Chapter 2- DNA and RNA: Composition and Structure Flashcards
Pneumococcus experiment
The first evidence that DNA is the genetic material was obtained in 1920 through an experiment involving two strains (R and S forms) of pneumococcus (a bacteria that causes pneumonia). The R strain forms rough colonies, and the S strain forms smooth colonies. S strains are more virulent and cause pneumonia, while R strains aren’t virulent. Treatment of the R-strain (non virulent) with DNA extracted from S-strain (virulent) resulted in their transformation into the S-form. This demonstrated that DNA was a transforming agent, as the traits were inherited from the S strain. It also showed that DNA was responsible for the transmission of information from one generation to the next.
How much DNA does a human cell contain?
It contains enough information for the synthesis of about 25,000 proteins.
Traditional vaccines
Traditional procedure for making vaccines include attenuated (inactive) intact cells or dead organisms that will have body produce antibodies, which will provide immunity. Examples include successful vaccines that provided protection against diseases such as polio, smallpox, whooping cough, typhoid and diphtheria. However, in addition to the growing problem of antibiotic resistance, it is difficult to make vaccines for some pathogens like HIV and malaria via traditional methods, this prompted the use of alternate methods to make vaccines. A malaria vaccine was recently approved using protein fragments from the falciparum parasite.
How could DNA vaccines be made?
DNA vaccines would typically be a bacterial plasmid engineered to include the sequence of an antigenic protein from the pathogen. DNA can enter various cells and replicate followed by transcription and translation, acting similarly to viruses. These DNA contain a limited amount of genetic information and cannot become infectious. The mechanism of uptake and induction is still unclear. Promising results have been obtained against viruses and bacteria and may help fight HIV/AIDS, TB and malaria. Could be used for cancer in the future
Antigenic
Refers to the mechanism by which an infectious agent changes the proteins or carbohydrates on its surface. It helps the pathogen to avoid the host immune response
Pros of DNA and RNA vaccines (3)
- Cost effective
- Ability to be developed more quickly than traditional vaccines, which rely on actual inactivated viruses and can take years to develop.
- Can be made more readily available because they rely on genetic code – not a live virus or bacteria. This also makes the vaccines cheaper- only requires a plasmid of the DNA sequence to produce a protein
Protein vaccines
Protein vaccines have been used for decades- protect people from hepatitis, shingles, and other viral infections. The vaccine contains the protein or protein fragments from the bacteria or virus. Protein based vaccines are under currently investigation for COVID
Advantages of mRNA vaccines
If you know what protein you want to produce in the body, it is easy to synthesize corresponding mRNA. Proteins can be difficult molecules to use because they are large molecules, in contrast to DNA and RNA, which are smaller. Proteins also have more complicated structures than DNA and RNA do, and proteins have some stability issues. Also mRNA vaccines can be safer, and mRNA vaccines can be more readily translated into proteins. mRNA cannot be integrated into host DNA
Cons of DNA vaccines
With a DNA vaccine, there is always a risk it can cause a permanent change to the cell’s natural DNA sequence. However, there are ways to minimize risk. mRNA cannot get integrated into the DNA, and is readily translated into protein. mRNA vaccines are safer and mRNA can be more readily translated into proteins
Hybridization probe
Probes are single stranded DNA or RNA sequences that are complementary to the specific sequences we are interested in. The probe is tagged for easy detection of the hybrid double helix. Based on the association of complementary polynucleotide strands with the probe, this method has been developed for the detection and quantitation of specific sequences of target nucleic acid.
Use of hybridization probes (4)
- Determines whether a certain sequence occurs on the DNA of a particular organism
- Determines genetic or evolutionary relatedness between different organisms
- Determines the number of genes transcribed in a particular mRNA
- Determines the location of any given DNA sequence
Hybridization Experiments
A mixture of denatured DNAs is treated with a DNA probe bearing a label. The probe can then hybridize with those DNAs with complementary sequences, and the unhybridized probes can be washed away. Probe labels can be radioactive to help with detection. Detection of the double-helical complexes allows for detection and quantitation of DNA that contains the sequence of interest.
