MMT: Nucleic acids I Flashcards
Specify the following features of the B form of DNA: 3,5 ́phosphodiester bonds of the sugar-phosphate backbone, number of hydrogen bonds in complementary base pairings, major and minor grooves, handedness.
- DNA is composed of two complementary antiparallel strands, each running 3’ to 5’ with bases linked via phosphodiester bonds
- GC has 3 H bonds, AT has 2
- Major grooves and minor grooves -
alternate within the helix; this is due to the equidistance of bases in complementary strands - Handedness: right-handed around the central axis
Describe the following physical dimensions of the DNA double helix (B form): number of bases per turn, approximate distance between stacked bases, number of bases per turn of the helix.
Number of bases per turn 10
Distance between stacked bases 3.4
Number of bases per turn of helix: 36
Explain what is meant by polarity of complementary DNA strands.
Strands of DNA run 3’ to 5’, but are aligned in an anti-parallel format. This gives the strands polarity
Describe how supercoils (negative and positive) occur
when DNA is underwound, DNA gets stressed and creates negative supercoils to relax the torsional stress created by the underwinding. When DNA is overwound, DNA again gets stressed and creates positive supercoils to relax the torsional strain created by the overwinding. Supercoiling relieves torsional stress by bending the helical axis of DNA.
identify processes in which supercoiling/topoisomerase holds functional significance
genomic DNA, transcription, DNA replication, mitosis/meiosis
describe how supercoiling is significant in genomic DNA
negative supercoiling is especislly useful in terms of genomic DNA. it allows for long linear molecules with reduced torsional stress, it is easier to unwind negatively supercoiled DNA than positively supercoiled DNA, and it allows for stored energy needed to facilitate unwinding of DNA
describe how supercoiling is significant in transcription
when RNA polymerase unwinds DNA to form the transcription bubble, overwinding ahead of the bubble can cause positive supercoils, and negative coils form behind the transcription bubble
what is the function of topoisomerases?
relaxing supercoils
Specify the effects of Type I and Type II Topoisomerases on Linking Number (Lk).
if they are relaxing negative supercoils they’ll increase Lk, if they’re relaxing positive supercoils they’ll decrease Lk
describe how topoisomerase I and II carry out their function
Type I: change Lk by cleaving a single DNA strand, allowing it to rotate around the second strand, and resealing it. They operate as swivelases. Applicable to pos and neg supercoiling
Type II: ATP dependent and change Lk via cleaving both strands, passing intact DNA through the gap and resealing the strands. These are more efficient. Applicable to pos and neg supercoiling
describe the function of types I and II topoisomerase in prokaryotes
Type I: relaxes negative supercoils
Type II: relaxes positive supercoils
what is gyrase?
a type II topoisomerase in prokaryotes that creates negative supercoils, decreasing linking number
Describe the applications of targeting topoisomerase in medications
this is often seen in antibiotics and chemotherapy
Define melting temperature (T m) and how it can be used to predict G+C content of DNA.
Higher GC, content, higher melting point. More H bonds, so it is harder to break the GC base pair bond.
Describe how the properties of denaturation and reannealing are used for detection of specific DNA sequences
Denaturation: unwinding the strands of a double helix, which can be done via heat, pH, and denaturing reagents. The AT rich regions denature first, then as more agent is added the GC will start to unwind.
Reannealing: if you let the dna strands cool in solution, the DNA strands will come back together via complementary base pairing. You can add and label a probe sequence to detect a specific sequence during this process (hybridization).
