W27N Nucleic Acid and DNA

• Central dogma illustrates how genetic information flows
• Genetic material replicates before cell division
• Functional genome generates RNA
• Genome translates into working parts

What is DNA?

• Genetic material arranged into genes
• DNA is a duplex of anti-parallel strands in a helix formation
• Complementary base pairing allows duplex to act as replication templates

DNA

• Deoxyribonucleic acid stores life's information
• DNA is chemically simple and remarkably stable
• DNA directs its replication

RNA

• Ribonucleic acid has multiple functions
• RNA is relatively simple, forming stable structures
• RNA can be catalytic (ribozymes) and regulate gene expression

DNA and RNA Structure

• Both are polymers of repeating nucleotide monomers
• Nucleotides have 3 components

The Bases

• DNA and RNA bases are heterocyclic aromatic rings with substituents
• Purines are a class of bases composed of imidazole and pyrimidine

Purines

• Adenine is stronger base, protonating at N-1
• Guanine exists as a carbonyl tautomer

Hybridisation States

• Hybridization states are important for stability.

Tautomerism

• Tautomerization involves intramolecular proton transfer
• Catalyzed by acid and base

Tautomerism In Heterocyclic Systems

• Tautomerism can affect the properties of molecules.
• Adenine and Guanine are purines that can exist in different tautomeric forms

Nucleosides

• Nucleosides form when bases attach to the 1' position of a pentose sugar
• The sugar is ribose in RNA, deoxyribose in DNA
• Attachment points: N-1 of pyrimidines, N-9 of purines
• Glycosylic or glycosidic bonds link the base to the sugar

Nucleotides

• Nucleotides are nucleosides with phosphate groups at the 3', 5', or (in ribonucleotides) 2' positions
• Deoxynucleotides contain deoxyribose
• Nucleotides are phosphate esters
• Up to three phosphates can attach at the 5' position
• ATP (adenosine triphosphate) and cytidine monophosphate exemplify nucleotides

Ribose and The 2'OH Group

• Differences between DNA and RNA function are due to the 2'OH group
• RNA is more reactive and cleaves spontaneously
• RNA's catalytic functions depend on the 2'OH

Linking Nucleotides

• Phosphodiester bonds link nucleotides into polymers

• A phosphate group links the 5'-hydroxyl of one ribose to the 3'-hydroxyl of the next

• Phosphodiester bonds create a directional nucleic acid chain with free 5' and 3' ends

• Phosphate groups can attach at the 3' end

• Nucleic acids are strings of nucleotides linked by phosphodiester bonds

• Each phosphate group carries a single negative charge, making nucleic acids strong, highly charged polymers

• An example DNA sequence is 5'-GCAT-3'

DNA Structure

• Two DNA strands form the well-known double helix

• James Watson and Francis Crick determined the structure

• Two DNA chains coil around each other to form a right-handed double helix

• The B-form is the most common helical form with 10 phosphates per turn (360°)

• Sugar-phosphate backbones are on the outside, with bases stacked inside

• Phosphates are on the helix's outside, bases inside

• Major and minor grooves follow a helical path between strands

• Bases on opposite strands join non-covalently to form base pairs

• Non-covalent hydrogen bonds join strands together to form base pairs

• Sequence complementarity arises from base structures and DNA backbone constraints

• Strands are antiparallel, running in opposite 5' → 3' directions

• The faces of base pairs are relatively neutral (green), while the edges have positive (blue) and negative (red) regions

• Pairing G with C and A with T brings together oppositely charged regions in the DNA double helix

Significance of Double Strandedness

• Double stranded molecules are advantageous because of two complementary strands that serve as copying templates
• The second strand is a template for damaged strand repair
• The double helix is a chemically stable environment for genetic information
• DNA must be stable and protected

Different DNA Forms

• B-DNA is structure identified by Watson and Crick

• Most DNA in vivo adopts this idealized form

• B-DNA features a 10 bp/turn helical repeat (closer to 10.5 bp/turn in 'real' B-DNA)

