* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Organization and Synthesis of DNA
Metalloprotein wikipedia , lookup
DNA profiling wikipedia , lookup
Genomic library wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Butyric acid wikipedia , lookup
SNP genotyping wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Community fingerprinting wikipedia , lookup
Restriction enzyme wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Non-coding DNA wikipedia , lookup
Molecular cloning wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Point mutation wikipedia , lookup
Biosynthesis wikipedia , lookup
Biochemistry wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
DNA supercoil wikipedia , lookup
Organization of DNA Andy Howard Introductory Biochemistry 12 October 2010 Biochem: Nucleic Acid Structure II 10/12/2010 What we’ll discuss Restriction Enzymes Review of A,B,Z DNA Intercalation Denaturation and renaturation of DNA DNA density DNA tertiary structure Review of supercoiling Gyrases Nucleosomes Higher levels Bacterial organization 10/12/2010 Biochem: Nucleic Acid Structure II p. 2 of 63 Restriction Endonucleases Evolve in bacteria as antiviral tools “Restriction” because they restrict the incorporation of foreign DNA into the bacterial chromosome Recognize and bind to specific palindromic DNA sequences and cleave them Self-cleavage avoided by methylation Types I, II, III: II is most important I and III have inherent methylase activity; II has methylase activity in an attendant enzyme 10/12/2010 Biochem: Nucleic Acid Structure II p. 3 of 63 What do we mean by palindromic? In ordinary language, it means a phrase that reads the same forward and back: Madam, I’m Adam. (Genesis 3:20) Eve, man, am Eve. Sex at noon taxes. Able was I ere I saw Elba. (Napoleon) A man, a plan, a canal: Panama! (T. Roosevelt) With DNA it means the double-stranded sequence is identical on both strands 10/12/2010 Biochem: Nucleic Acid Structure II p. 4 of 63 Quirky math problem Numbers can be palindromic: 484, 1331, 727, 595… Some numbers that are palindromic have squares that are palindromic… 222 = 484, 1212 = 14641, . . . Question: if a number is perfect square and a palindrome, is its square root a palindrome? (answer will be given orally) 10/12/2010 Biochem: Nucleic Acid Structure II p. 5 of 63 Palindromic DNA Example: G-A-A-T-T-C Single strand isn’t symmetric: but the combination with the complementary strand is: G-A-A-T-T-C C-T-T-A-A-G These kinds of sequences are the recognition sites for restriction endonucleases. This particular hexanucleotide is the recognition sequence for EcoRI. 10/12/2010 Biochem: Nucleic Acid Structure II p. 6 of 63 Cleavages by restriction endonucleases Breaks can be cohesive (if off-center within the sequence) or non-cohesive (blunt) (if they’re at the center) EcoRI leaves staggered 5’-termini: cleaves between initial G and A PstI cleaves CTGCAG between A and G, so it leaves staggered 3’-termini BalI cleaves TGGCCA in the middle: blunt! 10/12/2010 Biochem: Nucleic Acid Structure II p. 7 of 63 iClicker question 1. Which of the following is a potential restriction site? (a) ACTTCA (b) AGCGCT (c) TGGCCT (d) AACCGG (e) none of the above. 10/12/2010 Biochem: Nucleic Acid Structure II p. 8 of 63 Example for E.coli 5’-N-N-N-N-G-A-A-T-T-C-N-N-N-N-3’ 3’-N-N-N-N-C-T-T-A-A-G-N-N-N-N-5’ Cleaves G-A on top, A-G on bottom: 5’-N-N-N-N-GA-A-T-T-C-N-N-N-N-3’ 3’-N-N-N-N-C-T-T-A-AG-N-N-N-N-5’ Protruding 5’ ends: 5’-N-N-N-N-G A-A-T-T-C-N-N-N-N-3’ 3’-N-N-N-N-C-T-T-A-A G-N-N-N-N-5’ 10/12/2010 Biochem: Nucleic Acid Structure II p. 9 of 63 How often? 4 types of bases So a recognition site that is 4 bases long will occur once every 44 = 256 bases on either strand, on average 6-base site: every 46= 4096 bases, which is roughly one gene’s worth 10/12/2010 Biochem: Nucleic Acid Structure II p. 10 of 63 EcoRI structure Dimeric structure enables recognition of palindromic sequence sandwich in each monomer QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. EcoRI pre-recognition complex PDB 1CL8 57 kDa dimer + DNA 10/12/2010 Biochem: Nucleic Acid Structure II p. 11 of 63 The biology problem How does the bacterium mark its own DNA so that it does replicate its own DNA but not the foreign DNA? Answer: by methylating specific bases in its DNA prior to replication Unmethylated DNA from foreign source gets cleaved by restriction endonuclease Only the methylated DNA survives to be replicated