DNA Repair Exam III PDF

DNA repair Double strand breaks Non-homologous end-joining, in which the broken ends are simply brought together and joined by DNA ligation, generally with the loss of one or more nucleotides joining at the site of joining. Nonhomologous end-joining alters the original DNA sequence w...

DNA repair Double strand breaks Non-homologous end-joining, in which the broken ends are simply brought together and joined by DNA ligation, generally with the loss of one or more nucleotides joining at the site of joining. Nonhomologous end-joining alters the original DNA sequence when repairing a broken chromosome. These alterations can be either deletions or short insertion.  Non homologous end joining A central role is played by the Ku protein, a heterodimer that grasps the broken chromosome ends. The additional proteins shown are needed to hold the broken ends together while they are processed and eventually joined covalently. Repairing double-strand breaks by homologous recombination is more difficult to accomplish but this type of repair restores the original DNA sequence. lt typically takes place after the DNA has been duplicated but before the cell has divided. https://www.youtube.com/watch?v=86JCMM5kb2A Mechanism of double-strand break repair by homologous recombination. This is the preferred method for repairing DNA double-strand breaks that arise shortly after the DNA has been replicated and the two sister chromatids are still held together. Once a strand invasion reaction has occurred, the point of strand exchange (the "branch point") can move through a process called branch migration.  Two types of DNA branch migration observed in experiments in vitro A) Spontaneous branch migration is a back-and-forth random-walk process, and it therefore makes little progress of very long distances. B) Protein-directed branch migration requires energy, and it moves the branch point at a uniform rate in one direction. https://www.youtube.com/watch?v=L61Gp_d7evo  Something to remember Exonuclease vs Endonuclease Do a research to see the differences between both type of enzymes  Homologous recombination as a genetic variability Is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. it's important because it's one of the sources of genetic variation that we see among offspring of a set of parents. DNA to Proteins Is this the same organism? Same Genome; different Proteome!!!  The Central Dogma The pathway from DNA to protein The flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation) occurs in all living cells.  Genes can be expressed with different efficiencies In this example, gene A is transcribed and translated much more efficiently than gene B. This allows the amount of protein A in the cell to be much greater than that of protein B. Mention the difference between DNA and RNA  The chemical structure of RNA RNA contains the sugar ribose, which differs from deoxyribose, the sugar used in DNA by the presence of an additional –OH group. RNA contains the base uracil, which differs from thymine, the equivalent base in DNA, by the absence of a –CH3 group. Whereas DNA always occurs in cells as a double-stranded helix, RNA is single-stranded.  RNA can fold into specific structures RNA is largely single-stranded, but it often contains short stretches of nucleotides that can form conventional base pairs with complementary sequences found elsewhere on the same molecule. These interactions, along with additional “nonconventional” base-pair interactions, allow an RNA molecule to fold into a three- dimensional structure that is determined by its sequence of nucleotides. RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds. The phosphodiester chemical linkage between nucleotides in RNA is the same as that in DNA.  Transcription Copying of one strand of DNA into a complementary RNA sequence by the enzyme RNA polymerase DNA transcription produces a single-stranded RNA molecule that is complementary to one strand of DNA. https://www.youtube.com/watch?v=gG7uCskUOrA Which enzyme is the responsible to carry out the transcription?  Transcription process RNA polymerases can start an RNA chain without a primer. The RNA polymerase (pale blue) moves stepwise along the DNA, unwinding the DNA helix at its active site. As it progresses, the polymerase adds nucleotides, one by one to the RNA chain at the polymerization site, using an exposed DNA strand as a template. The RNA transcript is thus a complementary copy of one of the two DNA strands. RNA polymerase superpower enzyme RNA polymerase is not classified as DNA helicase biochemically, it includes helicase activity and can unzip or unwind DNA but only locally and usually only for small lengths.  