DMD5025/CHS5042 Nucleic Acids → Chromosomes → Genome PDF

DMD5025/CHS5042 Nucleic Acids →Chromosomes →Genome Q. Quinn Li, PhD Office: DOC108 Office phone: 469-8523 [email protected] Sept 13, 2024 DNA, RNA and genome Learning Objectives...

DMD5025/CHS5042 Nucleic Acids →Chromosomes →Genome Q. Quinn Li, PhD Office: DOC108 Office phone: 469-8523 [email protected] Sept 13, 2024 DNA, RNA and genome Learning Objectives Understand features of DNA structure and function Understand RNA structure features and its application Explain the experimental implications of DNA complementarity Define genome, chromosome, and genes Describe how they relate to each other Define nucleosomes, chromatin and interactions between DNA and histones Relate chromosome packaging, karyotype and nomenclature  Nucleic Acids Store Genetic Information Deoxyribonucleic Acid (DNA): a “boring polymer”…. 1871: Friedrich Miescher isolated phosphate-rich chemicals that he called “nucleins” from nuclei of white blood cells from pus in bandages from local hospital Friedrich Miescher (1844-1895)  Evidence for DNA as Genetic Material Timeline of key experiments: Watson & Crick; Chargaff Franklin & Wilkins Griffith (1950) (1953) (1928) Base Secondary structure Transforming ratios of of DNA principle DNA 1928 -- Avery, Hershey & MacCarty & Chase (1951) McLeod DNA is genetic (1944) material in DNA is the phage transforming principle DNA has 5’-3’ Polarity The strand has polarity: One end chemically distinct from the other 5’ (5 prime) and 3’ ends Bases can be in any order: Polarity of strand means that bases have a specific order--A SEQUENCE--- from one end to the other By convention, base sequences are written 5’ to 3’, UNLESS OTHERWISE NOTED  DNA & RNA are linear polymers of nucleotides The deoxy-riboses or riboses are linked via phosphate groups The 3’-OH of one is linked to the 5’-OH of the next by a phosphodiester bridge (link, bond) This is called the backbone of the DNA strand Bases project away from the backbone  5’ 3’ Arrangement of bases in DNA Strands are antiparallel Bases arranged in pairs, base on one strand opposed to base on other Only G to C and A to T base pairs allowed (explains Chargaff’s Rule) The two strands are said to be complementary to each other 3’ 5’ DNA structure accounts for storage & transmission of genetic information Genetic information is stored (encoded) in the sequence of bases in the DNA helix Base sequence related to protein sequence Changes in DNA sequence (mutations) may or may not change protein sequence The DNA molecule can be replicated (duplicated) Semi-conservative replication  The DNA molecule is flexible Allows DNA molecule to be folded (important in packing into nucleus, cell) Allows molecule to wrap around a protein (e.g. histones) Most DNA are surrounded (bound) by proteins: histones and non-histones  The DNA molecule can supercoil stores energy in DNA molecule compacts the helix can be positive: strands overwound before linked (Makes it harder to unwind strands of helix) can be negative: strands underwound before linked (Makes it easier to unwind helix) DNA in cell is maintained in a negatively supercoiled state Supercoiling can be modified by enzymes Topoisomerase II: Relaxes +/- supercoiled DNA. Dimers hold double stranded DNA, each subunit transiently cleaves one strand and passes unbroken strand through it. Important during replication to prevent overwinding of DNA ahead of the replication fork. Energetically favorable reaction, driven by energy stored in supercoiling  Supercoiling can be modified by enzymes DNA gyrase in prokaryotes - a member of Topoisomerase II Converts relaxed DNA into a negatively supercoiled molecule. Requires energy from ATP Required for bacterial DNA replication, transcription, repair, and