Most methylations are of A & G, but sometimes C gets it too 10/12/2010 Biochem: Nucleic Acid Structure II p. 12 of 63 How this works When an unmethylated specific sequence appears in the DNA, the enzyme cleaves it When the corresponding methylated sequence appears, it doesn’t get cleaved and remains available for replication The restriction endonucleases only bind to palindromic sequences 10/12/2010 Biochem: Nucleic Acid Structure II p. 13 of 63 Methylases A typical bacterium protects its own DNA against cleavage by its restriction endonucleases by methylating a base in the restriction site Methylating agent is generally Sadenosylmethionine QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. HhaI methyltransferase PDB 1SVU 2.66Å; 72 kDa dimer QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 10/12/2010 Biochem: Nucleic Acid Structure II p. 14 of 63 Use of restriction enzymes Nature made these to protect bacteria; we use them to cleave DNA in analyzable ways Similar to proteolytic digestion of proteins Having a variety of nucleases means we can get fragments in multiple ways We can amplify our DNA first Can also be used in synthesis of inserts that we can incorporate into plasmids that enable us to make appropriate DNA molecules in bacteria 10/12/2010 Biochem: Nucleic Acid Structure II p. 15 of 63 Summaries of A, B, Z DNA 10/12/2010 Biochem: Nucleic Acid Structure II p. 16 of 63 DNA is dynamic Don’t think of these diagrams as static The H-bonds stretch and the torsions allow some rotations, so the ropes can form roughly spherical shapes when not constrained by histones Shape is sequence-dependent, which influences protein-DNA interactions 10/12/2010 Biochem: Nucleic Acid Structure II p. 17 of 63 Intercalating agents Generally: aromatic compounds that can form -stack interactions with bases Bases must be forced apart to fit them in Results in an almost ladderlike structure for the sugar-phosphate backbone locally Conclusion: it must be easy to do local unwinding to get those in! 10/12/2010 Biochem: Nucleic Acid Structure II p. 18 of 63 Instances of intercalators 10/12/2010 Biochem: Nucleic Acid Structure II p. 19 of 63 Denaturing and Renaturing DNA See Figure 11.17 When DNA is heated to 80+ degrees Celsius, its UV absorbance increases by 30-40% This hyperchromic shift reflects the unwinding of the DNA double helix Stacked base pairs in native DNA absorb less light When T is lowered, the absorbance drops, reflecting the re-establishment of stacking 10/12/2010 Biochem: Nucleic Acid Structure II p. 20 of 63 Heat denaturation Figure 11.14 Heat denaturation of DNA from various sources, so-called melting curves. The midpoint of the melting curve is defined as the melting temperature, Tm. (From Marmur, J., 1959. Nature 183:1427–1429.) 10/12/2010 Biochem: Nucleic Acid Structure II p. 21 of 63 GC content vs. melting temp High salt and no chelators raises the melting temperature 10/12/2010 Biochem: Nucleic Acid Structure II p. 22 of 63 How else can we melt DNA? High pH deprotonates the bases so the Hbonds disappear Low pH hyper-protonates the bases so the H-bonds disappear Alkalai is better: it doesn’t break the glycosidic linkages Urea, formamide make better H-bonds than the DNA itself so they denature DNA 10/12/2010 Biochem: Nucleic Acid Structure II p. 23 of 63 What happens if we separate the strands? We can renature the DNA into a double helix Requires re-association of 2 strands: reannealing The realignment can go wrong Association is 2nd-order, zippering is first order and therefore faster 10/12/2010 Biochem: Nucleic Acid Structure II p. 24 of 63 Steps in denaturation and renaturation 10/12/2010 Biochem: Nucleic Acid Structure II p. 25 of 63 Rate depends on complexity The more complex DNA is, the longer it takes for nucleation of renaturation to occur “Complex” can mean “large”, but complexity is influenced by sequence randomness: poly(AT) is faster than a random sequence 10/12/2010 Biochem: Nucleic Acid Structure II p. 26 of 63 Second-order kinetics Rate of association: -dc/dt = k2c2 Boundary condition is fully denatured concentration c0 at time t=0: c / c0 = (1+k2c0t)-1 Half time is t1/2 = (k2c0)-1 Routine depiction: plot c0t vs. fraction reassociated (c /c0) and find the halfway point. 