DNA AND Biotechnology

DNA Structure • Two chemically distinct forms of nucleic acids within eukaryotic cells: Deoxyribonucleic

acid (DNA) and Ribonucleic acid (RNA). DNA and RNA are polymers of one another

• Bulk of DNA is found in chromosomes in the nucleus

Nucleosides and Nucleotides • DNA is a polydeoxyribonucleotide which is composed of many

monodeoxyribonucleotides linked together.

• Nucleosides: composed of five-carbon sugar (pentose) bonded to a nitrogenous base and are formed by covalently linking the base C-1’ of the sugar

o Carbon atoms in sugar are labeled with a prime symbol to distinguish them from the nitrogenous base

• Nucleotides: formed when one or more phosphate groups are attached to C-5’ of a nucleoside.

o Often named according to the number of phosphates present ▪ E.g. – adenosine di-/triphosphate

o High energy compounds since there is repulsion between the closely associated negative phosphate groups.

• Nucleic acids are classified according to the pentose they contain o If pentose is ribose, then the nucleic acid is RNA o If pentose is deoxyribose (2’- OH groups replaced by –H), then it is DNA

Sugar-Phosphate Backbone • Backbone of DNA is composed of alternating sugar and phosphate groups

• This determines the directionality of the DNA and is always read from 5’ to 3’

• Backbone is formed as the nucleotides are joined by 3’-5’ phosphodiester bonds o Phosphate group link the 3’ carbon of one sugar to the 5’ phosphate group of the

next incoming sugar in the chain

• Since phosphates carry a negative charge, DNA and RNA strands have an overall negative charge.

• Each strand of DNA has a distinct 5’ and 3’ end, which creates polarity within the backbone

o The 5’ end of DNA will have an –OH or phosphate group bonded to C-5’ of the sugar

o The 3’ end of DNA will have a free –OH on the C-3’ of the sugar.

• Base sequence of nucleic acid strand will always be written and read in the 5’ to 3’ direction

o If written backwards, the ends must be labeled as “3’” and “5’” (e.g. – 3’–GTA-5’) o Position of phosphates may be shown (e.g. – pApTpG) o “d” may be used as shorthand for deoxyribose (e.g. – dAdTdG)

• DNA is generally double-stranded (dsDNA) and RNA is generally single-stranded (ssRNA)

Purines and Pyrimidines • Two families of nitrogen-containing bases found in nucleotides: purines and pyrimidines

• Overall, there are five common bases, but there may be exceptions in tRNA or in some prokaryotes and viruses

• Purines: Contain two rings in their structure. o Adenine (A) and Guanine (G). o Both are found in DNA and RNA

• Pyrimidines: contain one ring in their structure o Cytosine (C), Thymine (T), & Uracil (U) o Cytosine is found in both DNA and RNA, thymine is only in DNA and uracil is only

in RNA

• These are examples of biological aromatic heterocycles. Aromatic describes an unusually stable ring system that adheres to the following rules

o Compound is cyclic o Compound is planar o Compound is conjugated (has alternating single and multiple bonds, or lone

pairs, creating at least one unhybridized p-orbital for each atom in the ring)

o Huckel’s Rule: compound has 4n+2 electrons.

• Stability from aromatic compounds results from delocalized pi-electrons which are able to travel throughout the entire compound using available molecular orbitals

• Heterocycles are ring structures that contain at least two different elements in the ring o Both purines and pyrimidines contain nitrogen in their aromatic rings o Thus nucleic acids have exceptional stability

Watson-Crick Model • Proposed the three-dimensional structure of DNA in which the double-helical nature of

it was deduced and also proposed specific base-pairings that would be the basis of a copying mechanism.

• In the double helix, two linear polynucleotide chains of DNA are wound together in a spiral orientation along a common axis.

• Key features: o Two strands of DNA are antiparallel: strands oriented in opposite directions

▪ One strand has polarity 5’ to 3’ down the page while the other strand has 5’ to 3’ polarity up the page

o Sugar-phosphate backbone is on the outside of the helix with the nitrogenous bases on the inside

o Complementary base-pairing: Adenine (A) is always base-paired with a thymine (T) via two hydrogen bonds. A guanine (G) always pairs with a cytosine (C) via three hydrogen bonds.

