DNA Structure and Replication

DNA Structure and Replication Essentials

• Genetic material must contain organism-building information.
• Genetic material must be passed from parent to offspring to maintain heredity.
• Genetic material must be replicable to ensure hereditary passing.
• Genetic material must be capable of change to account for phenotypic variation.
• Purines possess a double-ring structure and include adenine and guanine.
• Pyrimidines possess a single-ring structure and include thymine, uracil, and cytosine.
• Adenine and thymine form two hydrogen bonds.
• Cytosine and guanine form three hydrogen bonds.
• Semi-conservative replication was proven through the Meselson and Stahl experiment.
• During replication, parental strands serve as a template for new strands.
• Bidirectional replication in E. coli begins at the origin (oriC) and proceeds in both directions.
• A replicon is an individual unit of replication.
• Linear DNA replication is coordinated by a large team of enzymes.
• Eukaryotic DNA replication initiates at multiple sites on each chromosome.
• Multiple initiation sites reduce total replication time.
• Eukaryotic origins of replication are about 40,000 base pairs apart, allowing for replication completion in 15–30 minutes.
• Replication bubbles from multiple origins merge into completely replicated chromosomes.
• E. coli's origin of replication is called oriC or origin of chromosomal replication.
• DnaA proteins bind to DnaA boxes at oriC.
• DnaB/helicase travels 5' to 3', unwinding DNA.
• DNA polymerase III builds onto the 3' end of an existing DNA strand.
• There is a loss of bases at the 5' end of eukaryotic DNA during each replication round.
• Chromosomes shorten with each replication, limiting cell divisions.

Telomeres and Mutation Dynamics

• Telomere DNA sequences contain moderately repetitive tandem arrays.
• Telomeres typically have a 3' overhang, 12–16 nucleotides long, with guanine and thymine nucleotides.
• Telomeres tend to shorten during active cell division.
• Telomere DNA is about 8000 base pairs at birth, shortening to about 1500 base pairs in the elderly.
• Genes near telomeres needing protection are important since cells become senescent as telomeres shorten.
• Losing ability to divide happens when cells become senescent as telomeres shorten.
• Inserting active telomeres can block senescence.
• Cancer cells increase telomere activity, preventing shortening and senescence.
• Telomeres may be a target for anti-cancer drug treatments.
• Mutations are inherited DNA sequence changes that are either genic or chromosomal.
• Point mutations, rearrangements, duplications, and deletions are examples of mutations.
• Mutations are a fundamental property of DNA and can be spontaneous or mutagen-caused.
• Replication errors/background irradiation are examples of spontaneous mutations.
• Chemical mutagens or increased radiation are examples of induced mutations.
• Germ line mutations are inherited, while somatic mutations are not.
• Somatic mutations during embryogenesis yield mosaic organisms.
• Somatic mutations are common and occur in about 1 x 10^6 cell divisions in humans.
• Forward mutations change a wild-type allele to a new variation.
• Reverse mutations revert a mutant allele back to wild-type.
• Deleterious mutations decrease survival chances, with lethal mutations as the extreme.
• Beneficial mutations enhance survival or reproductive success.
• Loss-of-function mutations inactivate genes, often recessively.
• Gain-of-function mutations qualitatively alter gene action, sometimes dominantly.
• Hypomorphic mutations reduce gene expression or activity.
• Hypermorphic mutations increase gene expression.
• Conditional mutations show no phenotype under permissive conditions but are apparent under restrictive conditions.
• Essential genes can only be inactivated as conditional mutants.
• Temperature-sensitive mutations alter phenotype only at certain temperatures due to protein conformation changes.
• Molecular gene mutations in protein-coding regions can be insertions or deletions.
• Frameshift mutations shift the mRNA reading frame (insertions/deletions not multiples of three nucleotides).
• In-frame mutations do not shift the mRNA reading frame.
• Transitions are only between adenine/guanine and cytosine/thymine; other changes are transversions.
• Missense mutations in protein-coding regions replace one amino acid with another, which may or may not change function.
• Neutral substitutions do not change protein function.
• Synonymous (silent) substitutions in DNA do not change the amino acid sequence due to genetic code redundancy.
• Nonsense mutations create new stop codons.
• Chromosomal rearrangements can cause a breakpoint within a gene or alter its expression through position effects.
• Position effects can result from movement to regulatory sequences or heterochromatic regions.
• During trinucleotide repeat expansion, DNA polymerase may slip off and form a hairpin loop.
• DNA polymerase then reattaches, replicating repeat regions and leading to expansion.
• Molecular gene mutations are dynamic depending on the instability of the region of DNA involved like trinucleotide repeat mutations.

