Lecture 1 DNA Structure, Function, Metabolism and Replication v1.pdf

Welcome to Molecular Diagnostics MDLS 5202 For DMS and MLS Class 90 Molecular Pathology  Recognized as an area of pathology practice  First molecular pathology board exam for pathologists-2001  ASCP has a specialist examination for technologists (Molecular Biology exam; Molecular Diagnostic Sc...

Welcome to Molecular Diagnostics MDLS 5202 For DMS and MLS Class 90 Molecular Pathology  Recognized as an area of pathology practice  First molecular pathology board exam for pathologists-2001  ASCP has a specialist examination for technologists (Molecular Biology exam; Molecular Diagnostic Scientist-Practicianer)  Clinical practice fundamental to almost every aspect of healthcare delivery including assisting with diagnosis, therapeutic choice, outcome monitoring, prognosis, prediction of disease risk, directing preventative strategies, beginning-oflife choices, patient and specimen ID, and clinical epidemiology. Our Focus  Fundamentals of molecular biology theory as relevant to molecular diagnostics and molecular medicine  DNA replication  RNA synthesis and Transcription  Protein synthesis (Translation)  Post-translational modifications  Inhibitors of these processes  Molecular Diagnostic Tools  Molecular Basis of Diseases and/or Disorders in Genetics, metabolism, hematology, immunology, microbiology, virology and cancer etc. So, let’s get to it! Lecture 1 DNA Replication Chromosome and Nucleic Acid Structure, Function, Metabolism and degradation Diseases associated with DNA replication process Unit 5 Nucleotide Structure and DNA Replication Adapted from Virtual Cell Biology Classroom on ScienceProfOnline.com Chromosome & gene, Graham Colm, National Human Genome Research Institute Many other Human Genome • The human genome consists of large amounts of the chemical deoxyribonucleic acid (DNA) • DNA carries within its structure the genetic information needed to specify essentially all aspects of what makes a human being a functional organism. • • • • embryogenesis, development, growth, metabolism, Reproduction • Every nucleated cell in the body carries its own copy of the human genome, which contains, depending on how one defines the term, approximately 20,000 to 50,000 genes Genome, Chromosomes & Genes • Genome - Complete set of an organism’s DNA. • Cellular DNA is organized in chromosomes. • Genes have specific places on chromosomes. Components of Nucleic Acids (Nucleotide) Phosphate Group O O=P-O Nitrogenous base (A, G, C, or T) 5 CH2 O O N Three Components: -Nitrogenous Base -Pentose -Phosphate Sugar (Pentose) C4 C3 C2 C1N-glycosidic bond Two Types of Pentose Sugars in Nucleic Acids Both retain a 3’ hydroxyl group Sugars in dNTP Analog Dideoxy nucleotide (ddNTP) Comparison of Three Different Ribose What are they and what function does each have? Nitrogenous Bases • Pyrimidine base • T, C, U • Purine base • A, G Nucleotide Structure • Nucleoside is a nucleobase linked to a sugar • Nucleotide is composed of a nucleoside and 1 or more phosphate groups • dNTP is deoxyribonucleotide triphosphate (in DNA) • NTP is ribonucleotide triphosphate (in RNA) • Mono-nucleotide • dNMP • Di-nucleiotide • dNDP • Tri-nucleotide • dNTP Functions of Nucleotides • Nucleic acid synthesis: DNA/RNA • Energy currency of cell • Second messengers in cellular communication • Ingredients of co-enzymes • Regulators of metabolic reactions Chargaff’s Rule • Total mole percentage of purines is approximately equal to that of the pyrimidines that is (%G + %A) = (%C +%T). • The mole percentage of thymine is nearly equal to that of adenine (%T / %A=1). • The mole percentage of cytosine is nearly equal to that of guanine (%G / %C=1). • This ratio is the same for DNA of different types of tissues of the same animal and doesn't differ with the age within the same species • In human body (or somatic cell): A = 30.3% T = 30.3% G = 19.5% C = 19.9% DNA: 3’ to 5’ Phosphdiester Linkage of polynucleotides Summary: Major Linkages in DNA 5 P 3’ to 5’ phosphodiester bond O Hydrogen bond 3 5 O C G 1 4 2 3 P 5 P 3 O 3 O 5 2 1 T A n-glycosidic Bond P 3 4 O 3 O 5 5 P P Double Stranded DNA Structure • Double stranded complementary sequences forms helix molecule. • The complementary linkage from Watson-Crick Bonds (hydrogen bond). • Two deoxyribose-phosphate chains as the “side rails” • Base pairs, linked by hydrogen bonds, are the “steps” • One strand of DNA goes from 5’ to 3’ (sugars), the