MCB 502A-2015. Lecture #5!DNA degradation (continued). DNA synthesis.— The rate of DNA degradation— Polarity of ss-specific exo— Polarity of ds-specific exo— Processivity of exonucleases— Measuring the processivity— Endonucleases. The cleavage specificity (position)— Substrate-specificity of endonucleases— The biological roles of exo- and endonucleases DNA synthesis— The oops of Ochoa vs Kornberg’s DNA polymerase— DNA depolymerization vs DNA degradation — The need for a primer— The ghost of the non-templated synthesis— Nick-translationCharacterization — where to start?— For example, E. coli cells are home to the following major classes of nucleases, most of them containing more than one entry:DNA substrate ClassSSL SSC DSL DSC assignment>95 <5 <5 <5 ss-exo~15 <5 >95 <5 ds-exo>95 <5 >95 <5 ss/ds-exo>95 >95 <5 <5 ss-endo<5 <5 >95 >95 ds-endo>95 >95 >95 >95 ss/ds-endo%TCA-soluble substrate in reactions run to completionFrom this perspective, the cell definitely looks like a bag of DNA-degradation enzymes, rather than a bag of DNA-replicating enzymes.The rateThe rate of DNA degradation-1— … degrade only linear ssDNA,… at least five of these in E. coli. — … first …:… is how fast the enzyme degrades its substrate DNA. — … how many nucleotides …are removed in a unit of time, … (substrate) a uniformly-labeled DNA strand.Rate of DNA degradation is measured in nt/sec releasedThe rate of DNA degradation-2— To measure the rate of degradation, the enzyme … over the substrate. — If the opposite is true, …The rate of DNA degradation-7— … why not the substrate in excess over enzyme?... — In a typical … determination of the enzyme rate,… the unlimited substrate minimizes the non-occupancy of the enzyme. — The difference is due to the relative sizes in these enzyme-substrate pairs…. — In fact, a typical enzyme is 100-1,000 times bigger than its substrate molecule…The rate of DNA degradation-8— For the DNA-processing enzymes … substrates are sometimes 10,000-times bigger than the enzymes …. — Since in the reaction the smaller component should be in excess, … and the rate is then calculated from the amount of the limiting component, …. — The amount of active enzyme … equals … the known amount of the limiting DNA substrate.The rate of DNA degradation-3— In principle, one could use excess of substrate-over-enzyme … — However, while it is straightforward to measure the total amount of a pure protein, …. Saturating the substrate with an enzyme is easier.— In fact, one does not have to know the exact amount of enzyme, as long as it is saturating in the reaction …. — When using saturating amounts of enzyme, … the amount of active enzyme = the (known) amount of the available substrate. Enzyme (ng)Reaction ratetotalactivePrep #1Prep #2The rate of DNA degradation-4— For example, 100 pM of the substrate is reacted for 100 seconds with 1 nM of the protein …. — A refresher: in chemistry the amount of substance is expressed in Moles (M) or fractions of Mole (1 mM = 10-3 M). — 1 M of any substance comprises… (Avogadro's number). — … in biochemistry, … biopolymers are huge µM (10-6 M), nM (10-9 M) pM (10-12 M) moleµmolnmolpmolAmedeo AvogadroThe rate of DNA degradation-5— With a 10-fold excess of enzyme over the substrate, …— Once one enzyme molecule falls off, … — This is what the enzyme excess is for: … — One stops the reaction, … TCA precipitation … the amount of the released radioactivity.The rate of DNA degradation-6— For example, one finds that, in 100 seconds, exactly 100 nM of nucleotides are released from the substrate by 100 pM of the active fraction of enzyme (which equals the amount of the substrate). — Therefore, the rate of DNA degradation by this nuclease is 10 nt/sec. — How come …? — Because 1 M of DNA substrate may contain… — …100 nM/100 sec = 1 nm/sec1 nm/sec / 100 pM enzyme = 10 nt/secPolarity (Directionaliy)Polarity of ss-specific exo-1— … we can ask whether they degrade linear DNA strands in any particular direction. — DNA strands have chemical polarities… — Therefore, the two directions …5'—>3'3'—>5'Polarity of ss-specific exo-2— To test the suspected polarity… … end-specific radioactive labeling… — … DNA strand labeled throughout with 3H-Thy, … and … polynucleotide kinase (PNK) … with 32P-phosphate at the 5’-end, … or … terminal nucleotidyl-transferase (TNT)… with 32P-nucleotide at the 3’-end. — The released 3H or 32P labels from the same DNA molecule …3H 3H 3H 3H 5' 3'3H 3H 3H 3H PNK32P5' 3'3H 3H 3H 3H 32PPNK5'3'Polarity of ss-specific exo-3— … three different ss-exo enzymes … the two end-labeled substrates. — With the 5’-32P-labeled substrate, …— … conclusion: enzyme #1 degrades DNA strands in the 3’—>5’ direction, while enzymes #2 and #3 degrade DNA strands in the 5’—>3’ direction.Time (min)% TCA-soluble#2#1#33H 3H 3H 3H 32P5' 3'Polarity of ss-specific exo-4— …verify … with the 3’-32P-labeled substrate. — You find that … — … conclusion: #1 degrades 3’—>5’, while #2 degrades 5’—>3’. — Enzyme #3, apparently, … degrades … from both ends. Time (min)% TCA-soluble#1#2#33H 3H 3H 3H 32P5'3'By the way, since we are discussing directionality,…Polarity of ds-specific exo-1— … can we apply this … to determine … polarity of dsDNA-specific exonucleases? — You must be surprised: … polarity of ds? … Rosalind Franklin …— As their name implies, dsDNA-specific exonucleases … 5' 3'3' 5'Polarity of ds-specific exo-2— … most dsDNA-specific exonucleases … degrade … one particular DNA strand…— With such enzymes, … duplex substrates with specifically-labeled ends. — … the problem: … the enzymes that we used to end-label DNA strands, do not work on duplex DNA. — …?5' 3'3' 5'5' 3'3' 5'Polarity of ds-specific exo-3— These days: … various (enzymes) or,… synthetic oligonucleotides… — … the complexity of natural DNAs makes the reassociation as a construction approach impractical.— Oligonucleotides were not known… NucleasesPolymerasesPolarity of ds-specific exo-4— … poly-dA and poly-dT … anneal with close to 100% efficiency. — … poly-dT200 throughout with 3H, then end-label it with 32P, …anneal it to
View Full Document
Unlocking...