10/15/2009biochemistry:nucleic acids iii organization and synthesis of dna andy howard introductory...

10/15/2009Biochemistry:Nucleic Acids III

Organization and Synthesis of DNA

Andy HowardIntroductory Biochemistry

15 October 2009

10/15/2009 Biochemistry:Nucleic Acids III

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What we’ll discuss Restriction Enzymes

(concluded) 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


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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


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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


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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


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Summaries of A, B, Z DNA


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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


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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!


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Instances of inter-calators


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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


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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.)


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GC content vs. melting temp High salt and no chelators raises the melting temperature


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How else can we melt DNA? High pH deprotonates the bases so the H-bonds 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


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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


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Steps in denaturation and renaturation


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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


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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.


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Typical c0t curves


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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


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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


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Density as

function of GC content


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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


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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


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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


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Examples with a tube


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How this works with real DNA


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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


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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


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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


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< 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


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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


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Cruciform DNA example


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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


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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


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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


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Chromosome structure: levels Each of the first 4 levels compacts DNA by a factor of 6-20; those multiply up to > 104


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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


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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


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Histones: table 11.2, plus…Histone #lys

,#arg

#acidi

c

Mr,kDa

Copies perNucleosome

H1 59, 3

10 21.2 1 (not in bead)

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)


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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


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Nucleosome core particle


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Half the core particle

Note that DNA isn’t really circular: it’s a series of straight sections followed by bends


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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


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Histone deactivation 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

NH3+

HN

O

O-

acylated lysineO


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Histone acetylation

Active histone + Acetyl CoA inactive (acetylated) histone + CoASH

Without the positive charges, the affinity for DNA goes down

CoASH

Histone H1PDB 1GHC8.3 kDa monomerChicken

Histone acetyltransfe

rasePDB 1QSO

66 kDatetramer

yeast


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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 peptidePDB 1SZD; 34 kDa “heterodimer”yeast


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Nucleosome structure

Core octamer is two molecules each of H2A, H2B, H3, H4

Typically wraps around~200bp of DNA

DNA betweennucleosomes is ~54 bp long

H1 binds to linker and to core particle; but in beads-on-a-string structure, it’s often absent


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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


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Next level of organization H1 interacts with DNA along linker region

Individual histones spiral along to form 30 nm fiber

See fig.19.25

Courtesy answers.com

Courtesy Johns Hopkins Univ


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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


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What about prokaryotes?

No actual histones Histone-like proteins involved

Bacterial DNA attached to scaffold in large loops (~100kb)

This makes a nucleoid


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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


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Synthesizing nucleic acids

Laboratory synthesis of nucleic acids requires complex strategies

Functional groups on the monomeric units are reactive and must be blocked

Correct phosphodiester linkages must be made

Recovery at each step must high!

10/15/2009biochemistry:nucleic acids iii organization and synthesis of dna andy howard introductory...

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