other strand is opposite in direction going 3’ to 5’ (relative to sugars) Central Dogma • The classic view of the central dogma of biology states that "the coded genetic information hardwired into DNA is transcribed into individual transportable cassettes, composed of messenger RNA (mRNA); each mRNA cassette contains the program for synthesis of a particular protein (or small number of proteins)." • However, many exceptions to this dogma are now known as a result of genomic studies in recent years. For example, much of the DNA that does not encode proteins is now known to encode various types of functional RNAs…. Synthesis and Degradation of Nucleotide Components Purine Synthesis of AMP and GMP Degradation of Purine Nucleotides Xanthine Oxidase deficiency (1) -Hypouricemia HGPRTase, PRPP to Purine (salvage pathway enzymes) (2) -Hyperuricemia (Lesch-Nyhan) Adenosine Deaminase deficiency (3) -Severe combined immunodeficiency (SCID) 3 HGPRT HGPRT 2 1 2. Hypoxanthine-Guanine Phosphoribosyltransferase Diseases Associated with Purine Metabolism • Hypouricemia • Level of uric acid in blood serum is below normal • Xanthine Oxidase deficiency • Hyperuricemia (Lesch-Nyhan Syndrome/Juvenile Gout) • HGPRTase, PRPP deficiency (salvage pathway enzymes) • Severe Combined Imunodeficiency Diseases SCID (missing body defense system T & B cells) • Adenosine Deaminase deficiency. • No expression of Adenosine Deaminase (bubble boy) • Accumulation of dATP inhibits ribonucleotide reductase and depletes DNA precursors • Over expression- Hemolytic Anemia • SCID can also be caused by a variety of other enzyme defects. Pyrimidine Synthesis • Do not require large Energy • EUK no significant salvage Degradation of Pyrimidines • Cytosine deaminated to uracil • uracil degraded to b-alanine • Thymidine degraded to beta-aminoisobutyrate (aa deritive) urea Synthesis of Deoxyribose • Ribose: RNA • Deoxyribose: DNA ribonucleotide reductase • Ribose deoxyribose This is why 3’to 5’ phosphodiester bond is formed, and DNA is linear, not-branched. Thymidylate (dTMP) Synthesis Inhibitors in cancer therapy: [B-12] - folate and B12 required - Involved in one carbon metabolism Nucleoside Analog AZT 5FU Uracil Amino Acid Methionine Synthesis (B 12 and Folate are also required) Inhibitors in Thymidylate Synthesis Chemotherapeutic Agents DHFR inhibitor, prevent one carbon metabolism Methotrexate Aminopterin Thymidylate synthase inhibitor 5-fluorouracil (5-FU)- Pyrimidine Analog Polynucleotide (DNA) Synthesis (Replication) • Primary Structure • nucleobases linked by 3’ to 5’ phosphodiester bonds • chain growth is always 5’ to 3’ • Secondary Structure • double stranded; anti-parallel; twists into helix • bases base paired through hydrogen bonding (WatsonCrick bond) •A T •C G DNA: 3’ to 5’ Phosphodiester Linkage of polynucleotides Watson-Crick Bonds Stabilize Double Helix Most DNA are Right-handed helical DNA Double Helix “Rungs of ladder” Nitrogenous Base (A,T,G or C) “Legs of ladder” Phosphate & Sugar Backbone copyright cmassengale DNA Synthesis • A process to put nucleotide together- called polymerization • Requires monomers (building blocks: Nucleotide) and energy. • Triphosphate deoxyribonucleotide provides both. • These building blocks of DNA bring their own energy for polymerization. • Requires enzymes, proteins and others Characteristics of DNA Strand Synthesis • Strands unwind and separate (helicase , gyrase) • Replication fork formation (multiple forks present in eukaryotic replication process) • 5’ to 3’ direction of synthesis. one strand is continuous (Leading strand), another strand is discontinuous (Lagging strand) • RNA primer required • strands must be rewound • Bidirectional • Both new strands synthesized simultaneously • Semiconservative • One new strand is made from one parental template DNA Replication Forks Okazaki Fragment Prokaryotic DNA Synthesis • Synthesize new DNA as soon as cell is large enough to divide • 3 prokaryotic polymerases • Pol I:(polA) replication and repair • Pol II: implicated in repair • Pol III: main processive replicative enzyme (de novo synthesis of DNA) Prokaryotic DNA Synthesis: Sequence of Events • • • • • • • • Topoisomerase unwraps and separates strands Helicase breaks the base pairing SSB binds to ssDNA Primase lays RNA primer (gives 3’-OH) DNA pol III synthesizes DNA (high processivity) Pol I cuts out primers and re-synthesizes DNA Ligase reseals strand nicks after Pol I Gyrase (type II