▪ 3 hydrogen bonds make the base pair interaction stronger ▪ hydrogen bonds and the hydrophobic interactions between bases

provides DNA stability o Amount of purines will be equal to total pyrimidines due to complementary

base-pairing

• Double helix of DNA is a right-handed helix forms what is called B-DNA o Contains 10 bases per every turn (every turn is 3.4 nm) o Has major and minor grooves, which are usually the site of protein binding

• Another form of DNA is called Z-DNA, which is a left-handed helix o Contains 12 bases with each turn (every turn is 4.6 nm) o Usually formed when there is a high GC-content or a high salt concentration o No biological activity since it is unstable and difficult to research

Denaturation and Reannealing • For replication and transcription, it is necessary to gain access to the DNA

• Double helical nature of DNA can be denatured by disrupting the hydrogen bonding and base-pairing which results in the “melting” of the double helix into two single strands

o No covalent link between the nucleotide in the backbone of the DNA break o Heat, alkaline pH, and chemicals like formaldehyde and urea are commonly used

to denature DNA

• Denatured DNA can be reannealed if the denaturing conditions are slowly removed

Eukaryotic Chromosome Organization • DNA is divided up among the 46 chromosomes found in the nucleus of the cell

• DNA double helix provides some compaction, but further compaction is necessary

Histones • DNA that makes up a chromosome is wound around a group of small basic proteins

called histones, which forms a chromatin.

• Five histone proteins in eukaryotic cells

Chargaff’s Rules

• Two copies each of the histone proteins H2A, H2B, H3, and H4 from a histone core o 200 base pairs of DNA can wrap around this protein complex o Nucleosome: histone protein complex with its DNA base pairs

▪ Look like beads on a string

• The last histone protein H1, seals off the DNA as it enters or leaves the nucleosome

• Histones are an example of nucleoproteins: proteins that associate with DNA o Most others are acid-soluble and stimulate processes

Heterochromatin and Euchromatin • Chromosomes have a diffuse configuration during interphase of the cell cycle

• Cell will undergo DNA replication during the S phase of interphase and the DNA is uncondensed during this process to make it more accessible

o Small percentage of chromatin remains compacted during interphase and is called heterochromatin

• Heterochromatin: appears dark under light and does not partake in transcription o Consists of DNA with highly repetitive sequences

• Euchromatin: dispersed chromatin and appears light under light microscopy o Contains active DNA

Telomeres and Centromeres • DNA replication cannot extend all the way to the end of a chromosome. As such, the

end of each chromosome is lost during replication. o Solution to this is to have a repeating unit at the end of DNA

• Telomere: simple repeating unit at the end DNA in order to prevent lost information o TTAGGG o Some of the sequence is lost in each round of replication and can be replaced by

the enzyme telomerase ▪ More highly expressed in rapidly dividing cells

o Set number of replications possible, shortening of telomere contributes to aging o High GC content in telomere creates exceptionally strong strand attractions at

the end of chromosomes ▪ Prevents unravelling of chromosome

• Centromere: region of DNA at the center of chromosome o Sites of constriction because they form noticeable indentations o Composed of heterochromatin (repeat sequences with high GC content which

promotes strong attraction) o Functions to keep sister chromatids connected until microtubules separate the

chromatids during anaphase.

DNA Replication Strand Separation and Origins of Replication

• Replisome or Replication Complex: set of specialized proteins that assist the DNA polymerases

• Origins of Replication: Points where DNA begins to unwind. Beginning of replication process

• Replication Forks: generation of new DNA proceeds in both direction, which creates forks on both sides of the origin.

• Bacterial chromosome is a closed, double stranded circular DNA with a single origin of replication.

• Eukaryotic replication must copy many more bases and is a slower process o Each eukaryotic chromosome contains one linear molecule of double-stranded

DNA that has multiple origins of replication. ▪ This allows the chromosomes to duplicate efficiently

o As replication forks move forwards towards each other, sister chromatids are created. Chromatids remain connected at the centromere.

• Helicase: enzyme responsible for unwinding the DNA, generating two single-stranded

template strands ahead of polymerase.

• Once opened, the unpaired strands want to hydrogen bond with other molecules (there are free purines and pyrimidines)

o Proteins are required to hold the strands apart. Single-stranded DNA-binding proteins bind to the unraveled strand which prevents the reassociation of the DNA strands and prevents the degradation of DNA by nucleases

• Supercoiling: wrapping of DNA on itself as its helical structure is pushed further toward the telomeres during replication.

o Occurs as helicase unwraps DNA. o This strains the DNA helix and increases the chances of strand breakage.

• DNA topoisomerases: introduces negative supercoils which alleviates the torsional stress o Work ahead of helicase and “nick (cut)” one or both strands. This allows

relaxation and the cut strands are then resealed.

