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127 Cards in this Set
- Front
- Back
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What is the ratio of phosphate to sugar?
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1:1
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What is the ratio of C to G?
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1:1
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What is the ratio of A to T?
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1:1
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When you’re looking at the diffraction of DNA, and you have a new strand with a longer helical pitch and a longer distance between bases on the strand, but the width is unchanged, then how will the diffraction affect the diffraction of the new form?
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The distance between the two dark overlap places will become shorter. The distance between the short horizontal stripes will be shorter.
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If the two lattices are close, what happens to the diffraction interference pattern?
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It becomes wider
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What does it mean that “the sequence of bases on a single chain doesn’t appear to be restricted in any way”?
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The bases are chosen arbitrarily and you can make any sequence
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What odes it mean that “it has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”?
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The structure suggests how it replicates. Hydrogen bonds can be “easily” figured out from there.
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Nucleoside
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Base and ribose
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Deoxynucleoside
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Base and deoxyribose
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Nucleotide
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Nucleoside + phosphate
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Purine
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A and G
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Pyrimidine
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C, U, and T
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A, B or Z. What form can RNA take?
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A
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A, B or Z. What form is important for DNA?
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B form
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What does a C2’ endo sugar select for?
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B form of DNA structure
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What does a C3’ endo sugar select for?
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A form of DNA structure
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What is the rise per base pair?
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3.4 A
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What is the base pairs per turn of helix?
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10.4
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What is the pitch per turn of helix?
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35.4 A
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In one turn of the helix, how much does it raise in height?
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35.4 A. (Base pairs per turn * rise in base pair)
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What are the typical motifs of DNA binding proteins?
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Helix turn helix, Leucine zipper, Zinc finger
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What do the majority of DNA binding proteins recognize major grooves with?
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Alpha helixes.
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How many base pairs can a single alpha helix read?
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3-4 on avg
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Do DNA binding proteins need to unwind dsDNA to read the sequence? Why?
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No. Because it will interact with the major groove and react with specific charged atoms on the pairs. They are specific in shape (It’s a very distinctive shape)
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DNA base pairs every how many Angstroms?
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3.4
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The amino acid side chains are facing which direction of the helix?
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Outiside
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Does allosteric regulation necessarily change the structure of each DNA binding domain?
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No
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What do inducers change?
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The arrangement, being the spacing or angle, of binding domains to fit 2 binding sites on DNA
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What kind of DNA binding activity do type 2 restriction enzymes have?
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Non-specific
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What mechanism vends dsDNA structure to expose cutting sites?
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Induced fit
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Are restriction enzymes working dimers?
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Yes
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What do restriction enzymes target?
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Inverted repeats.
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Does a type 2 restriction enzyme have a helix turn helix motif?
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No.
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What groove is used to be probed for base pairing?
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Major groove.
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How can you describe DNA binding proteins?
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Sequence-specific
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Regulation of DNA binding controls?
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Dimerization/conformation
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What do amino acid side chains of alpha helix make?
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Sequence specific DNA binding proteins
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Are transcriptional regulations occurring at the protein-DNA binding step in Eukaryotes?
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HAHAHA. no.
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Where are transcriptional regulations occurring at the protein-DNA bidning step?
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Protein-protein interaction
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Can RNA make a stable base pair at the B form? Why?
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No. There will be steric hindrance with phosphate
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DNA or RNA: Whhich has non-watson crick base pairs?
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RNA
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What pairs with I?
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A, C, U
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What bases cause wobble?
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GU and I
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What does it mean that a base wobbles
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There are base pairings that are allowed that otherwise wouldn’t be.
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Define semi-conservative replication
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each turn of replication we get 1 old and 1 new strand
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Can each strand of DNA be a template for the other?
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YES
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Which end does DNA polymerase extend?
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3’
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What enzyme do we use in vivo to remove a phosphate so we can incorporate new bases?
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Pyrophosphatase
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Is the extension of DNA reversible?
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Nope!
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Describe how a new base is added?
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At the 3’ end a Nucleoside tri-phosphate will come in and interact with the strand. Pyrophosphatase will remove 2 phosphate, and the remaining phosphate will be used to make the phosphodiester bond.
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What does DNA polymerase need?