Huntington's Disease and Causes of Mutation

• Huntington's disease has an incidence of 3-7/100,000.
• Huntington's pathology includes neuronal loss and brain atrophy, with abnormal cellular metabolism detectable before MRI changes.
• Huntington's disease clinical symptoms include progressive dementia, involuntary movements (chorea), and psychiatric disturbances with average onset at 35-45 and death within 10 years.
• Huntington's disease is autosomal dominant, fully penetrant, and about 90% familial.
• About 5% of Huntington's cases become symptomatic in the first decade of life, and approximately 50% show symptoms by 40 years, with anticipation.
• Huntington's disease is caused by an unstable, expandable CAG repeat in the open reading frame of the huntington gene coding for glutamine.
• Polyglutamine expansion in the huntington protein causes a new, toxic function, with larger expansions leading to earlier onset, and all expansions occur when inherited from the pre-mutation father.
• Endogenous factors like DNA polymerase error, strand slippage, unequal crossover, reactive oxides, and transposons cause mutations.
• Two common types of DNA damage are depurination and deamination.
• Depurination involves the loss of about ~10,000 purine bases/day in mammalian cells and is the most common spontaneous DNA mutation.
• Spontaneous deamination of cytosine to uracil occurs at ~100 bases per cell per day.
• A DNA molecule loses a purine base in depurination/a spontaneous chemical change
• The DNA strand with the missing base could have any base incorporated opposite of it during the next replication round, where ¼ of the bases are incorrect and A is most common.
• Deamination is a spontaneous chemical change where a DNA base loses an amino group/can also be by chemical change.
• Tautomeric shifts are temporary changes in base structure.
• The common, stable forms of thymine and guanine is the keto form
• Rarely, T and G convert to an enol form
• The common, stable forms of adenine and cytosine is the amino form with rare conversion to an imino form.
• Here rare forms promote mistakes in base pairing like TG and CA
• Tautomeric shifts must occur just before replication to cause mutations
• Aerobic organisms produce reactive oxygen species (ROS) like hydrogen peroxide, superoxide, and hydroxyl radicals.
• The body tries to block ROS build up via Enzymes, such as superoxide dismutase and catalase antioxidants.
• Oxidative stress is an imbalance between ROS production and an organism's ability to break them down.
• Mutagens increase the mutation rate and alter DNA. They bias the types of damage to the DNA that they produce.
• Most mutations induced by a mutagen CAN also occur spontaneously.
• Mutagens can result in permanent genome changes leading to cancer (somatic) or birth defects (germ line).

Mutagens

• Base modifiers covalently modify bases and some disrupt pairing via alkylating agents.
• Intercalating agents cause insertion/deletion of single base pairs
• Base analogs similar to a DNA base and prone to tautomerization
• Physical mutagens include ionizing radiation, UV light, and heat.
• Ionizing radiation often causes double-strand breaks in DNA causing inappropriate end-joining which can be problematic , and creates free radicals altering base structures and causes adjacent pyrimidine bases on a DNA strand to bond and form pyrimidine dimers.
• UV radiation (non-ionizing) similarly causes adjacent pyrimidine bases on one DNA strand to bond and form pyrimidine dimers.
• Heat leads to base loss (AP sites).

