MCB 502A-2015. Lecture # 8. Unwinding of DNA and keeping it single-stranded. Superhelicity. DNA unwinding: helix-destabilizing proteins— Alberts protein— The mechanism of helix destabilizationDNA unwinding: DNA helicases— The discovery of helicases— The substrate to detect the helicase polarity— Helicases are enzymes, but revealing this requires help from SSB— Models of active DNA unwindingSuperhelicity— The linking number of a DNA helix— Positive and negative supercoiling.— Detection of DNA supercoilingTopoisomerases— Topo IWhat we have learned so far— We learned a lot about DNA organization and replication in E. coli:— Chromosomes are circular in prokaryotes, linear in eukaryotes. — A single replication bubble moves fast duplicating the circular prokaryotic chromosome. Multiple bubbles in eukaryotic chromosomes move much slower. — DNA replication is semiconservative.— DNA polymerases synthesize DNA in the 5'—>3’ direction and use RNA primers. — The DNA at the replication forks is synthesized discontinuously, in Okazaki pieces, requiring multiple priming events by primase. Prokaryotic primases are RNA polymerases, eukaryotic primases are RNA/DNA polymerases. — The chromosome in E. coli is replicated by DNA pol III and several other factors, defined by the dna mutants. — DNA pol I and DNA ligase are required for maturation of Okazaki fragments.What we still have not discussed— … principles of DNA replication ….: … how the template DNA strands get primed and replicated. — …(but) how the template DNA strands are generated ….(?) — … (the scare) … of the 1950s: how this tightly-wound structure is unwound and rotated during replication.5'RNA DNA5'3321Two new questions— … three questions:— What are the mechanisms of 1) parental DNA unwinding and … 2) keeping it single-stranded so that it can serve as template for DNA replication? 3) What are the mechanisms of torsional stress removal and duplex winding after the synthesis?DNA unwinding: helix-destabilizing proteinshttp://www.materials360online.com/newsDetails/10620;jsessionid=821186BC8554BD2A1362EE6660C50852Chromatography-1— … identification of helix-destabilizing proteins, … chromatography. Ion-exchange (charged resins) Gel filtration (neutral resins)Chromatography-2— … an efficient molecular separation technique … two stages. — … (adsorbtion),… mixture … bound to a particular sorbent … — … (elution), … from the sorbent … changing conditions … differences in the specific quality between the bound substances…Chromatography-3— …. separation power … "rushing for the exit" … — … under destabilizing conditions … the sudden surge … still bound …. resist dissociation. — … amplifies the effect … "all or nothing" effects … tight band … — …chromatography can be also used to concentrate …Chromatography-4— … Mikhail Tsvet in the early 1900s … … plant pigments …— … different colors (of these pigments) … — the name. — The principles and basic techniques: …. 1940s and 50s. Михаил Семёнович ЦветAlberts protein-1— … to be replicated … unwound. — … DNA polymerases … (cannot unwind) … . … Kornberg polymerase… can displace … — … dedicated … to unwind DNA. — … a characteristic … to isolate them? — … logical: … protein … should be able to bind to ssDNA … to move along it…. A few nucleotidesssDNAAlberts protein-2— Bruce Alberts : … ssDNA-binding proteins…. ssDNA-affinity chromatography. — "Affinity" is a high specificity. … iron key from a mixture … a general sorbent (a magnet) … , … or an affinity sorbent…. . Bruce AlbertsAlberts protein-3— … attached ssDNA to … (cellulose) to produce …. ssDNA-cellulose … clarified cell lysates… — … developed the column … (a gradient) … dextran-sulfate, …. Protocol of Albert's procedure1) Lyse E. coli cells infecetd with T4 bacteriophage, clear debris by centrifugation. 2) Apply the lysate to ssDNA-cellulose column. Wash the column with an excess of the application buffer to remove the unbound stuff. 3) Develop the column with a gradient of application-elution* buffers, collecting fractions. * The elution buffer in this case is the application buffer supplemented with a high concentration of dextran-sulfate (DNA mimic). OD280 = [protein]Time[dextran-SO4]dextran-sulfateAlberts protein-4— … T4-infected E. coli … — T4 … replication machinery. — … a massive metabolic effort… phage replication proteins … — Alberts isolated several proteins…— E. coli cells typically have only two replication forks. Even when growing extra fast, E. coli cells have only six forks. — In contrast, T4 replicates its DNA at ~60 replication forks in the host cells.Alberts protein-5— … protein … failed to elute … flushed out … with 2 M NaCl …. — … so tightly-bound to ssDNA, … lysates, generated by infection of non-suppressing E. coli cells with various sus mutants in T4 DNA replication. — … gene 32 … (no) protein. … gp32 (gene product 32). OD280 = [protein]Time[dextran-SO4]Cleaning with 2M NaClAlberts protein-6— … protein… does not bind to duplex DNA, … binds tightly to ssDNA. — … the protein catalyzes reassociation …. — … the optimal renaturation temperature … … (90°C for natural DNA)… — … around 65°C. gp32Ta = Tm – 25°CAlberts protein-7— At 25°C (room temperature),… will take weeks …. — gp32 accelerates … at 25°C …— … in vivo function of gp32… ? — … drops the melting temperature of a duplex DNA some 40°C… the optimal renaturation temperature … 25°C. — … a prototype of helix-destabilizing proteins.20° 30° 40° 50° 60° 70° 80° 90°meltingannealinggp32gp32The mechanism of helix destabilization-1— The mechanism of DNA helix destabilization… two characteristics …: 1) high specificity: … 2) high cooperativity … — Cooperativity is a characteristic of protein binding to polymers. — If a molecule of gp32 binds ssDNA, …— … high cooperativity … protein "polymerization" in the presence of its polymeric substrate. Non-cooperative protein binding Highly-cooperative protein bindingThe mechanism of helix destabilization-2— The high cooperativity … a peculiar binding pattern of sub-saturating … — … 30% of all binding sites… …
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