topoisomerase) rewraps and recoils Models of DNA Replication in E.coli Replication Complex Sum Replication Enzymes Replication Enzymes Function Helicase Breaks hydrogen bonds linking the two strands of double helix. Topoisomerase Mitigates the supercoiling effect that occurs in advance of the replication fork. topoisomerases bind to DNA and cut the phosphate backbone of either one or both the DNA strands. This intermediate break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed again. Acts as a retractor, preventing the single strands of the DNA double helix from rejoining Single-strand binding proteins RNA primase Synthesizes RNA primers used in DNA daughter strands formation. Certain DNA polymerases also act as part of the DNA repair machinery DNA polymerase Synthesizes DNA daughter strands. Certain DNA polymerases also act as part of the DNA repair machinery DNA ligase Links newly synthesized DNA fragments (Okazaki fragments) Eukaryotic DNA Synthesis • Replication in S phase of cell cycle • Unwrap DNA from histone proteins • Larger genome, multiple origins of replication on each eukaryotic chromosome • Humans can have up to 100,000 origins of replication across the genome • Number of DNA polymerases in eukaryotes: 14 are known • pol α, pol β, pol γ, pol δ, and pol ε. are known to have major roles during replication and have been well studied DNA Replication in Eukaryotic Cell • S phase during interphase of the cell cycle • DNA is located in Nucleus of eukaryotic cells DNA replication takes place in the S phase. S phase G1 interphase Mitosis -prophase -metaphase -anaphase -telophase copyright cmassengale G2 Note: 1. Bacterial primases are monomer. dnaG, is the central gene. The N-terminal domain has a zincfinger motif . The central catalytic domain binds single-stranded DNA and catalyzes RNA polymer initiation and elongation complementary to it. The Cterminal domain interacts with other proteins such as helicase etc. 2. Archaeal/eukaryotic primase resides in eterotetramer, consisting of a small primase subunit, a large primase subunit, a regulatory phosphoprotein, and DNA polymerase alpha. www.slidewshare.net https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/primase Eukaryotic DNA Synthesis Enzymes • DNA helicase - unwinds and separates double stranded DNA as it moves along the DNA. It forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA. • DNA primase - a type of RNA polymerase that generates RNA primers. Primers are short RNA molecules that act as templates for the starting point of DNA replication. • DNA polymerases - synthesize new DNA molecules by adding nucleotides to leading and lagging DNA strands. • Topoisomerase and/or DNA Gyrase - unwinds and rewinds DNA strands to prevent the DNA from becoming tangled or supercoiled. • Exonucleases - group of enzymes that remove nucleotide bases from the end of a DNA chain. • DNA ligase - joins DNA fragments together by forming phosphodiester bonds between nucleotides. Reece, Jane B., and Neil A. Campbell. Campbell Biology. Benjamin Cummings, 2011. Put the Picture Together! Exercise 1 ?’ 5’ ?’ ?’ RNA Primers ?’ ?’ Replication Fork ?’ ?’ Replication Fork Now lets look at how replication of the leading and lagging strands occurs at each of the two replication forks within the replication bubble: 1. Label each end of the parent strands as either 5’ or 3’. 2. Start a RNA primer for each daughter strand and label its 5’ and 3’ ends. 3. Show how new strands are built (continuously or discontinuously). From the Virtual Cell Biology Classroom on ScienceProfOnline.com Exercise II Inhibitors of DNA Synthesis • Quinolines-inhibit DNA gyrase • Norfloxacin, Ciprofloxin, Novobiocin • Methotrexate and Aminopterin, Trimethiprin- inhibit dihydrofolate reductase (inhibits DNA synthesis via thymidylate synthesis) • Chemotherapeutic Agents • Methotrexate • AZT-DNA chain terminator (no 3” OH) • 5’Fluorouricil-inhibits thymidylate synthesis Nucleoside Analog AZT 5FU Uracil Chromosomal Structure and Cell Cycle Polynucleotide and Chromatin Structure • Tertiary Structure • Prokaryotes-circular DNA supercoiled into compact rings • Eukaryotes-DNA • • • • wrapped around histone proteins- supercoiling packed into solenoid condensed into chromatin wrapped on scaffolding proteins Chromosomal Organization DNA double helix of about 2.0 nm thick is wrapped around a core of 4 pairs of histone molecules to form nucleosomes Linker DNA (C) connects between nucleosomes. Nucleosomes