Compounds necessaries to do the transcription process A σ factor is a bacterial protein that transiently associates with the RNA polymerase (RNAP) core enzyme complex to direct binding of RNAP to gene promoters, thereby initiating transcription. How RNA polymerase recognize where to start and end the transcription process?  Promoter: Specific nucleotide sequence in DNA to which RNA polymerase binds to begin transcription. Terminator Signal in bacterial DNA that halts transcription. The transcription cycle of bacterial RNA polymerase https://www.youtube.com/watch?v=1b-bRVgqof0  RNA’s and RNA polymerases Eukaryotic RNA polymerase II Bacteria RNA polymerase III A single type of RNA polymerase RNA polymerase I RNA polymerase III  Bacterial RNA polymerase requires only a single additional protein (σ factor) for transcription initiation Eucaryotic RNA polymerases require many additional proteins, collectively called the general transcription factors. Transcription Factors Are proteins involved in the process of converting, or transcribing, DNA into RNA. Transcription factors include a wide number of proteins, excluding RNA polymerase, that initiate and regulate the transcription of genes. Activator Repressor  Enhancer Regulatory DNA sequence to which gene regulatory proteins bind, increasing the rate of transcriptional and structural gene that can be thousand of base pairs away.  Transcription initiation by RNA polymerase II in a eucaryotic cell Transcription initiation in vivo requires the presence of transcriptional activator proteins. These proteins bind to specific short sequences in DNA. Consensus sequences found in the vicinity of eucaryotic RNA polymerase II start points  Transcription in Eukaryotes Transcription is only the first of several steps needed to produce an mRNA. The mRNA needs to be processed. 1. Capping on the 5’ 2. Removal of intron sequences (splicing) 3. Polyadenylation of the 3’  Capping on the 5’ As soon as RNA polymerase II has produced about 25 nucleotides of RNA, the 5’ end of the new RNA molecule is modified by addition of a cap that consists of a modified guanine nucleotide. The 5’-methyl cap signifies the 5’ end of eucaryotic mRNAs, and this landmark helps the cell to distinguish mRNAs from the other types of RNA molecules present in the cell.  Removal of intron sequences Eucaryotic genes were found to be broken up into small pieces of coding sequence (expressed sequences or exons) interspersed with much longer intervening sequences or introns. Both intron and exon sequences are transcribed into RNA. The intron sequences are removed from the newly synthesized RNA through the process of RNA splicing.  Splicing Removal of introns from a pre-mRNA transcript by splicing together the exons that lie on either side of each intron. Structure of two human genes showing the arrangement of exons and introns. A) The relatively small β-globin gene, which encodes one of the subunits of the oxygen-carrying protein hemoglobin, contains 3 exons. B) The much larger Factor VIII gene contains 26 exons; it codes for a protein (Factor VIII) that functions in the blood-clotting pathway. The most prevalent form of hemophilia results from mutations in this gene.  Alternative splicing Production of different RNAs from the same gene by splicing the transcript in different ways https://www.youtube.com/watch?v=aVgwr0QpYNE  Spliceosome The basic process of constitutive splicing starts with the recognition of splice sites by the spliceosome. U1 and U2 are attached to the 5´ splice site and 3´ splice site, respectively. Splice site recognition is followed by the complete assembly of the spliceosome. After complete assembly of the spliceosome, splicing occurs in two main steps: first, 5´ splice site cleavage and lariat formation, and then, 3´ splice site cleavage and exon ligation.  Identification of splices sites The consensus nucleotide sequences in an RNA molecule that signal the beginning and the end of most introns in humans. Only the three blocks of nucleotide sequences shown are required to remove an intron sequence; the rest of the intron can be occupied by any nucleotides. Here A, G, U, and C are the standard RNA nucleotides; R stands for purines (A or G); and Y stands for pyrimidines (C or U). The A highlighted in red forms the branch point of the lariat produced by splicing. Only the GU at the start of the intron and the AG at its end are invariant nucleotides in the splicing consensus sequences.  Polyadenylation of the 3’ Addition of a long sequence of A nucleotides (the poly-A tail) to the 3’ end of a nascent mRNA molecule. Cleavage and polyadenylation specificity factor (CPSF). Cleavage stimulation factor (CTsF). poly-A polymerase (PAP). Steps leading from gene to protein in Eucaryotes and Bacteria RNA to Proteins mRNA molecules that serve as intermediaries on the pathway to proteins.  