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78 Cards in this Set
- Front
- Back
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Between what stages of the cell cycle does dna replication happen?
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Between G1 and G2
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What experiment tells us that dna is semi conservative?
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1958 Meselson and Stahl
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What are the 3 stages of DNA replication?
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initiation, elongation, termination
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What is it call where the two replication complexes meet?
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the termination site
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What does the 3 polymerases do?
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I -repairs DNA and participates in DNA synthesis of lagging strand
II- role in DNA repair III-work horse- leading and lagging strand |
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Who discovered polymerase I?
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Arthur Kornberg
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4 things you should know about DNA pol III?
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1. its a dimer -- 1 protein for each strand
2. the alpha subunit is where replication takes place 3. beta subunit is the sliding clamp 4. gamma puts everything together |
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What type of reaction happens in elongation?
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nucleotidyl group transfer reaction - terminal 3' OH attacks alpha phosphhorous of incoming nucleotide to form new phosphodiester linkage
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What is require for DNA pol III to add nucleotides?
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1. template
2. terminal 3' OH |
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What is the anchor for the other proteins in DNA pol III?
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the Beta clamp - 2 beta subunits associate to form a head to tail dimer that complete surrounds the dna
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What size are the okazaki fragments and how long does it take to make one?
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1000 bp and 1 coded per second
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Why are primers required to start DNA replication?
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Provides the terminal 3' OH
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What puts a primer on? what are they made out of?
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RNA polymerase-which contains primase enzyme which synthesizes short pieces of rna -made out of RNA
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What is the replisome made out of?
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the primosome and DNA pol III
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What joins together the okazaki fragments?
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the action of DNA pol I and DNA ligase
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What are the 3 steps that connects okazaki fragments?
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1. removal of the rna primer by DNA pol I
2. synthesis of replacement dna by nick translation (dna pol 1) 3. sealing of adjacent dna fragments -dna ligase |
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What bonds does DNA ligase form?
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the formation of a phosphodiester linkage between 3' OH and 5' phosphate of adjacent Okazaki fragments
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What is a difference between Euk and pro dna ligase?
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Euk enzymes use atp cosubstrate
Pro dna ligase uses NAD+ as a cosubstrate |
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1. How does ssBinding proteins bind?
2. what do they do to the DNA? |
1. they bind cooperatively
2. they produce an extended relatively inflexible DNA conformation - ideal for DNA replication |
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Which strand is ahead? by how much?
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the leading - about 1000 nucs -about 1 okazaki fragment
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What does processive mean? what protein is processive and why?
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the protein stays on the dna until the replication is complete
-DNA pol III because of sliding beta clamp |
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Which direction is DNA pol III exonuclease activity? DNA pol I?
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III - 3-5
I - 5-3 |
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Dna A-
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origin binding protein in E. Coli - helps control replication by controlling initiation frequency
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Where does termination happen? what protein helps?
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Termination happens at the ter site. Tus (terminator utilization substance) binds to the ter site and inhibits helicase activity
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Why does ddNTPs stop replication? Who invented this method to sequence?
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Because it does not have a 3' OH - Frederick Sanger
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How many DNA polmerases does Euk have? pro?
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euk - 5
pro- 3 |
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What euk structure is similar to the pro beta subunit? how is it different?
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the PCNA -its a trimer instead of a dimer in pro
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How is replication regulated in euk?
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the origin recognition complex -binds to each origin and when phosporlated replication can begin -- ORC is signaled to come off when s phase protein kinase reaches a threshold level
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When does ORC bind to the origin?
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during M and G1
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What is a disadvantage with reverse transcriptase ?
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no proofreading capabilities
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Whats the difference between direct repair and excision repair?
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direct just changes the nucleotide while excision changes the entire base
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What does Dimerization do? what causes it?
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adjacent pyrimidines (T and C) bind to each other - UV light
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How does humans repair pyrimidine dimers?
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excision repair -by dna photolyase-photoreactivation- weird because most organisms can use direct
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What proteins are involved in excision repair?
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endonuclease, polymerase, ligase, and helicase
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What is one of the most common type of dna damage? What enzyme fixes it?
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hydrolytic deamination - C to U. dna glycosylases and repaired by direct repair
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homologous recombination-
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exchange of dna from 1 chrom to another that have closely related sequences (crossing over during meiosis )
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What proteins are needed in recombination and what is their function?