recombination Targets of antibiotics: binding to prevent resealing DNA break Ciprofloxacin Example: Fluoroquinolones class: – Ciprofloxacin, Levofloxacin, Norfloxacin, Ofloxacin a Fluoroquinolone  The two DNA strands can be separated Main energy holding helix together is from many hydrogen bonds between bases Heat breaks hydrogen bonds, separates strands: denaturation Cool down, bonds reform between complementary strands: renaturation, or annealing Allow PCR primers to bind  Separation of DNA strands occurs over a relatively narrow temperature range DNA is said to melt DNA “melts” Midpoint of temperature range is called the melting temperature, Tm GC base pairs have 3 hydrogen bonds, vs. 2 for AT, so the higher the GC content, the higher the Tm Hyperchromism Hyperchromism and Hypochromism Hypochromism Bases absorb UV radiation A solution of single stranded DNA will have a certain UV absorbance If helix is denatured, base stacking is destroyed, UV absorbance goes up: hyperchromism If strands are allowed to anneal, base stacking reduces UV absorbance: hypochromism  The The total amount of DNA within a cell Genome Chromosomal DNA Extrachromosomal DNA bacterial plasmids eukaryotic mitochondrial and chloroplast DNA  Genome sizes  Measured in base pairs of DNA (1000 base pairs = 1 kilobase pair)  Size varies with species – Viruses: smallest; ~1-150 kb – Bacteria: intermediate; ~5 x 103 kbp (E. coli) – Human: ~3 x 106 kbp (haploid genome); diploid genome = 6 billion bp  Genome size does not correlate well with species or evolution complexity  bp 5x106 5x107 5x108 5x109 5x1010 Flowering plants Birds Mammals Reptiles Amphibians Bony Fish Cartilag. Fish Echinoderms Crustaceans Insects Mollusks Worms Molds Algae Fungi Gram(+) bacteria Gram(-) bacteria 107 108 109 1010 1011  Viral  Can be double-stranded (ds) or Genomes single-stranded (ss) RNA or DNA  Physical form varies –single linear chromosome-like form –1 or more circular chromosome- like forms –several nucleic acid segments  Physical form may change during virus life cycle Bacterial 1. Bacterial chromosome Genomes – always double-stranded (ds) DNA – mostly single, circular chromosome 2. Extrachromosomal DNA ….PLASMIDS – non-essential; not all bacteria have them – always ds DNA, circular arrangement – often carry genes for antibiotic resistance (R factors) B A C T E R Plasmid I A L D N A  Comprised of: 1. Chromosomal DNA Eukaryotic – always ds DNA; linear arrangement – wide range of sizes 2. Mitochondrial and Chloroplast DNA Genomes – always ds DNA; circular arrangement  Human  22 pairs of autosomes Chromosomes numbered from largest (250 million bp) to smallest (50 million bp)  2 sex chromosomes ◦ (XX = female; XY = male)  if stretched to full length ~ 1 meter/haploid genome or 2 meters/somatic cell ◦ VERY COMPACTED in cells  Human body averages 1014 cells With 2 m DNA/cell…. Did you know…. 1014 cells/human x.002 km DNA/cell = 2 x 1011 km DNA/human !!!! For perspective: Circumference of earth = 4 x 104 km Distance from earth to sun = 1.5 x 108 km Could make almost 70 round trips to the sun & back Chromatin Composition DNA + proteins = chromatin Proteins are critical for DNA compaction Histones: major structural proteins – 5 types: H1, H2A, H2B, H3, and H4 – small (MW = 11-21 k daltons) – very basic (lots of Lys, Arg) Unraveled DNA…. If the protein scaffold of the chromatin is digested away, DNA unwinds How do you squish 2 meters of DNA into a cell nucleus? DNA COMPACTION!  