Cellular Biology: DNA, RNA & Genetic Processes

Cell Division and Mitosis

• The completion of one set of cell activities signals the start of the next
• Stages include prophase, prometaphase, metaphase, anaphase, and telophase
• Chromosome pairs condense and attach to fibers during mitosis
• These fibers pull sister chromatids to opposite sides of the cell
• Cytokinesis is the process where the cell divides, producing two identical daughter cells
• Mitosis and cytokinesis often occur together, which leads to the interchangeable use of the terms "mitosis" and "mitotic phase"
• Mitosis and cytokinesis can occur separately resulting in single cells with multiple nuclei
• This happens mostly in fungi and slime molds, but also in animals under certain circumstances
• Errors in mitosis can cause apoptosis or mutations which may lead to cancer

DNA Replication Overview

• DNA replication is essential for biological inheritance and DNA copying occurs in all living organisms
• It is "semi-conservative" because each original DNA strand serves as a template for a complementary strand's reproduction
• Results in two identical DNA molecules from one double-stranded DNA molecule
• Proofreading mechanisms help ensure accurate DNA replication
• DNA replication starts at specific genome locations called “origins”
• Unwinding DNA and synthesizing new strands at the origin forms a replication fork
• DNA polymerase synthesizes new DNA by adding matched nucleotides to the template strand
• Other proteins assist with the initiation and continuation of DNA synthesis at the fork
• DNA replication can be performed in vitro using isolated DNA polymerases and artificial DNA primers
• Polymerase chain reaction (PCR) amplifies a specific DNA fragment from a DNA pool using artificial synthesis in a cyclic manner

DNA Replication Process:

• Helicases unwind the parental double helix
• Single-strand binding proteins stabilize the unwound parental DNA
• The leading strand is synthesized continuously by DNA polymerase in the 5' to 3' direction
• The lagging strand is synthesized discontinuously
• DNA ligase then joins the Okazaki fragments
• After RNA primers are replaced by DNA, DNA ligase joins the Okazaki fragment to the growing strand

DNA Polymerase and Replication

• DNA polymerases are a family of enzymes involved in all forms of DNA replication
• A DNA polymerase can only extend an existing DNA strand paired with a template strand, but cannot initiate new strand synthesis
• A primer, a short DNA or RNA fragment, must be created and paired with the DNA template to begin synthesis
• The DNA polymerase then synthesizes a new strand of DNA by extending the 3' end, adding new nucleotides to match the template
• Phosphodiester bonds are created and this requires energy
• Energy is supplied from two of the three phosphates attached to each unincorporated base
• Free bases with attached phosphate groups are called nucleoside triphosphates
• Two phosphates are removed when a nucleotide is added to a growing DNA strand
• Created energy forms a phosphodiester bond with the phosphate left on the DNA strand
• Energetics explain the direction of synthesis: energy comes from free nucleotides, implying synthesis is from 5' to 3' direction
• DNA polymerases are accurate, with less than one error/10^7 nucleotides
• Some DNA polymerases have proofreading ability, and mismatched bases can be removed from the end of a strand
• If proofreading removes a 5' nucleotide, the triphosphate end and energy source are lost

DNA Replication Within the Cell

• A cell must replicate its DNA before dividing
• Initiated at "origins," which are targeted by proteins that separate strands and start DNA synthesis
• Origins contain DNA sequences recognized by replication initiator proteins
• Initiator proteins recruit other proteins to separate DNA strands at the origin, forming a bubble and replication forks
• Origins tend to be "AT-rich" for easier separation due to weaker hydrogen bonding
• RNA primers are created on template strands
• The leading strand receives one RNA primer per active origin
• The lagging strand receives several RNA primers, called Okazaki fragments
• DNA polymerase extends the leading strand continuously, and the lagging strand discontinuously, due to Okazaki fragments
• RNase removes RNA fragments and DNA polymerase fills the gaps
• Ligase fills in nicks on the leading strand (one nick) and lagging strand (several nicks)
• DNA synthesis continues, as original DNA unwinds, forming replication forks
• In bacteria, which have a single origin and a circular chromosome, this creates a "theta structure"
• Eukaryotes have longer, linear chromosomes and initiate replication at multiple origins

Replication Fork Details

• Original DNA splits in two during replication, forming two "prongs" resembling a fork–replication fork
• DNA has a ladder-like structure
• The ladder is broken vertically, where each half requires a new matching half
• DNA polymerase can only synthesize a new DNA strand in a 5'-3' manner
• Therefore, the two DNA helix strands replicates differently

Leading and Lagging Strand:

• Leading strand is oriented in a 5'-3' direction
• DNA polymerase "reads" the leading strand and continuously adds nucleotides
• This polymerase is DNA polymerase III (DNA Pol III) in prokaryotes and presumably Pol ε in eukaryotes
• Lagging strand that is oriented in a 3'-5' direction
• Because its orientation conflicts with the 5'-3' working direction of DNA polymerase III, replication of the lagging strand becomes more complex than leading strand
• Primase "reads" the lagging strand and adds RNA in short, separated segments
• In eukaryotes, primase is intrinsic to Pol aDNA
• DNA polymerase III or Pol 8 lengthens primed segments, forming Okazaki fragments; primer removal in eukaryotes is also performed by Pol 8
• In prokaryotes, DNA polymerase I "reads" fragments, removes RNA using its flap endonuclease domain, and replaces RNA nucleotides with DNA nucleotides
• RNA and DNA use slightly different kinds of nucleotides
• DNA ligase then joins the fragments together

