L5. Topoisomerases_Nucleases_2024.pdf

L5. Topoisomerases and nucleases 1 DNA supercoiling is increased or decreased by topoisomerases Topoisomerases are classified into Typestrands I and type II 2 cut both...

L5. Topoisomerases and nucleases 1 DNA supercoiling is increased or decreased by topoisomerases Topoisomerases are classified into Typestrands I and type II 2 cut both Type : Type 1 : nicks a strand (Relaxes positive supercoils) 2  How does Type I topoisomerase work? Mechanism of bind-cleavage-passage-religation by Type I topoisomerase: Hep used No Binds to a segment of the double-stranded DNA. Cleaves one strand of the DNA, forming a single-stranded break. Passes the other strand of the DNA through the break. Religates the cleaved DNA strand. Each round of catalysis changes the DNA by one helical turn, relaxing by ONE negative supercoil Simplified overview of ”bind-cleavage- passage-religation” type I topoisomerase mechanism DNA 3 How does Type I topoisomerase work in detail? after this run -- mechanism is continued on next slide 4 How does Type I topoisomerase work in detail (part 2)? & relax" he supercoils per rxn Step 4 is is just the ‘reverse reaction’ of step 1 5  > - introduce the syercoils How does Type II topoisomerase (=DNA gyrase) work? Mechanism of bind-cleavage (double)-passage-relegation by Type II topoisomerase: used Alp Binds first to one then to two segments of the DNA Cleaves across both strands of one DNA segment, forming a double-stranded break. Passes the other segment of the DNA through the break. Religates the cleaved DNA segment (both strands). Each round of catalysis changes the DNA by two helical turns, generating two negative supercoils. Type II topoisomerase can also untangle DNA. supercoils Imagine human DNA as a very long and thin rope: (1 cm diameter, 105 km long, easy to get tangled up) Simplified overview of ”bind-cleavage (double)-passage-religation” mechanism 6 How does Type II topoisomerase (=DNA gyrase) work in detail? S = DLk = Lk – Lko & [DNA Binding ] site is introducer 7  (i. e. B : A DNA) Cellular DNA usually is negatively supercoiled (i.e. the DNA is underwound). Underwound DNA promotes strand separation à easier to start DNA replication and transcription. BUT… In some thermophilic organisms (living in near boiling water), DNA is positively supercoiled (i.e. DNA is overwound). Hot spring in Yellowstone park DNA usually is denatured at these high temperature Overwound (positively supercoiled) DNA is structurally more tight, thus more resistant to heat-denaturation. Reverse gyrase has been found in thermophilic organisms to generate positive supercoils in DNA. Electron micrograph of thermophilic bacteria from hot spring 8  less An experiment showing relaxation Supercoiling of DNA of circular DNA supercoils: Islow , compact) electrode The circular relaxed DNA (shown to the right) + has no supercoil. ve Supercoiled DNA was treated with topoisomerase I, - (to attract eged 2 Relaxed DNA: one helical turn per 10.5 bp DNA- tes Agarose gel electrophoresis (migrates from top to bottom). die e GNA na phosp DNA bands were visualized by staining If the circular with isethidium relaxed DNA bromide. overwound to (made to have more helical turns), it forms positive supercoil. Lane 1: Circular plasmid DNA extracted from E. coli If the circular relaxed DNA is underwound (made to have less helical turns), it forms negative supercoil. Lane 2: The DNA was pre-treated with topoisomerase I. Supercoiled DNA isFor relaxed by one supercoil DNA supercoiling to occur, per round of I fast , more catalysis,producing both strands various of the DNA partially must be relaxed intact.(Each DNA DNAs compact) band differs from itsi.e. Cannot have neighbor by aone break (nick) in either DNA strand. supercoil.) Lane 3: Same as Lane 2, pre-treated for a longer time. Electron micrographs showing circular DNA. More supercoiled DNA Panelshas frombecome more left to right: relaxed relaxed DNA to highly supercoiled DNA. Migrates more slowly (because it is less compact) From previous lecture) 3 ‘Decreasing Lk’ toward the bottom of the gel, because the DNA is negatively supercoiled, DNA has more negative supercoils nearer the bottom. S = Lk – Lko ‘Decreasing Lk’ makes S a larger negative number, hence more negative supercoils. 9 Nucleases: degradation of nucleic acids Ø food digestion, RNA degradation in gene regulation, DNA degradation in apoptosis Nucleases are enzymes that catalyze breakage (hydrolysis) of phosphodiester bonds in DNA or RNA. Substrate Specificity: - ribonuclease (RNase) degrades RNA. - deoxyribonuclease (DNase) degrades DNA. - non-specific nuclease degrades both. - single-strand, double-strand, or both. (1 exact position) Mode of Attack: - endonuclease hydrolyzes interior phosphodiester bond - exonuclease acts sequentially from one end (either 5’ à 3’ or 3’ à 5’) ↳ pacman Nature of Phosphodiester Bond Cleavage: - produces 5’-OH, 3’-P (e.g. RNase A and RNase T1) - produces 3’-OH, 5’-P (e.g. DNase I) Base Specificity: - specific for a single type of base (e.g. RNase T1, specific for G) - specific for a particular class of base (e.g. RNase A, specific for pyrimidine (C or U) - non-specific 10 5 Practice Exercises G Provided to you that RNase T1 is specific for G (i.e. cleaves after G), produces 5’-OH, 3’-P If RNA 5’ pApGpCpUpCpGpUpC 3’ is digested with RNase T1, what are the products? products are: pApGp O + CpUpCpGp + UpC Provided to you that RNase A is specific for pyrimidine, produces 5’-OH, 3’-P If RNA 5’ pApGpCpUpCpGpUpC 3’ is digested with RNase A, products are: pApGpCp + Up + Cp + GpUp + C 00 Provided to you that DNase I is non-specific for bases, produces 3’-OH, 5’-P - If an RNA-DNA fusion molecule 5’ pApUpCpApCpGpApC 3’ (ribonucleotides are underlined) & 00000000 is digested with DNase I, products are: pApUpCpA + pC + pG + pA + pC won't clearl RNA 11 Alkaline hydrolysis of RNA : Ø treatment with solution of concentrated NaOH hydrolyzes RNA OH- u mixture of both Alkali (e.g. NaOH) does not hydrolyze DNA (DNA lacks 2’-OH) Alkali denatures DNA (Alkali breaks H-bonds in DNA) 12