DNA Arrays
New methods are being developed to monitor gene expression and to analyze genes rapidly for mutations. The arrays consist of a number of gene-specific DNA probes immobilized at specific sites on a chip. Chips can then be treated with labeled target DNA or RNA derived from cells of an organism. Arrays can contain thousands of DNA probes. Hybridization of the targets with complementary probe sequences allows for immobilization of the label at specific sites on the chip.
Rapid screening of disease associated mutations is an advantage of this technique. They profile gene expression by determining the amount of mRNAs, which may further assist in detection of cancer and individualized treatments.
Uses of DNA arrays (5)
- To detect mutations leading to ataxia telangiectasia,
- To detect recurrent respiratory infections
- To detect dilated blood vessels in the skin and eyes
- To detect mutations in the hereditary breast and ovarian cancer gene BRCA
- To identify pathogens present in the sample.
Conformations of Double helical DNA
A-, B-, and Z-DNA are different DNA conformations that are associated mainly with variation in the conformation of the nucleotide constituents of DNA.
Depending on the conditions and base sequence, the double helix can acquire various geometries
Noncanonical DNA structures
Formed when DNA interacts with certain proteins. This structure of DNA is not straight, it bends and forms unusual structures such as cruciforms or triple-stranded arrangements as it interacts with proteins. These variations in DNA conformation are an important recurring theme in the process of molecular recognition of DNA by proteins and enzymes. Variations in DNA structure or conformation are favored by specific DNA sequence motifs such as inverted repeats, mirror repeats, and direct repeats.
Bent DNA
Noncanonical DNA structure. DNA sequences with runs of 4 to 6 adenines separated by 10 base-pair produce bent conformations. This structure is important for DNA replication and transcription. Bending variations in DNA structure are associated with different DNA sequences or motifs. DNA damage may also lead to bending, which helps the cell to recognize it needs to initiate repair
DNA bending purpose
A fundamental element in the interaction between DNA sequences and proteins that catalyzes replication and transcription. Bending also occurs because of photochemical damage or mispairing of bases and serves as a recognition signal for initiation of DNA repair.
Cisplatin
Tetracoordinate platinum complex, which is used as a chemotherapy drug in ovarian, bone, testicular and lung cancers. It forms inter and intrastrand cross-links in double stranded DNA and the adduct (bond) itself represents 90% of DNA lesions or damaged sites of DNA. The bonds are formed from displacement of chloride ligands on platinum. Structural studies of intrastrand cross-linked DNA adducts show the double helix is bent
How are cisplatin-DNA adducts recognized in the cell?
Bent structures of the cisplatin-DNA adduct recognized by (1) several DNA-binding proteins that helps in repair (nucleotide excision repair - NER proteins), and (2) nonhistone DNA binding proteins - high mobility group (HMG) proteins, which regulate transcription
How does cisplatin cause cell death?
Transcription and apoptosis are affected by cisplatin-DNA adducts. Nucleotide excision repair proteins are recruited but excision repair is prone to produce DNA strand breaks and accumulation of these breaks will ultimately induce apoptosis as the DNA becomes too damaged to function.
High mobility group (HMG) domains and cisplatin
These are nonhistone DNA binding proteins, and are considered high mobility as they exhibit high mobility on gel electrophoresis. These proteins also help to regulate transcription. In binding to DNA, the HMG domain prefers single-stranded or bent double-stranded structures. HMG-domain proteins may affect the antitumor properties of cisplatin by repairing cisplatin DNA adducts
Cruciform DNA
Cross shaped DNA, which is another noncanonical DNA structure. Disruption of hydrogen bonds between the complementary strands and formation of intra- strand hydrogen bonds within the region of an inverted repeat produce a cruciform structure.
The loops generated by cruciform formation require the unpairing of 3 to 4 bases at the end of the “hairpin.” Bases base pair with other complementary bases to form a branch of the cross
Function of cruciform DNA
Cruciform structures at origins of DNA replication in mammalian cells have been shown to recruit cruciform binding proteins that function during the initiation of DNA synthesis. Depending on the sequence, these structures may be only slightly destabilizing because residues in the loop can remain stacked at the end of the helix.
Triple-stranded DNA
Noncanonical DNA structure- some polynucleotides such as poly(dA) and poly(dT) combine to form triple-stranded complexes rather than the expected double helices. Intramolecular triple helices can be formed by disruption of double-helical DNA with polypurine sequences in mirror repeats.
