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Purdue BIOL 10200 - Enzymes, Checkpoints and Mitosis Notes

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Enzymes:Before I get into enzymes, let me clarify some terminology● Kinases are enzymes that phosphorylate other molecules● Phosphorylate is just a phrase that refers to “activating” different cells in the cell cycle byadding a phosphate groupThere are a few enzymes we need to look at to help with the cell cycle. I covered these in myChapter 10 Bio Notes, but they are:● Cyclins (named so because they are regulatory proteins in a cyclical fashion) thatincrease going into mitosis and degrade coming out of mitosis.● Cyclin-dependent kinases (Cdks) are a type of kinase that need cyclin● Complexes of cdk (the enzyme) and cyclins (the regulator) function to trigger differentstages of cell cycle by phosphorylating other molecules to activate them● Cyclins + cyclin-dependent kinase. This means that when cylins increase in quantity,MPF does tooI should point out, MPF is just one example of cyclins + Cdks that occurs during the end of the Sphase. The anaphase-promoting complex is another one, and it receives signals to make surethe spindles are at the right spotCdk + cyclin complexes can phosphorylate chromosomal proteins to initiate Mitosis; it canphosphorylate microtubule-associated proteins to activate the spindle; and it can phosphorylatean enzyme that degraded cyclin, which leads to cyclin concentrations decreasingCheckpoints:Checkpoints are points in the cell cycle where the condition of the cell is assessed todetermine if it should move to the next phase. If it fails the test, cell division is halted for repairCheckpoints are sensitive to internal and external factorsThe checkpoints are:1. G1/S (aka START): the start of the restriction point, the commitment to divide and beginDNA replication. Growth factors are important here2. G2/M: the checkpoint is where MPF comes in to make sure the replication is completed3. Anaphase: the checkpoint where chromosomes separate during anaphase. Onceseparated they must segregate into new cells. The anaphase-promoting complex(above) makes sure hereThese are irreversible stepsWe need to check to make sure everything is good before DNA replication because if the DNAdivides with incompletely replicated DNA, it would probably be lethal. If it divides with damagedDNA, it could be lethal or cancerous. Therefore, the cell assesses itself with checkpointsDNA and Bases:In 1869, Swiss chemist Friedrich Miescher discovered a white substance from the nuclei thatwas slightly acidic, so he called it nucleic acid. The two main kinds of nucleic acids are DNAand RNA, and nucleic acids are made of nucleotides.DNA contains 3 main parts1. A 5-carbon sugar2. A phosphate group3. A nitrogenous (nitrogen-containing) base.The big twist of DNA is called the major groove, and it is where the backbones of the twostrands are far apart. The smaller portion of the DNA is called the minor groove, and it is wherethe backbones are close together and the bases are connecting.The bases are:● Large Purines: A and G● Small Pyrimidines: C, T (DNA only) and U (RNA only)A and T form two hydrogen bonds (and are therefore easier to melt)G and C form three hydrogen bonds (and are therefore harder to melt)We refer to the five carbons on a 5-carbon sugar with the prime (‘) symbol. In DNA, thephosphate connects to the 5’ carbon atom and the hydroxyl (-OH) connects to the 3’The 5’ end has a phosphate and the 3’ end has the sugar.The phosphodiester bond links the hydroxyl of the 3’ atom to the phosphate of the 5’ atom. Itis called that because the phosphate is now linked by way of ester bonds. This forms long acidpolymersErwin Chargaff discovered that DNA isn’t constant. It’s complex, and it varies a lot, but italways has the same amount of A and T, and it always has the same amount of C and G.Chargaff’s rule states C=G and A=T (or A=U for RNA)The two strands of the helix are made of repeating sugar and phosphate units, which we call thephosphodiester backboneA single phosphodiester strand has inherent polarity because the 3’ side ends with OH and the5’ side ends with PO4. Thus, they are referred to as having 3-to-5 polarity or 5-to-3 polarity.DNA strands are antiparallel and run in opposite directions (one running 3-5, and the otherrunning 5-3).The H bonds between bases on the interior hold each individual strand together. The twostrands are then connected by “ladders” of covalent bondsThe new DNA strand is copied by pairing of complementary bases using the old strand as atemplate. (AKA semiconservative replication.)1. Two sides of the double helix have to separate2. Each strand is a template for a new strand with complementary bases (A-T, C-G)3. New nucleotides only added to existing nucleotides4. Strands grow from 5’ end to 3’ end5. Enzymes link nucleotides at their sugar-phosphate groupsThis will require a lot of enzymes1. Primase2. Helicase3. DNA polymerase4. LigaseThe three stages are:1. Initiation process begins2. Elongation majority of building blocks are added3. Termination process endsDNA Replication Phases:A large protein complex--the replication complex/replisome--interacts with template strandsAll chromosomes have an origin of replication which is a base sequence in DNA wherereplication begins.The proteins in the replisome bind to the DNA sequence in the origin.*Side Note: A DNA molecule gets copied at several points simultaneously, and the copied DNAgoes in lots of different directions. But for the purposes of this course, we only ever focus on one“snapshot” (one copied strand) going in one direction*DNA replication begins with a short primer, a starter strand of RNA made by the enzymeprimase. The primer is complementary to the DNA template (one of the strands that split) andthe DNA polymerases (an enzyme that accelerates this reaction), and the primer attracts thepolymerase to itThis process occurs from the 5’ end to the 3’ endDNA unwinds at the replication fork, where the unwound DNA is exposed to bases with the aidof the enzyme helicase● One new strand, the leading strand, is oriented to grow at its 3’ end as the form opens● One strand, the lagging strand, is oriented so that it’s exposed 3’ end gets farther fromthe forkAfter they unwind, proteins prevent the DNA template strands from reforming via hydrogenbonds. This allows elongation of the leading and lagging strands without the template DNAcoming back togetherDNA polymerases work very fast. They are processive, meaning they can catalyze


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