Translation Process by which the sequence of nucleotides in a mRNA molecule directs the incorporation of amino acids into protein. Occurs on a ribosome. 20 aminoacids The sequence of nucleotides in the mRNA molecule is read in consecutive groups of three. RNA is a linear polymer of four different nucleotides, so there are 4 × 4 × 4 = 64 possible combinations of three nucleotides. Each group of three consecutive nucleotides in RNA is called a codon, and each codon specifies either one amino acid or a stop to the translation process. The nucleotide sequence of a gene, through the intermediary of mRNA, is translated into the amino acid sequence of a protein by rules that are known as the genetic code. The genetic code Most amino acids are represented by more than one codon, and that there are some regularities in the set of codons that specifies each amino acid. Codons for the same amino acid tend to contain the same nucleotides at the first and second positions and vary at the third position. There are 64 different codons: 61 specify amino acids and 3 are used as stop codons. A long open reading frame is often part of a gene (that is, a sequence directly coding for a protein). RNA sequence can be translated in any one of three different reading frames. Only one of the three possible reading frames in an mRNA encodes the required protein. The three possible reading frames in protein synthesis In the process of translating a nucleotide sequence (blue) into an amino acid sequence (red), the sequence of nucleotides in an mRNA molecule is read from the 5’ end to the 3’ end in consecutive sets of three nucleotides. In principle, therefore, the same RNA sequence can specify three completely different amino acid sequences, depending on the reading frame. In reality, however, only one of these reading frames contains the actual message. 5’ - GCU -AGA - UCG –UAC- AAG - CCU 3’ Translation of mRNA into protein depends on adaptor molecules that can recognize and bind both to the codon and, at another site on their surface, to the amino acid. These adaptors consist of a set of small RNA molecules known as transfer RNAs (tRNAs), each about 80 nucleotides in length. Eucaryotic tRNAs are synthesized by RNA polymerase III.  Anticodon, a set of three consecutive nucleotides that pairs with the complementary codon in an mRNA molecule. A tRNA molecule The cloverleaf structure showing the complementary base-pairing that creates the double-helical regions of the molecule. The anticodon is the sequence of three nucleotides that base-pairs with a codon in mRNA  Wobble position More than one tRNA for many of the amino acids or that some tRNA molecules can base-pair with more than one codon. Wobble base-pairing explains why so many of the alternative codons for an amino acid differ only in their third nucleotide. Some of the modified nucleotides lie within the anticodon—most notably inosine, produced by the deamination of adenosine.  Recognition and attachment of the correct amino acid depends on enzymes called aminoacyl-tRNA synthetases, which covalently couple each amino acid to its appropriate set of tRNA molecules. Most cells have a different synthetase enzyme for each amino acid (that is, 20 synthetases in all); one attaches glycine to all tRNAs that recognize codons for glycine, another attaches alanine to all tRNAs that recognize codons for alanine, and so on. The structure of the aminoacyl-tRNA linkage. The carboxyl end of the amino acid forms an ester bond to ribose. The fundamental reaction of protein synthesis is the formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming amino acid. Three sites in the ribosome structure: aminoacyl (A), peptidyl (B) and exit (E) https://www.youtube.com/watch?v=5bLEDd-PSTQ  Ribosomes are complexes of rRNA molecules and proteins, and they can be observed in electron micrographs of cells. Sedimentation Coefficient: a measure of the rate at which a molecule (as a protein) suspended in a colloidal solution sediments in an ultracentrifuge usually expressed in svedbergs. Protein synthesis is performed by the ribosome, a complex catalytic machine made from more than 50 different proteins (the ribosomal proteins) and several RNA molecules, the ribosomal RNAs (rRNAs). A typical eukaryotic cell contains millions of ribosomes in its cytoplasm, and it takes approximately 1 minute to synthesize an average-sized protein.  