10/12/2010 Biochem: Nucleic Acid Structure II p. 27 of 63 Typical c0t curves 10/12/2010 Biochem: Nucleic Acid Structure II p. 28 of 63 Hybrid duplexes We can associate DNA from 2 species Closer relatives hybridize better Can be probed one gene at a time DNA-RNA hybrids can be used to fish out appropriate RNA molecules 10/12/2010 Biochem: Nucleic Acid Structure II p. 29 of 63 GC-rich DNA is denser DNA is denser than RNA or protein, period, because it can coil up so compactly Therefore density-gradient centrifugation separates DNA from other cellular macromolecules GC-rich DNA is 3% denser than AT-rich Can be used as a quick measure of GC content 10/12/2010 Biochem: Nucleic Acid Structure II p. 30 of 63 Density as function of GC content 10/12/2010 Biochem: Nucleic Acid Structure II p. 31 of 63 Tertiary Structure of DNA In duplex DNA, ten bp per turn of helix Circular DNA sometimes has more or less than 10 bp per turn - a supercoiled state Enzymes called topoisomerases or gyrases can introduce or remove supercoils Cruciforms occur in palindromic regions of DNA Negative supercoiling may promote cruciforms 10/12/2010 Biochem: Nucleic Acid Structure II p. 32 of 63 DNA is wound Standard is one winding per helical turn, i.e. 1 winding per 10 bp Fewer coils or more coils can happen: This introduces stresses that favors unwinding Both underwound and overwound DNA compact the DNA so it sediments faster than relaxed DNA 10/12/2010 Biochem: Nucleic Acid Structure II p. 33 of 63 Linking, twists, and writhe T=Twist=number of helical turns W=Writhe=number of supercoils L=T+W = Linking number is constant unless you break covalent bonds 10/12/2010 Biochem: Nucleic Acid Structure II p. 34 of 63 Examples with a tube 10/12/2010 Biochem: Nucleic Acid Structure II p. 35 of 63 How this works with real DNA 10/12/2010 Biochem: Nucleic Acid Structure II p. 36 of 63 How gyrases work Enzyme cuts the DNA and lets the DNA pass through itself Then the enzyme religates the DNA Can introduce new supercoils or take away old ones 10/12/2010 Biochem: Nucleic Acid Structure II p. 37 of 63 Typical gyrase action Takes W=0 circular DNA and supercoils it to W=-4 This then relaxes a little by disrupting some base-pairs to make ssDNA bubbles 10/12/2010 Biochem: Nucleic Acid Structure II p. 38 of 63 Superhelix density Compare L for real DNA to what it would be if it were relaxed (W=0): That’s L = L - L0 Sometimes we want = superhelix density = specific linking difference = L / L0 Natural circular DNA always has < 0 10/12/2010 Biochem: Nucleic Acid Structure II p. 39 of 63 < 0 and spools The strain in < 0 DNA can be alleviated by wrapping the DNA around protein spool That’s part of what stabilizes nucleosomes 10/12/2010 Biochem: Nucleic Acid Structure II p. 40 of 63 Cruciform DNA Cross-shaped structures arise from palindromic structures, including interrupted palindromes like this example These are less stable than regular duplexes but they are common, and they do create recognition sites for DNA-binding proteins, including restriction enzymes 10/12/2010 Biochem: Nucleic Acid Structure II p. 41 of 63 Cruciform DNA example 10/12/2010 Biochem: Nucleic Acid Structure II p. 42 of 63 Eukaryotic chromosome structure Human DNA’s total length is ~2 meters! This must be packaged into a nucleus that is about 5 micrometers in diameter This represents a compression of more than 100,000! It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments 10/12/2010 Biochem: Nucleic Acid Structure II p. 43 of 63 Chromatin Discovered long before we understood molecular biology Seen to be banded objects in nuclei of stained eukaryotic cells In resting cell it exists as long slender threads, 30 nm diameter From answers.com 10/12/2010 Biochem: Nucleic Acid Structure II p. 44 of 63 Squishing the DNA If the double helix were fully extended, the largest human chromosome (2.4*108bp) would be 2.4*108 *0.33nm ~ 0.8*108nm=80 mm; much bigger than the cell! So we have to coil it up a lot to make it fit. Longest chromosome is 10µm long So the packing ratio is 80mm/10µm = 8000 10/12/2010 Biochem: Nucleic Acid Structure II p. 45 of 63 Chromosome structure: levels Each of the first 4 levels compacts DNA by a factor of 6-20; those multiply up to > 104 10/12/2010 Biochem: Nucleic Acid Structure II p. 46 of 63 Nucleosome Structure Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins Histone octamer structure has been solved without DNA: Moudrianakis, 1991 with DNA by Richmond Nonhistone proteins are regulators of gene expression 10/12/2010 Biochem: Nucleic Acid Structure II p. 47 of 63 Histone types H2a, H2b, H3, H4 make up core particle: