA Template Nucleotides (nucleoside triphosphates) Substrates Adenine, Guanine, Cytosine, Thymine Ori C Initiation Sequences Gyrase, Helicase, Primase Initiation Enzymes DNA Polymerases Synthetic Enzymes Phosphodiester, Hydrogen Types of Bonds 2 (bidirectional) # Directions of Synthesis Collision of Replication Forks Termination Process Nucleus Where is product used? Almost 100% Percentage of Template Copied Telomerase finishes replication; editing; 3D Modifications to Product compaction into chromosome Replication Transcription Nucleus Where in the cell? S phase When during cell cycle? ds DNA Product ds DNA Template Nucleotides (nucleoside Substrates triphosphates) A, G, C, T Ori C Initiation Sequences Gyrase, Helicase, Primase Initiation Enzymes DNA Polymerases Synthetic Enzymes Phosphodiester, Hydrogen Types of Bonds 2 (bidirectional) # Directions of Synthesis Collision of Replication Forks Termination Process Nucleus Where is product used? Almost 100% % of Template Copied Telomerase finishes replication; Modifications to Product editing; 3D compaction into chromosome Replication Transcription Nucleus Where in the cell? nucleus S phase When during cell cycle? G1 and G2 ds DNA Product ss RNA ds DNA Template ds DNA (select genes) Nucleotides (nucleoside Substrates A, U, C, G triphosphates) A, G, C, T Nucleotide triphosphates Ori C Initiation Sequences Promoter Gyrase, Helicase, Primase Initiation Enzymes RNA polymerase (sigma, core) DNA Polymerases Synthetic Enzymes Core RNA polymerase Phosphodiester, Hydrogen Types of Bonds Phosphodiester 2 (bidirectional) # Directions of Synthesis Unidirectional Collision of Replication Forks Termination Process Rho protein, loop Nucleus Where is product used? Cytoplasm Almost 100% % of Template Copied Varies Telomerase finishes replication; Modifications to Product MUCH!!!! editing; 3D compaction into chromosome Replication Transcription Nucleus Where in the cell? nucleus S phase When during cell cycle? G1 and G2 ds DNA Product ss RNA ds DNA Template ds DNA (select genes) Nucleotides (nucleoside Substrates A, U, C, G triphosphates) A, G, C, T Nucleotide triphosphates Ori C Initiation Sequences Promoter Gyrase, Helicase, Primase Initiation Enzymes RNA polymerase (sigma, core) DNA Polymerases Synthetic Enzymes Core RNA polymerase Phosphodiester, Hydrogen Types of Bonds Phosphodiester 2 (bidirectional) # Directions of Synthesis Unidirectional Collision of Replication Forks Termination Process Rho protein, loop Nucleus Where is product used? Cytoplasm Almost 100% % of Template Copied Varies Telomerase finishes replication; Modifications to Product MUCH!!!! editing; 3D compaction into chromosome