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45 Cards in this Set

  • Front
  • Back
During replication, DNA polymerase initially incorporates 1 wrong nucleotide every ~106bp. However, the overall accuracy is much higher (1 error per 109bp). Why?
Repair mechanisms are present to decrease the incidence of an error during replication
In which direction is DNA being synthesized biosynthetically?
5’ – 3’ direction. If replication occurred 3’ – 5’, could not proofread because no PPP.
Double-stranded DNA is fairly stable and strand separation usually requires high energy. Instead of high temperature, how do organisms cope with this “problem” during replication?
Through the use of enzymes
What is an Okazaki fragment and in which process does it play a role?
An Okazaki fragment is a short fragment of DNA created on the 3’ – 5’ lagging strand during DNA replication. It’s role is to allow the lagging strand to be replicated in the 5’ – 3’ direction.
Name three activities of DNA polymerases.
Polymerase (gap filling)
5’ – 3’ exonuclease activity (proofreading)
3’ – 5’ exonuclease activity ( primer removal, RNAse H)
Name a process in which DNA ligase plays a role.
Ligation of Okazaki fragments
Name a process in which DNA topoisomerase is important.
Untwisting of parental strands in DNA replication
Which functional group of DNA ligases and topoisomerases is thought to play a catalytic role?
Phenolic hydroxyl group on the Tyr residue.
What is the importance of the 5’ → 3’ exonuclease activity of DNA polymerase I in E. coli?
Primer removal, RNAse H activity
What is the difference between processive and distributive enzymes and name an example for each.
Processive- carries out multiple nucleotide additions before disassociating Ex. Pol III
Distributive- readily dissociates from DNA Ex. Pol I
Does DNA synthesis occur in one or two directions from the ori?
In both directions from the ori.
Drugs that inhibit DNA synthesis usually cause cell cycle arrest at which phase?
What are telomeres?
TTAGGG repeats added to the ends of linear chromosomes to protect the ends from shortening and losing genetic information.
Why is it advantageous that thymine rather than uracil is a DNA base?
If U was used instead of T in DNA, it would increase the risk of mutation. C can easily be oxidized to form U. If this occured, then U would base pair with A instead of C with G which would result in unrecognized errors in transcription.
Which promoter sequences are being recognized by bacterial RNA polymerase?
-35 region and the Pribnow Box
How can promoter sequences be identified?
The -35 region and Pribnow box are both highly conserved regions that the promoters can recognize.
Where does the σ factor play a role?
Specific σ factors recognize classes of genes in Prokaryotes. Along with a Polymerase they bind to the promoter to initiate transcription.
Name a characteristic of high promoter efficiency.
Several RNS polymerases can initiate transcription simultaneously.
How is it ensured that transcription occurs in one direction?
The -35 region is asymmetrical and the enzyme can recognize the direction of transcription.
What is a difference between DNA and RNA polymerase with respect to the necessity for primers?
DNA requires a primer, RNA does not require a primer.
Which nucleoside analog do you expect would inhibit elongation: 3’-deoxyadenosine or 5’-deoxyadenosine and why?
3’- deoxyadenosine because it is missing the PPP (triphosphate)
Which process do intercalating chemotherapeutics interfere with?
They interfere with replication and transcription.
What is the basis for ρ-independent termination of transcription in prokaryotes?
GC rich palindrome upstream of 6-7 U (A on template DNA). RNA forms a stem-loop secondary structure which results in termination.
Would the use of ITP instead of GTP during in vitro transcription increase, decrease or not affect termination efficiency?
ITP would diminish termination efficiency because I:C pairs are weaker than G:C pairs (2 vs. 3 H bonds)
What is the TATA box?
Promoter sequence for transcription in Eukaryotes.
What are common characteristics of transcription factors?
Contain both DNA recognition helix and protein interaction domains
 What is a Holliday junction and in which process is it thought to be an intermediate?
o Homologous recombination
o Holliday junction formed from 4 DNA strands
o Forms Holliday intermediate
 What is a heteroduplex in homologous recombination?
o Regions where one strand was from one duplex, the other from the homologous duplex; caused by reciprocal inversion
 How can you recognize a transposon in the genome?
o Insertion sequence has inverted repeats at both ends
o Insertion sequences are flanked by direct repeats in the same orientation in target sequence
 Name different types of DNA repair processes.
o Direct repair
 Cyclobutane thymine dimmer photoreactivation
• Formed by [2+2] cycloaddition
• Photolyase binds
• Light absorption (300-500nm)  cleavage
• Photolyase dissociates
• Demethylation
 Demethylation- alkyltransferases (methyl guanine methyl transferase) reverse this process
o Excision Repair
 Base Excision Repair
• Part of DNA excised by DNA glycosylase
• Removal of damaged base by specific glycosidases
• Repair of AP sites left after removal of damaged base (or by spontaneous hydrolysis);
o AP endonucleases & lyases excise abasic sugar
o Gap filling (DNA polymerase, pol 
o Ligation (DNA ligase)
 Nucleotide excision repair
• Excises several nucleotides
• Excision repair of photodimers
• Endonucleases excise
• Gap filling by DNA polymerase
• Ligation
o Special Cases of Nucleotide Excision Repair
 Transcription coupled repair- only occurs if you need proteins around
• Lesions also block transcription
• Transcribed regions are repaired faster
 Mismatch repair
• If wrong base is incorporated (undamaged)
 What is the importance of DNA methylation in DNA repair?
o How to recognize newly synthesized strand (and not “repair” parental strand)?
 A: Look at methylation pattern! Higher degree of methylation in parental strand.
 Sometimes damaged DNA is not repaired yet by direct or excision repair when the replication complex approaches a lesion. Which repair mechanism(s) can come into play?
o Translesion synthesis, recombination repair by daughter strand gap repair, double-strand break reapir, or repair of replication forks
 What are bypass polymerases, what statement can you make about their processivity?
o Bypass polymerases are only induced upon extensive DNA damage (SOS response) and readily dissociates from DNA (distributive)
 Does coupled transcription-translation occur in prokaryotes eukaryotes, or both?
o prokaryotes
 What statement can you make regarding the degree of RNA modification in prokaryotes versus eukaryotes?
o More dramatic
 Name features of eukaryotic mRNA.
o 5’-terminal cap
o 3’-terminal poly(A) tail
o Methylated internal variants
o Splice variants
 Name 3 unique structural features of tRNA.
o Nucleosides are highly modified
o Anticodon
o 3’-CCA acceptor arm for aminoacylation
o many non-Watson-Crick base pairs
 What is splicing?
o excision of intron sequences
o Creation of several splice variants possible ie. Exon between two introns-> modified functionality ie. Depending on cell type
o Increased protein diversity relative to prediction from genome, more variability for organism
o Other ways to generate protein diversity: posttranslational modifications
 Why could not DNA be spliced by the mechanism discussed in class?
o No 2’-OH
 What is a putative advantage of splicing?
o Creation of several splice variants possible- modified functionality depending on cell
o Increased protein diversity relative to prediction from genome
 Which sequence is for the attachment of amino acids onto tRNAs?
o CCA required for attachment of amino acids
 Why is rRNA originally transcribed as one unit?
o want unit 1:1:1 ratio (of the S segments)
 How can you distinguish prokaryotic from eukaryotic rRNA?
o Prokaryotes
 28S rRNA 5.8S rRNA 18S rRNA
o Eukaryotes
 23S rRNA 16S rRNA 5S rRNA
 What are small interfering RNAs?
o 21-23 ribonucleotides long
o Facilitate sequence specific gene suppression via RNA interference (of homologous genes)