Ribosomes are the sites in a cell in which protein synthesis takes place. Cells have many ribosomes, and the exact number depends on how active a particular cell is in synthesizing proteins. A ribosome is composed of two subunits: large and small. During translation, ribosomal subunits assemble like a sandwich on the strand of mRNA, where they proceed to attract tRNA molecules tethered to amino acids (circles). A long chain of amino acids emerges as the ribosome decodes the mRNA sequence into a polypeptide, or a new protein. Translating an mRNA molecule Each amino acid added to the growing end of a polypeptide chain is selected by complementary base-pairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain. In step 1, an aminoacyl-tRNA molecule binds to a vacant A-site on the ribosome and a spent tRNA molecule dissociates from the E-site. In step 2, a new peptide bond is formed. In step 3, the large subunit translocates relative to the small subunit, leaving the two tRNAs in hybrid sites: P on the large subunit and A on the small, for one; E on the large subunit and P on the small, for the other. In step 4, the small subunit translocates carrying its mRNA a distance of three nucleotides through the ribosome. Membrane Structure  Cell membrane or Plasma membrane Encloses the cell, defines its boundaries and maintain the essential differences between the cytosol and the extracellular environment. 5 to 10 nm wide. Cell membranes serve as barriers and gatekeepers. They are semi-permeable, which means that some molecules can diffuse across the lipid bilayer, but others cannot. Plasma membranes are selectively permeable (semipermeable)—they allow some substances to pass through, but not others. Non-polar and lipid-soluble material with a low molecular weight can easily slip through the membrane's hydrophobic lipid core. Fat-soluble vitamins A, D, E, and K readily pass through the plasma membranes Oxygen and carbon dioxide molecules have no charge and pass-through membranes by simple diffusion.  Diffusion Is a passive process of transport. A single substance moves from a high concentration to a low concentration area until the concentration is equal across a space.  Cell membrane a) An electron micrograph of a plasma membrane see in a cross-section b-c) Two dimensional and three-dimensional views of a cell membrane and the general disposition of its lipids and protein constituents.  Composition The lipid bilayer provides the basic structure for all cell membranes, it is an amphiphilic The most abundant membrane lipids are phospholipids  The main phospholipids in most animal cell membranes are phosphoglycerides, which have a three-carbon glycerol backbone. The subregions of a glycerophospholipid molecule; phosphatidylcholine is shown as an example. The hydrophilic head is composed of a choline structure The parts of a phosphoglyceride molecule (blue) and a phosphate (orange). This head is connected to a glycerol (green) with two hydrophobic tails (purple) called fatty acids.  Ethanolamine 7H 2C 1O 1N Four major phospholipids in mammalian plasma membranes. The lipid molecules shown in (A-C) are phosphoglycerides which are derived from glycerol. The molecule in (D) is sphingomyelin, which is derived from sphingosine (E) and is therefore a sphingolipid. In addition to phospholipids, the lipid bilayers in many cell membranes contain cholesterol and glycolipids. Eucaryotic plasma membranes contain especially large amounts of cholesterol around 20%. What other type of components has the plasma membrane? Proteins and Carbohydrates Membrane Proteins  Integral protein also called transmembrane Is a protein that is embedded in the membrane. Functions  Various ways in which membrane proteins associate with the lipid bilayer Most transmembrane proteins are thought to extend across the bilayer as 1) a single α helix 2) as multiple α helices 3) as a rolled-up β sheet (a β barrel). 4) Some of these are anchored to the cytosolic surface by an amphipathic α helix that partitions into the cytosolic monolayer of the lipid bilayer through the hydrophobic face of the helix. 5) Others are attached to the bilayer solely by a covalently attached lipid chain—either a fatty acid chain or a prenyl group—in the cytosolic monolayer or, 6) via an oligosaccharide linker, to phosphatidylinositol in the non cytosolic monolayer. 