Mirror repeats
A mirror repeat is a region such as AGGGGA that has the same base sequence when read in either direction from a central point. Refolding generates a triple-stranded region and a single-stranded loop in a structure called H-DNA. These repeats may contribute to the development of triple-stranded DNA
Function of triple-stranded DNA
Many sequences in eukaryotic genomes have the potential to form triple-stranded DNA structures. These potential triple helical regions may constitute as much as 0.5% of some eukaryotic genomes and are especially common near sequences involved in gene regulation. Because of this, it has been proposed that H-DNA may play a role in the control of RNA synthesis and including possible roles in initiation and termination of replication and recombination. The ability to interfere with transcription have also led to efforts to use intermolecular triple helices to control RNA and protein synthesis.
Hereditary Persistence of Fetal Hemoglobin (HPFH) pathophysiology
A group of conditions in which fetal hemoglobin synthesis is not terminated at birth but continues into adulthood. Under normal conditions, fetal hemoglobin synthesis would be terminated at birth. It is characterized by changes in RBC similar to those found in the genetic blood disorder β-thalassemia and by delayed production of adult hemoglobin. The condition results from failure to stop transcription of human γ-globin genes, leading to elevated levels of fetal hemoglobin.
Fetal hemoglobin
Involved in transporting oxygen from the mother’s bloodstream to the organs and tissues of the fetus. Produced at 6 weeks of pregnancy, levels remain high after birth for 2-4 months until adult hemoglobin is produced. γ-globin genes are expressed in the fetal liver and make fetal hemoglobin
Hereditary Persistence of Fetal Hemoglobin (HPFH) symptoms
Associated with mild clinical or hematologic abnormalities. Mild musculoskeletal pains may occur infrequently, but patients with HPFH are often asymptomatic.
Hereditary Persistence of Fetal Hemoglobin (HPFH) genetics
The Condition results from failure to stop transcription of human γ-globin genes, leading to elevated levels of fetal hemoglobin. The formation of an intramolecular DNA triple-helical structure located about 200 bp upstream from the initiation site for transcription of the globin genes acts as a brake for their expression. Hemoglobin genes of patients contain mutations in one or more positions in this region, decreasing the stability of the triple helix and reducing its ability to inhibit the protein synthesis.
Four-stranded DNA
Four-stranded DNA guanine nucleotides and highly G-rich polynucleotides form novel tetrameric structures called G-quartets that contain a planar array of guanines connected by hydrogen bonds. Polynucleotides can interact to form tetraplexes where G-quartets stack on each other to form a multilayered structure.
The ends of eukaryotic chromosomes (telomeres) contain repetitive G-rich sequences and contain 800–2400 copies of the hexameric repeat sequence d(TTAGGG)n.
Oligonucleotides with this sequence can form tetraplex structures.
Four stranded DNA implications
Telomeres are attracting attention as targets for new anticancer drugs and G-tetraplexes have been implicated in recombination of immunoglobulin genes and in dimerization of double-stranded RNA of the HIV.
Telomeres
The ends of linear eukaryotic chromosomes, which are critical for maintaining the stability of the genome.
How is telomerase linked to cancer?
Telomerase activity present in most tumor cell lines may be responsible for their immortalization and when increased, correlates to poorer clinical prognosis. Two approaches are being examined for selective inhibition of telomerase.
Telomerase as a Target for anticancer agents- 2 approaches
- The first involves targeting of the RNA-containing portion of the enzyme. The RNA portion is used as a template to extend the repeat sequences of the telomere. Nucleic acids with chemically modified sugar phosphate backbones bind to telomerase RNA in immortal human cells, inhibit activity, and ultimately cause cell death or apoptosis.
- Second approach involves drugs that bind to G-quadruplex DNA, such as large aromatic molecules like porphyrins and anthraquinones. The drugs selectively bind and stabilize G-quadruplexes DNA structure
Slipped DNA (SMP-DNA)
DNA regions with direct repeat symmetry can form slipped, mis-paired DNA. Formation involves unwinding of the double helix, realignment, and subsequent pairing of one copy of the direct repeat with an adjacent copy on the other strand. Can form single stranded loops, which are a single strand of nucleotides. Although SMP-DNA has not yet been identified in vivo, genetic evidence suggests that it is involved in spontaneous frameshift mutations that result in base addition or deletion.