two copies of each, so: octamer All histones are KR-rich, small proteins H1 associates with the regions between the nucleosomes 10/12/2010 Biochem: Nucleic Acid Structure II p. 48 of 63 Histones: table 11.2, plus… Histone #lys, #arg # Mr, acidic kDa H1 59, 3 10 21.2 H2A 13, 13 9 14.1 2 (in bead) H2B 20, 8 10 13.9 2 (in bead) H3 13, 17 11 15.1 2 (in bead) H4 11, 14 7 11.4 2 (in bead) 10/12/2010 Biochem: Nucleic Acid Structure II Copies per Nucleosome 1 (not in bead) p. 49 of 63 Unfolded chromatin Treat chromatin with low ionic strength; that disrupts higher level interactions so the individual nucleosomes are strung out relative to one another like beads on a string Image courtesy U. Maine 10/12/2010 Biochem: Nucleic Acid Structure II p. 50 of 63 Nucleosome core particle 10/12/2010 Biochem: Nucleic Acid Structure II p. 51 of 63 Half the core particle Note that DNA isn’t really circular: it’s a series of straight sections followed by bends (like the Advanced Photon Source ring!) 10/12/2010 Biochem: Nucleic Acid Structure II p. 52 of 63 Histones, continued Individual nucleosomes attach via histone H1 to seal the ends of the turns on the core and organize 40-60bp of DNA linking consecutive nucleosomes N-terminal tails of H3 & H4 are accessible K, S get post-translational modifications, particularly K-acetylation 10/12/2010 Biochem: Nucleic Acid Structure II p. 53 of 63 O HN Histone deactivation ONH3+ O acylated lysine Histones interact with DNA via +charges on lys and arg residues. If we neutralize those charges by acetylation, the histones don’t bind as tightly to the DNA Carefully-timed enzymatic control of histone acetylation is a crucial element in DNA organization 10/12/2010 Biochem: Nucleic Acid Structure II p. 54 of 63 CoASH Histone acetylation Active histone + Acetyl CoA inactive (acetylated) histone + CoASH Without the positive charges, the affinity for DNA goes down Histone H1 PDB 1GHC 8.3 kDa monomer Chicken Histone 10/12/2010 Biochem: Nucleic Acid Structure II acetyltransferase PDB 1QSO 66 kDa tetramer yeast p. 55 of 63 Histone deacetylation Type III deacetylases use a non-trivial reaction: Prot-lys-NAc + NAD+ Prot-lys-NH3+ + nicotinamide + 2’-O-acetyl-ADP-ribose Part of the NAD salvage pathway Histone/protein deacetylase + histone H4 active peptide PDB 1SZD; 34 kDa “heterodimer” yeast 10/12/2010 Biochem: Nucleic Acid Structure II p. 56 of 63 Other histone PTM Histones can be post-translationally modified in other ways as well Methylation: e.g. lysines 4,27 of H3 Phosphorylation: H2A phosphorylated at several sites near “hinge” These are correlated with acetylation and play a role in folding and function 10/12/2010 Biochem: Nucleic Acid Structure II p. 57 of 63 Nucleosome structure Core octamer is two molecules each of H2A, H2B, H3, H4 Typically wraps around ~200bp of DNA DNA between nucleosomes is ~54 bp long H1 binds to linker and to core particle; but in beads-on-a-string structure, it’s often absent 10/12/2010 Biochem: Nucleic Acid Structure II p. 58 of 63 How much does this coil up? 200 bp extended would be about 50nm The width of the core-particle disk is 5nm So this is a tenfold reduction Nucleosomal organization corresponds to negative supercoiling … so DNA ends up supercoiled when we take away the histones 10/12/2010 Biochem: Nucleic Acid Structure II p. 59 of 63 Courtesy answers.com Next level of organization H1 interacts with DNA along linker region Individual histones spiral along to form 30 nm fiber See fig.19.25 10/12/2010 Biochem: Nucleic Acid Structure II Courtesy Johns Hopkins Univ p. 60 of 63 Even higher… The 30nm fibers are attached to an RNA-protein scaffold that holds the 30nm fibers in large loops Typical chromosome has ~200 loops Loops are attached to scaffold at their base Ends can rotate so it can be supercoiled 10/12/2010 Biochem: Nucleic Acid Structure II p. 61 of 63 What about prokaryotes? No actual histones Histone-like proteins (HLPs) involved Bacterial DNA attached to scaffold in large loops (~100kb) This makes a nucleoid 10/12/2010 Biochem: Nucleic Acid Structure II p. 62 of 63 How many loops in bacteria? Typical bacterial genome (E.coli) has 3000 open reading frames ~ 3000 genes. Assume 500 amino acids per protein = 1500 bases per gene (ignores transcriptional elements) Then genome is 1500 bp/gene * 3000 genes = 4.5*106 base-pairs That’s (4.5*106 bp)/(1*105 bp/loop) = 45 loops 10/12/2010 Biochem: Nucleic Acid Structure II p. 63 of 63