• Parental strands will serve as templates for the generation of new daughter strands. o Process is termed semiconservative since one parental strand is retained in each

of the two resulting identical double-stranded DNA molecules.

Synthesis of Daughter Strands • DNA polymerase: responsible for reading the DNA template and synthesizing the new

daughter strand o Can read the template strand in a 3’ to 5’ direction while synthesizing the

complementary strand in the 5’ to 3’ direction ▪ The separated parental strands of the helix are antiparallel to each other,

so one strand is oriented in the right direction for polymerase while the other is not.

• Leading Strand: the strand in the replication fork that is copied continuously (parallel to the polymerase direction)

• Lagging Strand: strand that is copied in a direction opposite to the direction of the replication fork.

o Has 5’ to 3’ polarity but polymerase only reads 3’ to 5’ o Solution for lagging strand is to use Okazaki fragments

• First step in DNA replication Is to lay down an RNA primer since DNA needs another molecule to “hook on”

o Primase synthesizes a short primer in the 5’ to 3’ direction to start replication on each strand

▪ These primers are constantly being added to the lagging strand because each Okazaki fragment starts with a new primer

• Leading strand only requires one in theory

o DNA polymerase III (prokaryotes) or DNA polymerases ,, & (eukaryotes) will then synthesize the daughter strands of DNA in the 5’ to 3’ direction

▪ Incoming nucleotides are 5’ deoxyribonucleotide triphosphates: dATP, dCTP, dGTP, and dTTP

▪ As new phosphodiester bonds are made, a free pyrophosphate (PPi) is releases.

o DNA polymerase I (prokaryotes)/RNase H (eukaryotes) enzymes remove RNA from the sequence

o DNA polymerase I (prokaryotes)/DNA polymerase (eukaryotes) add DNA nucleotides where RNA primer used to be

o DNA ligase seals the end of the DNA molecule which creates one continuous strand of DNA.

• Eukaryotic synthesis is considered similar to prokaryotic DNA synthesis. However, the five DNA polymerases in eukaryotic cells should be noted:

o DNA polymerases ,, & work together to synthesize both the leading and lagging strands. Delta also fills in the gaps left behind when RNA primers are removed

o DNA polymerase replicates mitochondrial DNA

o DNA polymerase & are used in the process of DNA repair

o DNA polymerase & are assisted by PCNA protein. This assembles into a trimer to form a sliding clamp, that helps to strengthen the interaction between these DNA polymerases and the template strand

Replicating the Ends of Chromosomes • DNA polymerase cannot complete the synthesis of the 5’ end of the strand

o Thus each time the DNA synthesis is carried out, the chromosome becomes shorter

• Telomeres are used to lengthen the time that cells can replicate and synthesize DNA before necessary genes are damaged.

o Repetitive sequence with high GC-content, and located at tips of the chromosome.

DNA Repair • DNA can be damaged from exposure to chemicals or radiation

• If not repaired, the damaged DNA will be passed on to daughter cell

• And damage causes an increased risk of cancer

Oncogenes and Tumor Suppressor Genes • Cancer can result from mutated genes. These cells can proliferate excessively since they

are able to divide without stimulation from other cells o Able to migrate by local invasion or metastasis. Allows migration to distant

tissue by the bloodstream or lymphatic system

• Oncogenes: mutated genes that cause cancer o Primarily encode cell cycle-related proteins

• Proto-oncogenes: before oncogene genes are mutated o Abnormal alleles encode proteins that are more active than normal proteins

which promotes rapid cell cycle advancement

• Antioncogenes: tumor suppressor genes (like p53 or Rb) that encode proteins that inhibit the cell cycle or participate in DNA repair processes.

o These function to stop tumor progression o Mutation of these genes result in the loss of tumor suppression activity which

then promotes cancer o Generally, both alleles need to be inactivated since even one copy of the normal

protein is usually enough to inhibit tumor formation.

Proofreading and Mismatch Repair • DNA polymerase is usually 100% accurate, but it does occasionally make errors

Proofreading

• During synthesis, two double-stranded DNA molecules will pass through a part of the DNA polymerase enzyme for proofreading

• If the wrong bases are paired, the hydrogen bonds between the bases are unstable. This instability can be detected as the DNA passes through the specified part of polymerase.

o Incorrect base is removed and replaced with correct one. o Enzyme determines which is the template strand by analyzing level of

methylation. The template strand has existed in the cell for a longer period of time so it will therefore be more methylated.