• Base pairs lie almost perpendicular to the helix axis

• B-DNA has well-defined, deep major and minor grooves

• DNA can form an alternative A-form helix under low humidity

• A-form is right-handed but wider and compressed

• Base pairs tilt relative to the helix axis and lie off the axis

• The A-helix has a hole down the middle

• The A-form has a helical repeat of around 11 bp/turn

• DNA adopts the A-form (or something close to it) in vivo under unusual conditions

• The A-form helix is formed by RNA and DNA-RNA hybrids

• The 2'-OH of RNA cannot fit into the B-form

Packaging DNA (Prokaryotes)

• The E. coli chromosome exemplifies prokaryotic genomes

• Most of E. coli's DNA is a single, closed-circular molecule, 4.6 million base pairs long

• The DNA is packaged within the nucleoid

• The nucleoid has high DNA concentration (30-50 mg/ml) and proteins

• Under normal growth conditions, the genome replicates continuously, with about two copies per cell

• DNA from E. coli, isolated without proteins, reveals the nucleoid's organization under an electron microscope

• The DNA has 50-100 loops or domains, ends bound to membrane proteins

• Loops are about 50-100 kb in size

• Related genes are located on the same loop/domain.

Eukaryotic DNA Packaging

• Eukaryotic cells have discrete chromosomes (46 in humans)

• The DNA length of these chromosomes can be several centimetres long

• All DNA must fit in the nucleus, a space with approximately the same volume as a eukaryotic cell

• Chromatin, a DNA and protein complex, packages DNA

• Over 50% of chromatin mass is protein

• Chromosomes compactness varies through the cell cycle to influence genetic accessibility

• Eukaryotic chromatin proteins consist mostly of small histone proteins (10-20 kDa)

• Lysine and arginine give all histones a large positive charge

• Histones strongly bind negatively charged DNA to form chromatin

• Histones assemble into octamers around which DNA wraps

• Histone H1 stabilizes the point where DNA enters and leaves the nucleosome core

• The familiar chromosome picture only actually appears at mitosis (anaphase)

• Mitotic spindle pulls apart daughter chromosomes, and this action would shear any fragile chromosomal DNA without it being highly compact

• The X-structure illustrates two sister chromatids, which are products of replication

• Telomeres are the chromosome tips

• DNA maps linearly along the chromosome in a convoluted path.

• The centromere constricts the region where sister chromatids join in metaphase

• Telomeres are specialized DNA sequences on eukaryotic chromosome ends and telomere consists of up to hundreds of copies of a short-repeated sequence = protection of the ends

Genes

• A gene, the functional heredity unit, is a gene sequence that holds the information to make an RNA molecule (may then be turned into protein)

DNA Replication

• DNA Replication complex but simplistic mechanism

• Watson and Crick suggested a copying mechanism in their double helix paper

• The paper stated their notice of the specific pairing they postulated that immediately suggests a possible copying mechanism for the genetic material

Why is DNA a helix?

• Additional information can be found: https://www.liverpool.ac.uk/sbs/DNA_Topology/HelixL.htm

Hershey Chase Experiment

• DNA is genetic material
• Alfred Hershey and Martha Chase proved DNA was genetic material
• Viruses (T2 bacteriophage) grew in isotopic mediums for component labeling
• Radioactive sulfur (35S) labeled proteins and radioactive phosphorus (32P) labeled DNA
• Viruses then infected E. coli, separating via centrifugation
• Larger radioactive bacteria formed the solid pellet
• 32P viruses' bacterial pellet was radioactive; 35S viruses' was not
• Therefore, DNA transformed the bacteria

DNA Replication

• DNA replication is semiconservative

• There are 3 models proposed:

  • Semiconservative replication yields hybrid molecules with one old and one new strand
  • Conservative replication results in two completely new strands and 2 completely old strands
  • Dispersive replication results in a mixture of new and old strands

• Results and experimetns by Meselson and Stahl proved the semiconservative replication model

Meselson and Stahl

• Centrifugation separated DNA by density
• Results supported semiconservative replication

DNA Replication Initiation

• Broadly similar among prokaryotes and eukaryotes, but more complex in eukaryotes
• Replication initiates giving rise to two replication forks
• Multiple origins required because genome is bigger