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Template DNA, RNA/DNA primer, and a pre-existing 3’ end
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What does DNA polymerase look like?
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Right hand. It has “fingers” a “palm” a “thumb” and a “wrist”
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In DNA polymerase, what is the Palm made of?
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Beta sheets
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In DNA polymerase, what are the fingers and thumb made of?
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Alpha helixes
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What happens every time a finger closes?
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A new nucleotide is encorporated into the primer
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What is the function of the “fingers”
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Moves in to correctly position thetri-phosphate. Moves out to release it when the nucleotide is added |
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What is the function of 2 Mg2+ on 2 Asp
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they are used as a bridge by the Asp. They willactivate the 3’ hydroxyl group resulting in a reduced association between theoxygen and hydrogen facilitating its attack on the phsphoryl group of the dNTPsubstrate. |
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Describe proofreading in DNA polymerase
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the polymerase will change confirmation and transfer it to asecond active site on the polymerase where it will be removed. Then it flipsback to it’s original confirmation. |
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Where is right pairing check from?
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Outside of pairing
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What does the palm “look at” to recognize the paired bases? What is the problem with this method?
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Two H bond donors. But two H bond acceptors are present in the minor groove of any base pair.
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Is proofreading a reverse reaction of DNA polymerase?
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No
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Is there a clear conformational change in proof reading?
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Yes
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What is required in proof reading?
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A distinct site
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How much energy is required in proof reading?
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None.
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How much does proof reading increase the fidelity?
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100
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If a polymerase adds the next polymerase without deleting the mistake, can the mistake still be repaired by the polymerase?
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Nope.
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If a polymerase can no longer fix a mismatch, how does it get fixed?
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Mismatch repair system
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What is the overall fidelity of DNA polymerase?
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10^-10
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How are most wrong nucleotides deleted?
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Exonuclease activity of polymerase
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Can a mismatched 3’OH interact with a palm correctly?
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Nope
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How does exonuclease work?
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Mismatched 3’OH interacts with the palm wrong, it slows down the nucleotide addition, polymerase changes confermation and unwinds the bp a few steps and moves the screws up to the exonuclease domain. The 3’ end is removed in 1 nt.
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Replisome is?
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Entire enzyme replication complex
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What is the point of a sliding clamp?
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It helps DNA polymerase’s processivity
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Why is a sliding clamp considered stable?
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It has a ring structure so it cannot fall off the DNA
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Is the sliding clamp conserved?
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Yes
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What is the structure of a sliding clamp?
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A dimer or trimer
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What is a clamp loader
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an multi-subunit protein that binds ATP and binds the ring and slides open the ring so DNA can slide through.
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How does a clamp loader load the sliding clamp?
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ATP hydrolysis energy. Loaded on the 3’ end of primer.
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Does a clamp loader work with a DNA/RNA hybrid?
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Yes
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What does a clamp loader need?
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3’ end
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Does a clamp loader care if it is dealing with DNA or RNA?
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Nope.
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What does a clamp need?
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A loader
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What is the typical structure of a helicase in DNA?
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Heximeric ring
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What direction does a helicase move?
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5’ to 3’ direction
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What does helicase require?
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DNA double strand. Not blunt ends. (5’ is the bottom strand if it’s linear. If it’s Y then 5’ is on top)
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How does a T7 helicase work?
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2 DNA binding loops extend toward the center, each subunit has an ATP binding domain. They are at different phases of ATP-binding, hydrolysis and release. ATP binding of free subunit pushes other subuites to progress to the next phases. In otherwords, DNA is relayed into a hexamer, hydrolysis occurs, it’s unwound, and DNA is released.
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What is helicase associated with on a replication fork?
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Primase
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Do we need DNA polymerase for the leading strand to keep synthesizing?
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No
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Is the interaction with helicase and primase permanent?
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Oh I certainly hope not.
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Does primase need a primer?
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No
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What happens when a primer meets the single stranded RNA?
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It needs to be quickly recognized by the clamp. It’s very unstable.
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How does DNA polymerase 3 holoenzyme look?
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Praying mantis face. The compound eyes are the core enzymes of poly 3. The mouth is the sliding clamp. The mandibles are the beta sliding clamp. The piece above it is the gamma clamp loader.