Primary Structure: Nucleosomes  Histone core comprised of 2 ea. H2A, H2B, H3 & H4  140 bp DNA wrap around histone octamer  DNA + histone core = nucleosome  Nucleosomes are connected by 20-60 bp “linker” ◦ histone H1 associates with linker DNA  The Organization of the Eukaryotic Genome An expressing gene Misteli, Cell, 2020 Overview of DNA compaction Horng et al. Science 2017;357 Primary Structure: Nucleosomes Give the appearance of “beads on a string” Secondary structure: Solenoids or 30 nm fibers each solenoid contains ~ 6 nucleosomes  Tertiary Structure: Loops Solenoids loop out and attach to nuclear protein scaffold in 10-100 kbp/loop segments These looped segments are the primary form of chromatin found in interphase nucleus  Chromatin Compaction: Interphase 15 cm 1.5 cm 0.3 cm 50 m This is the conformation of interphase DNA ds helix nucleosomes solenoids looped segments  Cyclic condensation of chromatin during the cell cycle Loops cluster & condense to form rosettes that Metaphase attach to nuclear scaffold Coils Rosettes stack to form Cell cycle coils Metaphase chromosomes Interphase represent 10,000 fold decrease in DNA length Rosettes Chromosome 1 = 15 cm if Prophase stretched; at metaphase = 15m  RNA hairpin: stem-loop structure RNA Structures ▪ Mostly single-stranded, but internal base pairing can produce secondary structure. Mix of single and double strands, ▪ Some viruses use RNA for their genomes: mostly bound by proteins double-stranded, or single stranded RNA. ▪ Double-stranded RNA is structurally very similar to dsDNA. ▪ Biochemically unstable when in single strand. Heat, heavy metals or RNase can digest RNA  Transport RNA (tRNA) 3D Ribon Flatten cloverleaf model, tRNAAla  Nobel Prize for Physiology or Medicine 2023 mRNA > Protein > Antigens To be good mRNA as vaccine: 1. mRNA modifications：U > pseudo-U in vitro synthesized; non-inflammatory inducing 2. Stability and expressivity: base modifications and free of double stranded RNA 3. Delivery to the body: packaging with lipo- nanoparticles (LNP) uridine pseudouridine QUESTIONS??  All chromosomes require 3 specialized regions: Chromosomal – origins of replication (ori) Structures – telomeres – centromeres  Specific DNA sequence recognized by Origins of proteins required for replication (DNA polymerase, etc.) Replication Each eukaryotic chromosome has (ori) several thousand oris  Centromeres Replication single region of each chromosome serves as attachment point for proteins form the mitotic spindle specific attachment region is called the kinetochore replicated chromosome copies (chromatids) are connected at the centromere During cell division  regions at ends of chromosomes have many repetitive sequences some repetitive elements are added on to replicated chromosomes by a specific enzyme, telomerase Telomeres Telomeres serve several important functions: – “seal” chromosome ends to prevent fusion or loss – Attach chromosomes to nuclear envelope – Facilitate replication   Total nuclear chromosomal complement Karyotype of an organism  Metaphase chromosomes: duplicated & maximally condensed  Cytogenetic techniques use stains to identify banding patterns: – differential uptake of dyes – G bands, Giemsa stain (A/T rich) – R bands, reverse of Giemsa (G/C rich) Heterochromatin: densely stained, highly compact, mostly repetitive DNA sequences Euchromatin: poorly stained, less compact, contains transcribed genes  Human Giemsa-banding chromosomes These are whose chromosomes? 22 pairs of autosomes 2 sex chromosomes Chromosome Banding Patterns p arm q arm (short) (long) 19q13.2 13.3 13.2 13.1 12 11 12 13.1 13.2 13.3 13.4 (centermere)  What are in the human genome? Short interspersed elements (100- 300 bp); ~1.5 million/genome Long interspersed elements (6-8 kbp); ~850,000/genome Actually Simple sequence repeats coding for proteins Segmental duplications  Classes of Short Interspersed Elements (SINES) Satellite DNA: –  satellites: 171 bp repeats found in arrays of several million copies near the centromere of each chromosome – Mini satellites: 15-65 bp,