• DNA ligase does not have proofreading ability as it closes the gaps between Okazaki fragments. Thus, it much more likely to have a mutation in the lagging strand as compared to the leading strand.

Mismatch Repair

• The G2 phase of the cell cycle is able to perform mismatch repair

• Enzymes are encoded by genes MSH2 and MLH1, which detect and remove errors introduced in replication that were missed during the S phase of the cell

• In prokaryotes, the enzymes that do a similar function are MutS and MutL

Nucleotide and Base Excision Repair • Cell machinery is able to recognize two specific types of DNA damage in the G1 and G2

cell cycle phases and fixes them through nucleotide or base excision repair

Nucleotide Excision Repair

• UV light induces the formation of dimers between adjacent thymine residues in DNA. o These dimers interfere with DNA replication and normal gene expression and

distort the shape of the double helix.

• Thymine dimers are eliminated from DNA by a nucleotide excision repair (NER) o This is a cut and patch process. o Specific proteins scan the DNA molecule and recognize the lesion because of a

bulge in the strand o An excision endonuclease then makes cuts in the phosphodiester backbone of

the damaged strand on both sides of the thymine dimer and removes the defective oligonucleotide.

o DNA polymerase then fills in the by synthesizing DNA in the 5’ to 3’ direction (uses the undamaged strand as a template)

o The cut in the strand is sealed by DNA ligase

Base Excision Repair

• Cytosine deamination is the loss of an amino group from cytosine and results in its conversion to uracil. This is usually caused by thermal energy being absorbed by DNA

o This is easily detected since uracil should not be found in the DNA molecule

• Alteration to other bases can occur through other mechanisms as well, and detection systems exist for many of these bases as well. Base excision repair is used to repair these base abnormalities.

• Base Excision Repair: o Affected base is recognized and removed by a glycosylase enzyme. This leaves

behind an apurinic/apyrimidinic (AP) site also known as an abasic site. o AP site is recognized by AP endonuclease which removes the damaged sequence

from DNA o DNA polymerase and DNA ligase can then fill in the gap and seal the strand.

Recombinant DNA and Biotechnology • Recombinant DNA technology allows a DNA fragment from any source to be multiplied

by either gene cloning or polymerase chain reaction (PCR) o Provides a means of analyzing and altering gene and proteins o Provides reagents necessary for genetic testing (e.g. – carrier detection or

prenatal diagnosis of genetic diseases) o Can also provide a source of a specific protein, like insulin

DNA Cloning and Restriction Enzymes • DNA Cloning: can produce large amounts of a desired sequence

o Goal is to produce a large quantity of homogenous DNA o Piece of DNA that is of interest needs to be ligated into a piece of nucleic acid

called a vector, this forms a recombinant vector ▪ Vectors are usually bacterial or viral plasmids that can be transferred to a

host bacterium after insertion of the DNA

o Bacteria are then grown in colonies and a colony containing the recombinant vector is isolated.

▪ Can be done by ensuring that the recombinant vector includes a gene for antibiotic resistance. Antibiotics can then be administered and all other colonies will be killed

o Resulting colony can then be grown in large quantities o Bacteria can then be made to express the gene of interest or can by lysed to

reisolate the replicated recombinant vector

• Restriction Enzymes (restriction endonucleases): enzymes that recognize specific double stranded DNA sequences

o Sequences are palindromic (Read the same backwards and forwards) o These enzymes are isolated from bacteria (natural source) o Restriction enzyme can cut through the backbones of the double helix once a

specific sequence is identified o Some produce offset cuts which yields sticky ends on fragments

▪ Advantageous when the restriction fragment is joined with a vector DNA since they will fit together perfectly.

• DNA vectors contain at least one sequence that can be recognized by restriction enzymes.

DNA Libraries and cDNA • DNA Libraries: large collection of known DNA sequences which could potentially equate

to the genome of an organism. o Can consist of either genomic DNA or cDNA

• Genomic Libraries: contain large fragments of DNA and include both coding (exon) and noncoding (intron) regions of the genome

o Genomic libraries

• Complementary DNA (cDNA) Libraries: constructed by reverse-transcribing processed mRNA

o Lacks noncoding regions and only includes genes that are expressed in the tissue from with the mRNA was isolated

o Sometimes called expression libraries o Only cDNA libraries can be used to reliably sequence specific genes and identify

disease-causing mutation, produce recombinant proteins or produce transgenic animals

Hybridization • The joining of complementary base pair sequences. This can be DNA-DNA recognition or

DNA-RNA recognition

• Uses two single-stranded sequences

Polymerase Chain Reaction (PCR)

• Automated process that can produce millions of copies of DNA sequence without amplifying the DNA in bacteria.