DNA Replication Mechanistic Overview

• DNA synthesis occurs at the replication fork branch point in a replication eye

• A replication bubble contains one or two replication forks (unidirectional or bidirectional)

• DNA almost always replicates bidirectionally

• Prokaryotic and bacteriophage DNAs typically initiate replication at one point

• Reniji Okazaki illuminated the semidiscontinous model of DNA replication, and it all starts with - Prokaryotes, and Replication initiation

• DnaA proteins bind to 4 copies of a 9-bp sequence and then after occupying all bindidng sites, co-operatively recruit more DnaA proteins to then form the - DnaA barrel formation

• Next torsional stress opens the AT-rich region

• Replication forks generate

• DnaB recruits to replication fork and initiates a pre-priming complex

• DnaB (Helicase enzyme) breaks hydrogen bonds and base-pairs

• SSBs (single stranded binding proteins) cover the open DNA strands

• SSBs stop re-annealing of strands (base-pairing) and protect the DNA from attack

• DNA replication initiation now complete, next phase - elongation

• DNA synthesis proceeds by DNA-dependent DNA polymerase enzymes

• DNA in generated in the 5' to 3' direction

• Polymerase moves along the template strand in the 3' to 5' direction

DNA Polymerisation Reaction

• DNA synthesis proceeds by DNA-dependent DNA polymerase enzymes
• DNA in generated in the 5' to 3' direction
• Polymerase moves along the template strand in the 3' to 5' direction

DNA Polymerisation

Prokaryotic Elongation

• Primase enzyme (DnaG) primase starts to synthesise the RNA primer on the leading strand (Primosome) and binds near the helicase
• Single strand binding proteins stabilise the lagging strand
• And the dna polymerase III holoenzyme clumps to the leading stand whilst synthesising

Semi-Discontinuous Replication

• All DNA replication starts with a short RNA primer, which allows newly synthesised stand to start and proofread itself.
• In E-coli - Primase enzyme sets this in motion
• The Pol complex removes the primer
• And both replicated DNA strands are produced after the 2 DNA Plomerase enzymes are tethered together
• Continuous: in the 5' to 3' direction (leading strand) and Discontinuous : in the 5' to 3' direction on the lagging strand
• REPLISOME
• The lagging strand is looped so that both Plomerase move in same direction
• Okazaki fragments are joined together by a ligase
• DNA polymerase III will be replaced with another polymerase after the removal of of the Pol primer priot to ligation

DNA Replication Elongation

• replication forks can move without facing the topological problem of the of over-winding
• Topoisomerases alleviate this problem:
  • Type I: create break 1, and then proceed to add and pass DNA and then reseal
  • both strands of 2: type Break, then add the double helix prior to unsealing
• breaks made in DNA covalently attached, will lose its ends in the process

Overcoming Topological Problems

• These are overcome using the - DNA topoisomerase I nick strand during DNA Replication

DNA Replication/ Termination

• In E. coli the 2 replicons will be up to 180'away
• A regulatory point will be put into action to confirm that the stoping code is correct
• If that is correctly detected, than it will get there first so it allows for the DNA to finish as the replicon sequence is finished
• DNA chain replication must be in sync or it won't stop or overtake

DNA/ Eukaryotic Replciation

• Eukaryotic have many similarieis in DNA replication as Eukaryotic
• Eukaryotic are extremely long compared to prokayotics
• DNA eukaryotics can be checked by
  • Eukaryotic replciation will move to new location 50bp at a time
  • Eukaryotes and size will need extra components so it will divide so for instance 54 will be divided into thousands
• DNA will start at phase point when DNA/ S phase is contacted with protein - early S contact, late phase DNA strand, middle last centromic / chromic
• Chromic location for DNA reproduction minimum length is 11BP,
• DNA reproduction will occur at 11 BP
• Additional clones and models may also be needed
• Process
  • DNA must be connected by the organ in some sort of complex of a kinded DNA set to release
• EUK replicon origin needs DNA from a cell to separate a full or a non separate so division of DNA needs to happen in DNA cells
• Cells need time to reproduce
  • The place they do it:
  • set to copy by cell membrane
• To get the chain:
• To identify to get started
• For cells check
• For cell go, no issues detected