Regulation of DNA Replication

• Eukaryotic DNA replication is controlled within the cell cycle
• As the cell grows and divides, it progresses through stages in the cell cycle - DNA replication occurs during the S phase (Synthesis phase)
• Eukaryotic cell cycle progression is controlled by cell-cycle checkpoints
• Checkpoint progression is controlled through interactions between proteins, including cyclins and cyclin-dependent kinases
• G1/S checkpoint (or restriction checkpoint) regulates if eukaryotic cells enter DNA replication and subsequent division
• Cells that do not proceed through this checkpoint are quiescent in the "GO" stage (do not replicate)
• Replication of chloroplast and mitochondrial genomes occurs independent of the cell cycle, through the process of D-loop replication
• Most bacteria do not have cell cycle and continuously copy their DNA
• During rapid growth, replication may occur concurrently in multiple rounds
• Bacteria (E. coli) regulation of DNA replication mechanisms: hemimethylation and sequestering of origin sequence, ratio of ATP to ADP, levels of the protein DnaA
• E. coli methylates GATC DNA sequences DNA synthesis
• Results = hemimethylated sequences; Hemimethylated DNA is recognized by SeqA protein and origin sequence is sequestered
• DnaA (for replication initiation) binds less well to hemimethylated DNA
• Newly replicated origins are prevented from immediately initiating another round of DNA replication
• ATP builds up when the cell is in a rich medium, and triggers DNA replication when the cell reaches a certain size
• ATP competes with ADP to bind with DnaA: DnaA-ATP complex is allows initiation
• Number of DnaA proteins needed for DNA replication, The origin is copied where binding sites for DnaA double and requires DnaA doubles
• Synthesis is required to enable another replication initiation

Termination of Replication

• Chromosomes in bacteria are circular, so termination happens when two replication forks meet on opposite ends of the chromosome
• E. coli regulates this using termination sequences, which, when bound by Tus protein, enable only one direction can pass
• Replication forks meet within the termination region of the chromosome as a result
• Eukaryotes start DNA replication at multiple chromosome points: replication forks meet and terminate
• Eukaryotic termination is largely unregulated
• Eukaryotes DNA often fails replication synthesis to chromosome ends (telomeres), shortening the telomere
• Somatic cells undergo the normal process shortening, where cells are only able to divides number of times before DNA prevents any further division
• This limit is known as the Hayflick limit
• Germ cell line enzyme telomerase extends repetitive telomere sequences and prevents degradation, passing DNA to the next generation
• Telomerase can mistakenly become active in which somatic cells can causes cancer

DNA Interactions with Proteins

• DNA interacts with proteins and can influence DNA's function, protein interactions are nonspecific or specific to a single or multiple DNA sequence
• Copied DNA base sequences in transcription and DNA replication are particularly important in transcription, such as polymerases and enzymes
• DNA structural proteins are mostly nonspecific DNA
• DNA is held in chromosome complexes; structural proteins organize DNA into chromatin
• Eukaryote structure = DNA binds to histones while prokaryotes have multiple types of proteins
• Histones form a nucleosome, which involves two turns of double stranded DNA wrapped around a surface
• Histonse include amino acids that connect the acidic sugar phosphate backbone of the DNA, and are independent of the base sequence
• Histons undergo chemical residue modifications like methylation, acetylation, and phosphorylation
• These modifications can alter DNA's function making it more or less accessible to transcription factors

Single-Stranded DNA-Binding Proteins

• Humans contain replication protein A used for DNA replication, DNA Recombination, and Repair
• These binding proteins hold and stabilize DNA, and stop stem loops from forming and being hurt by nucleases

DNA Modifying Enzymes

• Nucleases enzymes cut DNA strands by catalyzing the hydrolysis of phosphodiester bonds
• Exonucleases are which hydrolyze nucleotides from ends of DNA and endonucleases that cut within the strand
• Restriction endonucleases cut DNA at specific sequences, and are the most used nucleases in bio
• EcoRV enzyme recognizes the 6-base sequence 5'-GAT|ATC-3', then makes cuts on a line
• ENzymes protect bacteria from phage infections or DNA destructions which are apart of restriction
• Enzymes used in technology for molecular cloning and DNA fingerprinting
• DNA ligases can mend break DNA strands and are important on lagging strands in DNA replication
• Ligases use a complted DNA template by putting short DNA segments together

Topoisomerases and Helicases

• Topoisomerases have both nuclease and ligase activity. These actions change the supercoiling amount in DNA
• Some work by cutting the helix and letting one part rotate, this lowers supercoling the enzyme then seals
• Other enzymes cut one part and transfer DNA down to the break, then rejoin helix. These processes are key to DNA replication
• Helicases are proteins help to unwind the double strand. which is key for accessing the DNA

DNA Methlyation

• DNA Methylation modification has a bunch of bio processes like gene imprinted, x enzyme inactivation, and suppressor tumor gene on cancerous stem
• DNA helps protect pathogens from endotuclease
• Base modification changes the packages with regions that have low low gene expression
• Cytosine Methlation makes a average amount in organisms, with worm that have cytosine and others have a smaller contain.
• Cytosine importance is that thymine base, methylated are more prone to mutations
• Other basics is that they can bactera and glycosation

DNA Muation

• DNA mutation id a change in dna seuence which copy erros, exposure radiations, viruses,
• It is found through oragnisms with reproductive cells. Also its subdivided by germ mutations or decendants through cell reproduce or somatic in dedicated cell which transmitted to descendants.
• Its also transmittied somatic through descendants and flower buds in part pf plants. The source are the mutagens from mutagens
• Mutations can high energy and radiions and rays. Thymine or oxidant which cross pyrminidine and hydrogen or free redicals that has double strand breaks cells then count to contain and lesion the double strand and point mutations. and translocations
• It is fit intercaation on the fit or arimatic pairs bases needs to be seperater by unwinding doubile its transript and reploications