• Used to identify criminals, family relationships, and disease causing bacteria/viruses

• Requires: primers that are complementary to the DNA that flank the region of interest, nucleotides (dATP, dTTP, dCTP, dGTP), and DNA polymerase

o Primer has high GC content which increases stability

• Also needs heat to cause the DNA double helix to denature o DNA polymerase from humans does not work at high temperature, so DNA

polymerase from a bacterium (Thermus aquaticus) is used.

• During PCR, DNA of interest is denatured, replicated and then cooled to allow reannealing of the daughter strands with the parent strands.

• DNA is doubled each cycle, and can be repeated as desired.

Gel Electrophoresis and Southern Blotting

• Gel electrophoresis is used to separate macromolecules like DNA and proteins, by charge and size.

• All DNA strands will migrate towards the anode of an electrochemical cell since all DNA molecules are negatively charged

• Preferred gel is agarose gel.

• The longer the DNA strand, the slower it will migrate in the gel

• Electrophoresis is often used while performing a Southern Blot: used to detect the presence and quantity of various DNA strands in a sample

o DNA is cut by restriction enzymes and then separated by gel electrophoresis o DNA fragments then transferred to a membrane (retain their separation) o Membrane is then probed by many copies of a single-stranded DNA sequence

▪ Probe will bind to its complementary sequence to form double-stranded DNA

▪ Probe is labeled to indicate the presence of a desired sequence

DNA Sequencing • Sequencing reaction requires template DNA, primers, DNA polymerase, and all four

deoxyribonucleotide triphosphates o Dideoxyribonucleotide is a modified base that is also added at lower

concentrations. ▪ Contain a hydrogen at C-3’ rather than on the hydroxyl groups ▪ ddATP, ddCTP, ddGTP, ddTTP

o When the modified base is incorporated, the polymerase cannot add to the chain

• Eventually the sample will contain many fragments that each terminate with a modified base

o As many fragments as the number of nucleotides in the desired sequence

• Fragments can then be separated by gel electrophoresis and the last base for each fragment can be read

• Bases are then sorted easily since electrophoresis was used (sequences the DNA)

Application of DNA Technology

Gene Therapy

• Offers potential cures for individuals with inherited diseases

• Intended for diseases in which a given gene is mutated or inactive, which gives rise to pathology

• Works by transferring a normal copy of the gene into the affected tissue o E.g. – severe combined immunodeficiency (SCID) was cured by placing a working

copy of the affected gene into a virus

• Need efficient gene delivery vectors o Most use modified viruses since viruses naturally inject their own DNA into a cell

▪ Virus genome is modified so that it can infect but not complete its replication cycle

• Poses a risk of integrating near and activating a host oncogene. (activated leukemia in some kids with SCID)

Transgenic and Knockout Mice

• If DNA has been isolated, it can be introduced into eukaryotic cells

• Transgenic mice are altered at their germ line by introducing a cloned gene into fertilized ova or into embryonic stem cells

o Transgene: cloned gene that is introduced

• If the transgene is a disease-producing allele, then the transgenic mice can be used to study the disease process form all stages of development

• Knockout Mice: genes are intentionally deleted

Common Methods for Developing Transgenic Mouse

• Most common procedure to develop transgenic mice is to microinject a cloned gene into the nucleus of a newly formed ovum. (Sometimes/rarely the gene can be incorporated into the nuclear DNA of a zygote)

o Ovum is then implanted into a surrogate mother and the resulting offspring should all contain the transgene (including their gametes)

o Transgene will then be passed to these mice’s offspring o This model is good for studying dominant gene effects since the transgene

coexists with the two naturally occurring genes. ▪ Not that good for recessive gene effects since the number of those

affected will be random.

• Can also use embryonic stem cells where the altered stem cells are injected into developing blastocysts and implanted into surrogate mothers

o Blastocyst is composed of two types of stem cells: ones containing the transgene and the original blastocyst cell

o Resulting offspring is a chimera: has patches of cells that are derived from each of the two lineages.

o Can often identify which mice are chimeras by using two cell lineages with different colour coats (mice will have patchy coats of two colors).

▪ Chimeras can be bred to produce mice that are heterozygous for the transgene and mice that homozygous for the transgene.

o Advantages of this method: ▪ Allow the cloned genes to be introduced in cultures (groups) ▪ Can select for cells where transgene is successfully inserted (not random)