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28 Cards in this Set
- Front
- Back
the basics of replication
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-DNA replicates in a semi-conservative fashion and is composed to two anti-parallel strands
-DNA replication USUALLY proceeds bidirectionally, but it can proceed in one direction too -bidirectional replication implies that there are TWO replication forks (going in opposite direction) for every origin of replication |
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What direction does DNA synthesis occur in? What direction is DNA read in?
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DNA is synthesized 5' --> 3'
the DNA template is read 3' --> 5' |
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how is a strand of DNA lengthened?
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the incoming nucleotide, a deoxynucleoside (dNTP), will lose two phosphates and attach via Hydrogen Bond* linkage to the opposing nucleotide on the template strand (i.e. A pairing with T), and via a Phosphodiester Linkage* to the adjacent nucleotide on the growing DNA strand that it adds onto
USING DNA POLYMERASE |
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Transition state during DNA elongation
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DNA polymerase will form a transition state involving THREE Aspartate residues and TWO Mg2+ ions
the Aspartates are coordinated with Mg2+ which functions to: 1. prepare the attack of 3'OH of the growing DNA chain on the alpha phosphate of the incoming dNTP, and 2. to stabilize the beta- and gamma-phosphates, which will be irreversible broken down into two inorganic phosphates |
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what do DNA Polymerases require?
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both a TEMPLATE to read off of, and a PRIMER to get started
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accuracy of DNA Polymerase
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the rate of mutation is super low
-DNA Polymerase MUST be accurate -has Proofreading ability -only A bound to T, or C bound to G, will fit perfectly into the active site of the polymerase. Any other mismatched pair will signal that somethings wrong. -also has editing function (exonuclease activity) |
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exonuclease activity of DNA polymerase I
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new DNA is synthesized from 5' -->3'
as soon as a mismatch arises, the DNA polymerase will backtrack in the 3' --> 5' direction to remove the mismatched nucleotide, and corrects it with the right one and will move forward again in the 5' --> 3' direction thus, DNA polymerase is capable of moving in both 5' --> 3' and 3' --> 5' directions |
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distinguishing the different classes of DNA polymerases in E. coli
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ALL have 3' --> 5' Exonuclease (proofreading) capabilities
DNA Pol I is the only one with 5' --> 3' Exonuclease activity DNA Pol III has the fastest rate of DNA synthesis |
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Structure of DNA Polymerase III
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THREE beta-clamps (dimer)
ONE clamp loader (pentamer) TWO core units (which carry out the actual DNA polymerase activity) (Note: Helicase is NOT part of the DNA polymerase, but it does interact with it later on to unwind DNA) two beta subunits form the beta clamp |
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Replication: Initiation
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The Origin of Replication is composed of two parts:
1. DNA Unwinding Element (DUE)** A section rich in Adenine-Thymine pairs 2. EIGHT binding sites for DnaA protein Steps: 1. DnaA, with the help of ATP, binds to ALL eight binding sites, creating a torsional strain 2. this strain will cause DNA to be denatured in the DUE region, so the strand separates 3. DnaB (aka HELICASE) binds to the newly opened DUE region with the help of DnaC-ATP 4. since helicase is docked and ready to do its thing, replication begins |
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DUE
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DNA Unwinding Element
three 13-bp sequences in a row at the start of the replication origin AT-rich region: this is important because for replication to occur you need to separate the strands. AT only have 2 hydrogen bonds, whereas CG have 3. So its easier to denature and separate AT sequences and be able to start replication. |
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DNA Gyrase ("DNA Topoisomerase II")
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when the DnaB helicase starts to unwind the DNA, it creates a torsional stress on the DNA strand.
DNA Gyrase relieves this stress |
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primase
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located next to the helicase
synthesizes a short RNA primer to serve as a starting point for replication (this RNA primer will need to be replaced because we ultimately want DNA, not RNA) |
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single stranded binding proteins (SSB)
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stabilize the single-stranded fragments that have not replicated yet
without this, the single-stranded DNA will be targeted for degradation |
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what happens after the RNA primer is laid down?