7, 8) Finally, many proteins are attached to the membrane only by noncovalent interactions with other membrane proteins. Something to remember  Proteins as a transporters Membrane transport protein that Membrane transporter protein that binds forms an aqueous pore in the to a solute and transports it across the membrane through which a specific membrane by undergoing a series of solute, usually an ion can pass. conformational changes. https://www.youtube.com/watch?v=kK-N8jUH5Xs  O, CO2 Passive and active transport compared Passive transport down an electrochemical gradient occurs spontaneously, either by simple diffusion through the lipid bilayer or by facilitate diffusion through channels and passive transporters. By contrast active transport requires an input of metabolic energy and is always mediated by transporter that harvest metabolic energy to pump the solute against its electrochemical gradient. https://www.youtube.com/watch?v=ufCiGz75DAk If a substance must move into the cell against its concentration gradient—that is, if the substance's concentration inside the cell is greater than its concentration in the extracellular fluid (and vice versa)— the cell must use energy to move the substance. Carrier proteins or pumps facilitate movement: there are three protein types or transporters. uniporter carries one molecule or ion. A symporter carries two different molecules or ions, both in the same direction. An antiporter also carries two different molecules or ions, but in different directions. The sodium-potassium pump is an example of primary active transport that moves ions, sodium and potassium ions in this instance, across a membrane against their concentration gradients. The energy is provided by the hydrolysis of ATP. Three sodium ions are moved out of the cell for every 2 potassium ions that are brought into the cell. This creates an electrochemical gradient that is crucial for living cells. https://www.youtube.com/watch?v=_bPFKDdWlCg  Proteins as a receptors Any protein that binds a specific signal molecule (ligand) and initiates a response in the cell. Some are on the cell surface, while others are inside the cell. A simple intracellular signaling pathway activated by an extracellular signal molecule. The signal molecule usually binds to a receptor protein that is embedded in the plasma membrane of the target cell and activates one or more intracellular signaling pathways mediated by a series of signaling proteins. Finally, one or more of the intracellular signaling proteins alters the activity of effector proteins and thereby the behavior of the cell.  Peripheral membrane proteins Are associated with the membrane but are not inserted into the bilayer.  Carbohydrates Are the third major component of plasma membranes. They are always found on the exterior surface of cells and are bound either to proteins (forming glycoproteins) or to lipids (forming glycolipids). These carbohydrate chains may consist of 2–60 monosaccharide units and can be either straight or branched These carbohydrates on the exterior surface of the cell—the carbohydrate components of both glycoproteins and glycolipids—are collectively referred to as the glycocalyx (meaning “sugar coating”). Microscope image of individual cells showing their hair-like surface covering of glycans (the”glycocalyx”).  Most transmembrane proteins in animal cells are glycosylated. Carbohydrates extensively coat the surface of all eukaryotic cells. These carbohydrates occur as oligosaccharide chains covalently (glycoproteins) bound to membrane proteins and lipids (glycolipids). A single-pass transmembrane protein. Note that the polypeptide chain traverse the lipid bilayer as a right-handed alpha helix and that the oligosaccharide chains and disulfide bonds are all on the non cytosolic surface of the membrane.  The carbohydrate layer on the cell surface A) Electron micrograph of the surface of a lymphocyte stained with ruthenium red emphasizes the thick carbohydrate-rich layer surrounding the cell. B) The carbohydrate layer is made up of the oligosaccharide side chains of glycolipids and integral membrane glycoproteins and the polysaccharide chains on integral membrane proteoglycans. Why sugars-carbohydrates are important in the cell membrane? Membrane carbohydrates are sites of cell recognition and adhesion, either cell-cell recognition or cell-pathogen interactions. Lectins serve as means of attachment of different kinds of cell as well as viruses to other cells via the surface carbohydrates of the latter. In some cases, cell-surface lectins bind particular glycoproteins, whereas in other cases the carbohydrates of cell surface glycoproteins or glycolipids serve as sites of attachment for biologically active molecules that themselves are lectins (e.g. carbohydrate-specific bacterial and plant toxins). Cancer cells have a unique spectrum of glycans, the complex, branched carbohydrate structures found on the outside of the cell. https://www.youtube.com/watch?v=RT61MUjogRo Thinking about membranes… What other organelles have a membrane? Nuclear membrane The nuclear membrane, also known as the nuclear envelope, consists of two lipid bilayers. The outermost layer is contiguous with the endoplasmic reticulum (ER). The innermost layer is lined by a fibrillar network consisting of nuclear intermediate filament proteins, known as nuclear lamins. The nuclear lamina provides structural support and acts as an anchoring point for chromatin, thus playing an important role in nuclear organization.  