Deletions and duplications of DNA segments that are longer than a single base can occur during DNA replication between direct repeats, causing slipped-looped structures.
DNA Triple Repeats and Human Diseases
The fragile X-syndrome, myotonic dystrophy, X-linked spinal and Kennedy’s disease, Friedrich ataxia, and Huntington disease are the genetic diseases involving presence of repeated three-base pair DNA sequences.
Associated with expansion of nucleotide triplet repeats.
Kennedy’s disease
Caused by a CAG repeat in the first exon of the androgen receptor gene. It is a neurological disease leading to muscular atrophy, muscle weakness, and poor muscle coordination. Motor neurons are lost
Friedrich ataxia
Caused by a GAA sequence found within an intron. Symptoms- difficulty walking, impaired speech, brain damage
Myotonic dystrophy
Caused by a CTG sequence found in the 3’-untranslated region. Symptoms- muscle weakness
Fragile X-syndrome
Characterized by expansion of GCC triplet of the FMR-1 gene, expanding from 30 to >2000 copies. Develops when normal expression of the FMR-1 gene is halted/stops. Causes intellectual disabilities
How do DNA triple repeats cause disease?
Overall, the expansion of the triplet interferes with normal functioning of the related protein. Loss of protein function but sometimes gain of deleterious function may occur, and diseases can increase in severity with each successive generation. Triplet expansion may result from slipped mispairing during DNA synthesis. Because of massive amplification that characterizes these diseases, repeated or multiple slippages would have to be involved. One possible mechanism - slippage of nascent DNA during lagging strand synthesis, which may be aided by formation of a stable hairpin structure by a slipped loop.
Topoisomerases
Regulate the formation of superhelices. These enzymes catalyze the concerted breakage and rejoining of DNA strands, producing a DNA that is more or less superhelical than the original. The precise regulation of the cellular level of DNA super-helicity is important to facilitate protein interactions with DNA.
Mechanism of action of topoisomerase I
If DNA is overwound, TP1 nicks one strand of DNA. The intact strand passes through the break and the ends of broken strands are ligated
Topoisomerase as Treatment
Topoisomerases are important targets of antimicrobial and antineoplastic agents. Trap complex between topoisomerase and DNA resulting in degradation of DNA, introduction of mutations, or inhibition of translation and replication - leading to apoptosis. Anti-cancer durg Camptothecin and its derivatives act on topoisomerase 1, while other drugs target topoisomerase 2
Drugs targeting topoisomerase 1
Anti-cancer drug Camptothecin and its derivatives act on topoisomerase 1. The activity of Camptothecin and its derivatives may be improved by increased levels of topoisomerase I in tumor cells (colon cancer).
Which drugs act on topoisomerase 2
- Amsacrine and etoposide
- Anthracycline (Adriamycin and doxorubicin), synthetic intercalators, ellipticines, and podophyllotoxins
- Ciprofloxacin
Amsacrine and etoposide mechanism
Act on topoisomerase II. These drugs kill cells by stabilizing covalent topoisomerase II-DNA cleavage complexes, which accumulates leading to permanent DNA breaks.
Which cancers are treating by targeting topoisomerase 2?
Lymphoid and nonlymphoid leukemias, high-grade non-Hodgkin lymphomas, and Hodgkin disease are treated mostly with combinations of topoisomerase II inhibitors with or without additional cytotoxic agents. Leads to DNA breaks and apoptosis
Ciprofloxacin
Topoisomerase 2 inhibitor that gained considerable attention for its use in the treatment of inhalation anthrax. It is representative of the quinolones, a class of powerful broad-spectrum antibacterial activities. Cell death occurs due to accumulation of lethal double stranded breaks in the bacterial DNA. Ciprofloxacin shows good activity against Gram-positive and Gram-negative bacteria.
Newer drugs show even broader activity, for example, Gemifloxacin is useful for treating respiratory infections including those by multidrug resistant Staphylococcus pneumoniae, mycoplasma and legionella.