DNA Repair, Recombination

• DNA repair systems are vital for organism survival due to the deleterious nature of most mutations.
• Living cells contain diverse DNA repair systems, often with overlapping abilities to fix similar problems.
• Estimated that less than 1/1000 DNA changes becomes a mutation
• DNA repair is generally a multistep process
• Irregular DNA structure is detected during DNA repair and the abnormal DNA is removed as normal DNA is synthesized, using one of the existing DNA molecules as a template
• DNA repair mechanisms can be direct repairs, AP endonuclease system repairs lost base nucleotide sites, and Excision/Mismatch repair .
• Damaged bases can be directly repaired and nucleotide covalent modifications are reversed by enzymes.
• Photolyase splits thymine dimers with visible light for photoreactivation and alkyltransferase repairs alkylated bases by transferring the methyl or ethyl group from the base.
• Both apurinic and apyrimidinic sites are repaired by AP endonuclease.
• During excision repair, A stretch of damaged DNA strand is removed from duplex molecular and replaced with re-synthesis
• BER uses DNA N-glycosylase enzymes
• NER is used if bulky damage is present and removes a stretch of damaged DNA
• BER will remove the base while NER will remove a stretch of damaged DNA strands.
• Mismatch repair is generated by mis-incorporation of standard bases during DNA replication.
• An excising of DNA during this process, followed by repair synthesis.
• Homologous recombination repair and non-homologous end joining fix double-strand breaks in DNA from ionizing radiation.
• DSB is processed by short digestion of DNA strands in homologous recombination repair and sister chromatid is used as template.
• Non-homologous end joining has broken ends recognized by end-binding proteins/formation of a cross-bridge/processing may result in deletion of small region.
• Defects in DNA repair can lead to diseases such as Xeroderma pigmentosum or Ataxia telangiectasia.
• Nucleotide excision repair mutations describes numerous freckle-like spots in sun-exposed areas, extreme sensitivity to sunlight, and predisposition to skin cancer
• Mutation hot spots are where mutations are nonrandom with respect to gene position.
• Sites of cytosine methylation and trinucleotide repeats are usually highly mutable unstable.
• Recombination is initiated by a break in DNA.
• The cell divides into two cells with DNA mismatch or with DNA gap synthesis.

Transcription and Translation Essentials

• RNA synthesis is complementary and antiparallel to the template strand, adding new nucleotides to the 3'-OH group, and transcription occurs in a 5' → 3' direction.
• Bacterial cells have only one RNA polymerase holoenzyme with six polypeptide chains, and eukaryotes possess RP1, RP2, and RP3.
• A transcription unit encodes an RNA molecule and contains regulator regions, a promoter, an RNA-coding region, and a terminator.
• The transcription process assembles the apparatus, elongates RNA, and terminates with template release.
• Promoter recognition by RNA polymerase is a prerequisite for transcription initiation.
• The rut sequence is recognized by the rho protein.
• Transcription termination sites are inverted repeats forming loops and Uracil rich sites that allow dissociation of DNA and RNA.
• Eukaryotic mRNA often contains an untranslated region before the polypeptide-specifying open reading frame (ORF) and an untranslated region after the ORF
• Eukaryotic core promoters contain the TATA box and transcriptional start site produce basal transcription.
• The first mRNA processing step adds 7-methylguanosine to the 5'-end and the presence of exons and introns are noncolinearity.
• The splicing mechanism requires consensus sequencess
• The translation system has mRNA, tRNA, ribosomes, aminoacyl-tRNA synthetases, and initiation/elongation/termination factors.
• Translation in prokaryotes has no cap and there is no scanning mechanism to locate 1st AUG
• In E. Coli the ribosome is bound with a special tRNA charged with tRNAfMet.
• Polycistronic mRNA has multiple amino acid sequences of different proteins
• Cistron: DNA sequence encodes a single polypeptide chain.

Prokaryotic Gene Expression

• Bacteria regulates when certain genes are used via genetic regulatory proteins binding to DNA.
• In attenuation, transcription terminates after it begins due to a terminator.
• Feedback inhibition occurs when the product of a metabolic pathway inhibits the first enzyme of the pathway.
• Positive, negative, or turning off and on occurs when regulation can occurs as the protein.
• Positive regulation has Transcription that is normally “off” until it's turned “on” by a transcriptional activator protein.
• Negative regulation has transcription that is normally “on” until turned “off” by a repressor.
• Activation and Repressor proteins have a DNA domain and an allosteric domain
• Operons are units where related enzymes are linked and coordinately expressed from a single promoter.
• The lac operon, is negatively regulated with Lactose must be present. The lac operons is positively regulated with Glucose.
• The E. coli and Lacl-, lacOc, and laclS are genetic mutations that cause mis-regulation when glucose is depleted.
• Trp Operon encodes the leader peptides that are involved in attenuation.

Eukaryotic Gene Expression

• Transcription factors combine with the promoter to initiate transcription.
• Eukaryotic activators, repressors, TFIID direct or through coactivators alter regulatory functions.
• Steroid hormones are the regulatory hormone for trancription and 5'-TGACGTCA-3' is the sequence of the CREB protein.
• Phosphorylated CREB binds to DNA and stimulates transcription.
• Chromatin structure is regulated via acetylation and methylation.
• Chromatin-remodeling complexes alter DNA, and DNa methylation is heritable.
• Uniparental disomy and methylation DNa sequences/genomic imprinting depends on the expression of genes.