Cell Signaling Communication between cells is mediated mainly by extracellular signal molecules.. A simple intracellular signaling pathway activated by an extracellular signal molecule. The signal molecule usually binds to a receptor protein that is embedded in the plasma membrane of the target cell and activates one or more intracellular signaling pathways mediated by a series of signaling proteins. Finally, one or more of the intracellular signaling proteins alters the activity of effector proteins and thereby the behavior of the cell. Cells in multicellular animals communicate by means of hundreds of kinds of signal molecules Amino Peptides Proteins acids Steroids Fatty Retinoids acids Gases  The binding of extracellular signal molecules to either cell-surface or intracellular receptors. A) Most signal molecules are hydrophilic and are therefore unable to cross the target cell’s plasma membrane directly; instead, they bind to cell- surface receptors, which in turn generate signals inside the target cell. B) Some small signal molecules, by contrast, diffuse across the plasma membrane and bind to receptor proteins inside the target cell— either in the cytosol or in the nucleus. Many of these small signal molecules are hydrophobic and nearly insoluble in aqueous solutions. https://www.youtube.com/watch?v=wJk7nRccLbc  Target cell responds by means of a receptor, which specifically binds the signal molecule and then initiates a response in the target cell. The extracellular signal molecules often act at very low concentrations and the receptors that recognize them usually bind them with high affinity. Receptors are transmembrane proteins on the target cell surface. When these proteins bind an extracellular signal molecule (a ligand), they become activated and generate various intracellular signals that alter the behavior of the cell. In other cases, the receptor proteins are inside the target cell, and the signal molecule has to enter the cell to bind to them: this requires that the signal molecule be sufficiently small and hydrophobic to diffuse across the target cell’s plasma membrane. Many signal molecules remain bound to the surface of the signaling cell and influence only cells that contact it. Such contact-dependent signaling is especially important during development and in immune responses. Contact-dependent signaling requires cells to be in direct membrane–membrane contact. C-type lectin receptors on various antigen-presenting cells. For every receptor, the known interacting bacterial species are indicated. Where known, the intracellular signaling motif or associated signaling adaptor molecule is stated. https://www.youtube.com/watch?v=jkNxmTrrZSk Signaling cells secrete signal molecules into the extracellular fluid. The secreted molecules may be carried far afield to act on distant target cells, or they may act as local mediators, affecting only cells in the local environment of the signaling cell. The latter process is called paracrine signaling.  Example paracrine signaling : Allergies White cells Drugs in allergies https://www.youtube.com/watch?v=YS2lipwoMgg&t=14s  Target cells 1. Muscle cell 2. Gland cells 3. Neuron cells Synaptic signaling is performed by neurons that transmit signals electrically or chemically along their axons and release neurotransmitters at synapses, which are often located far away from the neuronal cell body.  A typical vertebrate neuron The arrows indicate the direction in which signals are conveyed. The single axon conducts signals away from the cell body, while the multiple dentrites (and cell body) receive signals from the axons of other neurons. The nerve terminals end on the dentrites or cell body of other neurons or on other cell types such a muscle or gland cells.  The Synapse The synapse is a connection between a neuron and its target cell (which is not necessarily a neuron). The presynaptic element is the synaptic end bulb of the axon where Ca2+ enters the bulb to cause vesicle fusion and neurotransmitter release. The neurotransmitter diffuses across the synaptic cleft to bind to its receptor. The neurotransmitter is cleared from the synapse either by enzymatic degradation, neuronal reuptake. https://www.youtube.com/watch?v=eTYe1CtjJRE  Endocrine cells uses a quite different strategy for signaling over long distances. These secrete their signal molecules, called hormones, into the bloodstream, which carries the molecules far and wide, allowing them to act on target cells that may lie anywhere in the body. Example: Diabetes https://www.youtube.com/watch?v=OImJMiFJ8Qo