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136 Cards in this Set
- Front
- Back
Where does replication begin along a chromosome or plasmid?
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origin of replication
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Whose experiments lead to the finding that the replication fork was bi-directional?
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Cairns
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In Cairns experiment he observed tritium labeled ___________ was incorporated into ______ loops durind DNA replication in bacteria.
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thymidine, two
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In Cairns "pulse-chase" experiments he revealed that ___________ of the replication fork was ____________.
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movement, bi-directional
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Which image is correct?
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the bi-directional one
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How would you perform a "pulse chase" experiment?
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In biochemistry and molecular biology, a pulse-chase analysis is a method for examining a cellular process occurring over time by successively exposing the cells to a labeled compound (pulse) and then to the same compound in an unlabeled form (chase). Radioactivity is a commonly used label.
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Whose experiments concluded that chromosomes or plasmids have an origin of replication?
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Inman's experiments
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Inman's experiments used denaturation loops as reference marks on plasmids called ____________ ___________.
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denaturation mapping
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Inman concluded that for a given plasmid, replication ____________ at the same place every time. What is this called?
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originated, point of origin of replication
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Fill in the blanks.
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Fill in the blanks.
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DNA replication requires an unpaired _________ to copy, or what is called a __________ strand.
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template, parent
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DNA replication requires a primer to provide a free ______.
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3' OH
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DNA replication can add only onto the 3' OH so the strand always grows in a ____________ direction.
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5'--->3'
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Draw the DNA polymerase reaction mechanism. Include the amino acids involved and the metal ions.
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The primers used to start DNA synthesis are created by the enzyme ______________>
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primase
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Primers created from primase are RNA/DNA?
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RNA
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The primers used to start DNA synthesis are small ____ fragments, which are ________ nucleotides in length.
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RNA, 1-60
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Primase primers are ultimately (in the end of DNA synthesis) ____________, and replaced with _______.
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hydorlyzed, DNA
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Which replication strand needs multiple primers?
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Lagging strand
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In E. coli the error rate in DNA synthesis is about 1 per __________ nucleotides incorporated into DNA.
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10^9-10^10
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The level of fidelity of E. coli DNA polymerase is insured by the ability of the enzyme to discriminate between __________ and __________ base pairing.
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correct, incorrect
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This question is for E. coli DNA polymerase.
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When a nuclease hydrolyzes DNA/RNA it can yield what kind of ends?
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Exonucleases hydrolyze from ____________________ of the nucleic strand.
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one end or the other
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Can an exonuclease hydrolyze from either end of the nucleic strand?
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yes
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Exonucleases act on _________ strand and ___________ strand DNA/RNA.
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single, double
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Endonucleases hydrolyze from ___________ the strand.
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within
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Restriction __________ are used in cloning to cut the dsDNA.
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endonucleases
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DNA polymerases have nuclease activities for what functions?
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proofreading function, repair function
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The proofreading function of DNA polymerases does what?
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corrects immediate errors in replication by acting as a 3'--->5' exonuclease
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The repair function of DNA polymerases does what?
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corrects errors that lie ahead of the replication fork acting as a 5'----->3' exonuclease.
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The proofreading function and the repair function use what kind of nuclease?
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exonuclease
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What strand direction does the proofreading function work?
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3'--->5'
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What strand direction does the repair function work?
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5'---->3'
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Define nick translation?
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DNA poly1 is a ___________ polypeptide?
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single
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DNA helicase use the energy derived from _____ hydrolysis to _______ and ________ alond dsDNA.
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ATP, unwind, translocate
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The DnaB helicase of E. coli is a __________ structure with each subunit containing its own _______ domain.
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homohexameric, ATPase
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DNA helicases have a ________ effect, one subunit _______ when bound to ATP, while the next subunit _________ when bound to ADP.
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rotary
grabs pulls |
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The rotary effect of a DNA helicase pushes one strand thru the _______, and one strand ________ ________ _______.
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center of the enzyme, around the outside
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Coordination between adjacent subunits causes an ATP _________ cycle that pulls ssDNA through the __________ of the enzyme structure.
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hydrolysis, hole in the center
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All enzymes and proteins associated with DNA/RNA replication is called the?
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Replisome
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This man isolated DNA pol1 from E.coli in 1955.