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In DNA Poly 3 holoenzyme, is the core enzyme for the lagging or leading strand?
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Either or.
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Where is DNA replication in bacteria initiated?
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OriC
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What is DNA replication in bacteria triggered by?
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An initiator protein that is NOT polymerase.
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How many initiator proteins do we have?
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3. A, B, and C
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What does the initiator do in bacterial DNA replication?
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It associates at OriC and unwinds about 10-15 nt. And then recruits helicase
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What does topoisomerase do?
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It fixes any over-winding in the chromosome. It breaks the DNA strand and release over or underwinding and reseals DNA. (It also allows a segment of DNA through the break, obviously). It uses tyrosine residues to form a covalent intermediate with polynucleotides
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How many types of topoisomerase are there?
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2
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What does topoisomerase 1 do?
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Catalyze relaxation of supercoiled DNA. It does not require ATP. One end of the strand becomes free from the protein and the other end can rotate to make the most thermodynamically stable confirmation. Making or releasing intermediate is almost equally possibly. It can go back and forth. The number of times it can rotate depends on the type.
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What does topoisomerase 2 do?
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Uses free energy from ATP hydrolysis and changes the linking number by 2. It requires ATP. There are two DNA binding sites, and a DNA cleavage site and an active tyrosine site. One will cleave one end of the DNA one will cleave the other. Tyrosine cleaves and transfers phosphodiester bond. After cleavage, the structure will change. The two ends are held by enzymes. N and C terminus open and close in coordinated manner to pass DNA through. DNA binds in C gate. Goes in the direction of N gate to C gate. So basically: Strand 1 comes in, split, 2nd comes in, splits. First can come back together and then one can leave through C gate.
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Which topoisomerase can do decatenation?
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2
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Define linking number
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Over or underwounding; where it crosses over itself
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What should we know structurally about type 1 topoisomerase?
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Active site surrounds DNA and contains the 1 active tyrosine side chain that has OH group at the tip and OH group when it binds and cleaves DNA, goes into DNA and tyrosine receives phosphodiester bond
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What are the required proteins for DNA polymerase being loaded onto DNA?
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DnaA, DnaB, DnaC
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What is DnaA?
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The initiator
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What is DnaB?
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Helicase
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What is DnaC?
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Loader
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Describe initiation
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DnaA partially unwinds origin. Dna BC is loaded via DnaA-C interaction. DNA slides open. DnaC opens ring of DnaB and loads it on the ssDNA. helicase unwinds dsDNA and polymerase 3 clamp spontaneously attaches
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In bi-directional DNA replication what moves: DNA poly or DNA?
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DNA.
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Where is the replication mechanism and polymerase located in bacteria?
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At the center of the cell in the fork. It stays there and everything else is separated and moved to opposing sides of the cell.
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What are the 3 polymerases in Eukaryotes?
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Alpha, delta, epsilon.
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What is the point of polymerase epsilon?
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It does the leading strand
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What is the point of polymerase delta?
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It does the lagging strand
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What is the point of polymerase alpha?
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It is associated with the primase
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What is the name of the helicase in eukaryotes?
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MCM complex
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What is the name of the clamp loader in eukaryotes?
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RFC
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What is the name of the sliding clamp in Eukaryotes?
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PCNA
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Describe initiation of eukaryotic DNA replication
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ORC is recruited. Licensing: your part of the strand has yet to replicate. Cdc6 recurits MCM and associates it to DNA. MCM activation.
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When will CDC6 inactivate?
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When it assembles and it is phosphorylated. |
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What happens when CDC6 or CDT1 are phosphorylated?
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They’re destroyed. New licensing factors need to be made.
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Where is the pre-replication complex made in the cell cycle?
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G1
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What does MCM require for activation?
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S-CDK
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What part of replication occurs in the S phase?
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MCM activation, replication proteins are recruited to the orgin (all of the poly’s alpha-primase, PCNA, and RFC). Phosphorylated cdc6 and cdt1.
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Do we know how the primase primes and recognizes primers and clamp loader?
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Nope.
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What are telomeres and what do they do?
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Telomeres are the repeats at the end of the chromosome which is extended and maintained by telomerase
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What’s telomerase and what’s important?
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It’s basically reverse transcriptase with a built in RNA template. Also it is related to aging.
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