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DNA POlymerase III* comes in and starts adding on nucleotides to the primer to form Okazaki Fragments
nucleotides will be added on until it meets the RNA primer that was part of the PREVIOUS Okazaki fragment at this point, the primes will make another RNA primer, and the cycle continues. |
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DNA Polymerase and the Lagging Strand
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DNA polymerase will interact/bind with the helicase, and move together in the direction of the opening replication fork
the lagging strand is looped around and then fed into the Beta-Clamp and core |
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DNA Polymerase beta-clamps
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there are 3 beta-clamps on ONE DNA Polymerase Complex
-the beta-clamp associated with the leading strand will always stay there -the beta-clamp in the middle that is in the clamp-loading pentameric subunit will essentially receive each new primer that is made, and ultimately slide over to the lagging strand's beta-clamp positions and clamp down on the DNA so it can continue adding nucleotides -the old beta-clamp gets discarded |
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how do we replace the okazaki fragments?
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at some point, the RNA primers of the okazaki fragments will need to be replaced by the exonuclease function of the DNA Polymerase I*
-this exonuclease activity is a bit different from the 3' --> 5' exonuclease (which functions in proofreading and removal of mismatched base pairs) in that it removes nucleotides from the RNA primer and replaces them with DNA nucleotides in the 5' --> 3' direction*** |
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The DNA Polymerase III Clamp Loader
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there are 5 subunits of the clamp-loading complex
the complex binds 3 ATP, and also binds to a dimeric beta-clamp the binding to the beta clamp forces the beta-clamp to open at one of its two subunit interfaces, allowing it to interact with the DNA hydrolysis of the bound ATP allows the beta-clamp to close again around the DNA |
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DNA Ligase
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seals fragments with the help of ATP
creates phosphodiester bonds between the 2 nucleotides |
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Replication Termination
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In the case of E. coli DNA (circular chromosome), the bidirectionally of an origin of replication means that the two replication forks will eventually meet each other
when that happens, there will be Termination Sequences (Ter) that are bound by Terminus Utilization Sequences (Tus). WHen this complex forms, a so-called "polymerase trap" occurs, and you'll end up with "Catenated" Chromosomes (linked together) then DNA Topoisomerase IV comes in, and basically cuts one of the DNA strands so that the other replicated DNA strand can detach, ultimately forming two independent chromosomes |
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Topoisomerase IV
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. Replication of the DNA separating opposing replication forks leaves the completed chromosomes joined as catenanes, or topologically interlinked circles. The circles are not covalently linked, but because they are interwound and each is covalently closed, they cannot be separated—except by the action of topoisomerases
comes in during termination and targets Catenated chromosomes in prokaryotes cuts one of the DNA strands so that the other replicated DNA strand can detach, ultimately forming two independent chromosomes |
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Eukaryotic Replication
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same mechanisms, but some differences:
1. There are multiple Origins of Replication 2. The Origin Recognition Complex found in euks is analogous to the DnaA that binds to the specific origin replication sites. Both DnaA and ORC will require ATP 3. The Minichromosome Maintenance Protein (MCM) is analogous to the DnaB helicase found in proks 4. Together, the ORC and MCM make up the Pre-Replicative Complex. Once this complex forms, it becomes LICENSED. 5. DNA Polymerase delta is analogous to the bacterial DNA Polymerase III |
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Assembly of a Pre-Replicative Complex at a Eukaryotic Replication Origin
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**key step in the initiation of replication in eukaryotes**
(analogous to the loading of the bacterial DnaB helicase by DnaC protein) loading of the MCM helicase complex onto the DNA forms the pre-replicative complex (pre-RC) once this complex forms it becomes LICENSED, which basically means its all ready to go if it passes ONE last checkpoint that is dependent on cyclin-dependent CDK (Cell Division Kinases)*** |
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Eukaryotic DNA Polymreases
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DNA Polymerase delta is analogous to the bacterial DNA Polymerase III
the DNA Polymerase delta will be most prevalent in the S phase of the cell cycle, when DNA replication occurs |
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why is regulation so important in the initiation step of eukaryotes?
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for eukaryotes especially, you want to make sure that replication occurs only ONCE in each cell cycle
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Chromatin
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more condensed chromatin will replicate later in the S phase**
(at the 6-8 hour mark) compared to less dense chromatin that is replicated first (0-2 hour mark) |
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telomeres
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telomeres are nucleotide repeat sequences at the tips of the chromosome
they serve to prevent the DNA from having a recognizable "end", and thats why theres a little LOOPED structure where the DNA folds back onto itself so that the DNA can still be continuous and not just end abruptly at the tip **the enzyme Telomerase that catalyzes the addition of nucleotides uses an RNA (not DNA!!!!) template* in order to synthesize the growing telomere strand of DNA telomeres shorten naturally with constant cell divisions and ultimately with age, so telomerase tries to help out to extend the ends of the chromosome |