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RecBCD - binds to DNA cleaves 1 strand then unwinds DNA using ATP
Rec A - strand exchange protein RuvA/Ruv B- drives branch migration between 2 dsDNA RuvC- cleaves crossover to resolve holliday junction |
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What does BRCA1 and BRCA2 do?
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required for normal recombination repair of ds breaks in DNA -common mutation in these leads to breast cancer
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Why is recombination needeD?
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1.DNA repair mechanism
2. new gene combinations 3. evolutionary survival |
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gene-
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a dna sequence that is transcribed (includes genes that do not encode protein
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housekeeping genes-
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encode proteins or rna essential for normal activities of the cell --proteins that you always need
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what is the role of small RNA?
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metabolic events - many have catalytic events
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What does the alpha, sigma, and beta subunit of rna pol do in pro?
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alpha- regulate transcription
beta- pol site and dna binding sigma - role in initiation of transcription - helps find the promoter |
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What does dna pol have that rna pol doesnt?
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proofreading
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operon -
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transcription unit in which several genes are often cotranscribed from a single promoter in pro
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Does euk have operons?
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no - usually each gene has its own promoter
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Which way is coding strand written? template?
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Coding strand has almost the same code so written 5-3
template is the template for rna pol so 3-5 |
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Where are consensus sequences found? how do they help regulate transcription?
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upstream from the promoter -- the better the match with the consensus sequence the stronger the promoter
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What is the sigma subunit of rna polmerase required for?
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promoter recognition and formation of the complex
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What directs rna pol to the promoter site?
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the sigma
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What is the orientation of genes?
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5-3
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What would the rna pol do without sigma?
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It would bind the dna nonspecifically -- the sigma makes it search for the promoter for the gene of interest
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What is the rate limiting step in transcription?
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the unwinding of the dna is the rate limiting step- because the unwinding of dna at the initiation site requires a conformation change
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What are the 2 types of transcription termination?
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1. unstable elongation complex
2. rho-dependent termination |
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pause site-
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regions of the gene where the rate of elongation slows down or stops temporarily
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How does rho dependent termination sites work?
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rho binds to ssRNA chain, destabilizing the rna-dna hybrid and terminating transcription --rho then binds to the new rna
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What euk rna pol makes all mRNA?
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RNA pol II
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Which is more complex DNA or RNA pol?
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RNA - binding of RNA pol to promoters requires a number of initiation transcription factors
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When can negatively regulated gene be transcribed?
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When the repressor is removed.
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When can positively regulated genes be transcribed?
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When the activator is present
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Inducers--
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ligands that bind to and INactivate repressors
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Corepressors -
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ligands that bind to and activate repressors
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What does recomination start with?
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Generation of a single stranded DNA with a free 3' end
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Does recomination require ATP?
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YES
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REC A -
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strand exchange protein - essential for homologous(recognizes similarity) recomination and some repair --promotes the formation of the 3 strand intermediate
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REC BCD -
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makes the single stranded dna regions --acts as endonuclease -uses atp
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Ruv A and Ruv B-
RuvAB |
drive branch migration at the holliday junction between 2 dsDNA strand
--RuvAB -powers A and B by ATP hydrolysis |
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Ruv C -
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binds to the holliday junction and cleaves the crossover strand
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Recomination --
Tell the proteins involved in each step 1. Junction Binding 2. Branch migration 3. Resolution |
1. Junction Binding - Rec A
2. Branch migration - RuvB 3. Resolution - Ruv C |
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Without BRCA1 and BRCA2 what can you not do? -- what is most likely messed up?/
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Recomination because you cant seal ds breaks in DNA - most likely ligase protein mutation
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Where does BRCA bind?
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Where you have single and ds dna together - where recombination is trying to take place
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What are 2 exceptions to the central dogma?
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HIV and prions ( protein to protein)
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Can multiple RNA copies be made in transcription at the same time?
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YES both
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Why does the RNA polmerase need to change conformation during initiation?
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needs to change from RPclosed to the RPopen complex RP-RNA pol and promoter
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What helps the RNA pol come of the DNA when strand is complete?
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NUS A and the conformational change of RPc to RPo which causes sigma to dissociate
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What is an example of unstable elongation complex in terminiation?
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a formation of a hairpin stucture -- also fueled by pause sites
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What is another name for mRNA?
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hnRNA
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What percentage of RNA is coded into protein?
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20-40
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