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Arthur Kornberg
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After complete characterization of DNA pol1 it was realized or suggested that DNApol1 __________ be the only DNA polymerase involved in replication.
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can not
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What were three characteristics of DNApol1 that Arthur Kornberg stated that led to his suggestion of more than one DNA polymerase?
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What does high and low processivity mean?
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low processivity means that the polymerase frequently falls off and must rebind to the nucleic acid.
high processivity is a result of not losing this transition time of being off and on the nucleic acid |
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DNA pol3 has how many subunits?
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10 different types of subunits
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DNApol3 is considered fast due to high ____________.
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processivity
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DNApol3 lacks a nuclease activity, name it.
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It can proofread, but has no nick repair
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_____________ is the principle replication DNA polymerase in E.coli.
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DNApol3
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Mutations in DNApol1 are __________, but mutations in DNApol3 are __________.
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unlethal, lethal
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What is the functions of the core subunits of DNApol3?
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polymerization of the nucleotides, 3'-->5' proofreading exonuclease, stabilization of the proofreading subunit
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What is DnaB?
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helicase for DNApol3
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What is the function of the clamp loading complex of DNApol3?
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Stable template binding, core enzyme dimerization, clamp loader, clamp opener
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Compare and contrast DNApol1 with DNApol3.
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What is a Klenow fragment?
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The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin. First reported in 1970,[1] it retains the 5'-3' polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity.
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___________ experiments used denaturation mapping and concluded that for a given plasmid, the replication originated at _________________. He then call this ________________.
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Inman, the same place every time, point of origin
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In E.coli the specific site of the origin is _______. This site is a ______bp sequence.
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oriC, 245
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___________ binds to the oriC DNA sequence causing the DUE site to separate.
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DnaA-ATP
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DnaA-ATP bind to the oriC DNA sequence causing an adjacent 45 bp to separate. What is the site where the separation occurs called?
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DUE DNA unwinding element
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DnaA-ATP binding at the oriC site occurs only when both strand of ______ sequences are fully ___________________.
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GATC, methylated at N6 of adenine.
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DnaA-ATP binds to R- and I- sites in the origin and induces ___________________.
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supercoiling
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____________ binds to R- and I- sites in the origin and induces supercoiling.
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DnaA-ATP
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What happens as a result of DnaA-ATP inducing supercoiling in the oriC site?
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The DUE (DNA unwinding element) separates to relieve the torsional stress.
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What enzyme binds to and opens the DnaB helicase and loads one enzyme onto each of the separated DUE strands?
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DnaC-ATP
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What enzyme generates an RNA primer on each separated strand at the DUE?
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DnaG primase
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Which enzyme binds to the loaded DnaB helicases?
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DNA polymeraseIII holoenzyme
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DnaA is active when bound to ______ and inactive when bound to _______.
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ATP, ADP
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The full methylation of the GATC sequences at oriC is called?
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DAM methylation
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Where on the GATC sequences of oriC does methylatio happen?
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N6 of adenine
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___________ is the only phase of DNA replication that is thought to be regulated.
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Initiation
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___________ levels are regulated throughout the cell cycle and peak around the start of replication
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DnaA-ATP
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DnaA-ATP levels are regulated throughout the cellc ycle and peak around the ___________________.
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start of replication
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Loading of the DNA polymerase3 complex stimulates hydrolysis of ___________ to ___________.
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DnaA-ATP to DnaA-ADP
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Hydrolysis of DnaA-ATP results in the ______________________. (long answer)
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disassembly of the DnaA complex at the origin
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What event stimulates the hydrolysis of DnaA-ATP?
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the loading of the DNApol3 holoenzyme
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The conversion of DnaA-ADP back to DnaA-ATP takes a lot of time resulting in?
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a sufficient lag to allow replication to completeR
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Replication creates new sites for ___________ binding that lie outside of the origin, effectively titrating away ____________.
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DnaA-ATP
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Conversion of the __________________ strands that result from replication back to fully methylated strand takes time.
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hemimethylated
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The left over nick is repaired by?
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DNA ligase
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DNA ligase catalyzes formation of __________ bond by using _______. (only in E.coli)
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phosphodiester, NAD+
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Name this molecule.
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NAD+
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