|Lecture Notes: 9 March
© R. Paselk 2006
Topoisomerases and the Control of Supercoiling in DNA
Supercoiled DNA can relax by nicking (single strand cleavage such as by DNAaseI) creating a "swivel."
DNA must have the proper topological state for normal biological function (replication, recombination, transcription). Normal negative supercoiled state aids unwinding during replication and transcription.
Supercoiling is introduced and controlled by topoisomerases. These enzymes change the linking number (L) in DNA and thus its topology. Two main types:
- Type I topoisomerases operate by making single strand breaks.
- Type II topoisomerases operate by making double strand breaks.
Type I topoisomerases relax supercoiling incrementally (change L ±1) by reversibly catenating the DNA strands.
- Done by reversible formation of a tyrosine-phospho-DNA strand on the enzyme (prokaryotes: 5'P; eukaryotes: 3'P), thus maintaining the "high energy" bond of the DNA strand, so the DNA strand can be relinked without any requirement for ATP. (text Figure 24-21, p 936)
Type II topoisomerases or Gyrases, require an energy source:
- NAD in eukaryotes,
- ATP in prokaryotes.
These enzymes catalyze the stepwise negative supercoiling of DNA in prokaryotes (catalyze the relaxation only in the absense of ATP). (text Figure 24-22, p 937)
Eukaryotes only catalyze relaxation of supercoiling via other enzymes.
DNA Annealing & Cot Curve Analysis
Analysis of cot curves
- DNA renaturation curve where
- plot fraction of single-stranded DNA reannealed vs log cot (mol*sec/L)
- c = concentration of single-stranded DNA at time t
- co = concentration of completely denatured DNA at time 0, to
- Basically DNA reanneals rapidly if short, takes increasing times to anneal as non-repetitive lengths increase in logarithmic fashion (essentially a matter of collision/alignment probability).
- Follow spectrophotometrically, A260 decreases with annealing
When we look at cot curves find the cot values increase with complexity. thus
- Poly A + Poly U reanneal extremely rapidly.
- Mouse satellite (highly repetitive) DNA anneals very rapidly
- viruses next
- then procaryotes
- then eukaryotes
Analysis of cot values indicate that viral and prokaryotic DNA has few or no repeated sequences. On the other hand eukaryotes are quite complex with varying degrees of repetition. Thus a eukaryotic genome will have:
- Unique sequences coding proteins etc. (approximately 1 copy/haploid genome).
- Moderately repetitive (< 106 copies/haploid genome).
- Occurs in segments of 100 - several thousand repetitions interspersed with larger blocks of unique DNA.
- Some specifies repetitive DNA of rRNA, tRNA and histones.
- Some is also thought to participate in control.
- Highly repetitive (>106 copies/haploid genome).
- Highly repetitive sequences are clustered at the centromeres, in clusters of nearly identical sequences of up to 10 bp tending to repeat thousands of times. This DNA is isolated as the so-called satellite DNA because it sediments as a distinct satellite band in CsCl gradient as a result of its distinct base composition.
- Inverted repeats ranging from 100-1000 base pairs.
- These renatature with first-order kinetics, indicating self-complementary (inverted) sequences. (Other sequences should renature with second-order kinetics, since they must find each other.) Approximately 2 x 106 copies occur in the human genome.
- May be used to align homologous chromosomes during meiosis and to facilitate recombination.
- Histones and nucleosomes (text Figure 24-26, p 939), (text Figure 24-27, p 940), (text Figure 24-29, p 941)
- 30 nm fiber (text Figure 24-30, p 942)
- scaffold (text Figure 24-31, p 942)
- Schematic chromosome (text Figure 24-33, p 943)
Last modified 11 March 2009