attach together by peripheral histone (HI) and condense forming a fiber of about 30 nm thick The nucleosomal fibers form loops radiating from scaffolding nonhistone protein to form the DNA protein complex of chromatin fibers during cell division As a result of DNA replication, each chromosome becomes two sister chromatids attaching at centromere and of about 1400 nm in thickness in metaphase. Karyotype of Homologous Chromosomes Each Homologous set is made up of 2 Homologues. Homologue Homologue • Chromosomes are most condensed (thickened) and highly coiled in metaphase, which makes them most suitable for visual analysis. • The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. • Metaphase chromosomes make the classical picture of chromosomes (karyotype). Types of Chromatin • Chromatin makes up chromosomes. changes to chromatin's structure can prevent or allow certain regions of the genetic code to be read and expressed. • Euchromatin is the genetically active type of chromatin involved in transcribing RNA to produce proteins. • The predominant type of chromatin found in cells during interphase, euchromatin is more diffused • Heterochromatin is genetically inactive type of chromatin. • tends to be most concentrated along chromosomes at certain regions of the structures, such as the centromeres and telomeres. The Centromere Types • The chromosome region that attaches to a spindle fiber at metaphase of mitosis or meiosis and moves to the spindle pole at anaphase • The position of the centromere is constant for a particular chromosome, but variable between chromosomes, which are called metacentric, acrocentric, or telocentric, depending on whether their centromeres are more or less central, near the end, or terminal Telomere and Telomerase • A repetitive nucleotide sequences, called the telomeres present in the buffer zone, serves as termination signal • In humans the sequence is TTAGGG (called “cap” sequence) repeated several thousand times. • Telomeres erosion does not affect cell function but protects against lose of functionally important genetic material. • Telomerase enzyme • Telomerase functions to add more nucleotides to the telomeres, regenerating these protective “cap ”DNA sequences to avoid vital region of DNA from damage. • Aging and Cell death • Lost /reduced telomerase activity may cause premature cell aging and death. • Over expression of telomerase may could help cancer cells to grow faster and live longer, potentially leading to more dangerous strains of cancer. Chromosome Nomenclature • Short chromosome arms are designated p (petit) • Long chromosome arms are designated q (next letter of alphabet) • Centromere and telomere • Regions (bands) in each arm numbered consecutively from centromere outward to telomere. Mutation Type • To designate a specific region of the chromosome … Chromosome number written first Location on the short or long arm Region of the arm (specific band) (exp. 18p22.31) - t (translocation) - del (deletion) - ins (dup) (insertion) - inv (inversion) • What is this? t(9;22)(q34;q11.2) band 34 on long arm of chromosome 9 has exchanged places with band 11.2 on long arm of chromosome 22. Schematic of Cell Cycle • • • • • G0 G1 Check point S G2 Checkpoint M • • • • • prophase, Metaphase Checkpoint anaphase, telophase cytokinesis Five Phases of Cell Cycle and the Checkpoints in the Cycle State Description Abbreviation quiescent/ Gap 0 G0 A resting phase where the cell has left the cycle and has stopped dividing. Gap 1 G1 Cells increase in size in Gap 1. The G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis. Synthesis S DNA replication occurs during this phase. G2 During the gap between DNA synthesis and mitosis, the cell will continue to grow. The G2 checkpoint control mechanism ensures that everything is ready to enter the M (mitosis) phase and divide. M Cell growth stops at this stage and cellular energy is focused on the orderly division into two daughter cells. A checkpoint in the middle of mitosis (Metaphase Checkpoint) ensures that the cell is ready to complete cell division. senescent Interphase Gap 2 Cell division Functions Mitosis Major Proteins that Control Cell Cycle • Control Proteins • Cyclin-dependent protein kinases (Cdks) • Cyclins • Complexes: Cdk-cyclin • ability of Cdk to “P” target is dependent on the cyclin that it forms a complex with • Chemical reactions of phosphorylation/dephosphoryation are the foundation of protein activation