Download DNA structure 2008

DNA Structure
Chapters 10&11
Biochemistry
by
Reginald Garrett and Charles
Grisham
Igor Chesnokov
Department of Biochemistry and Molecular Genetics
Office Phone # 934-6974
E-mail: [email protected]
Garrett and Grisham, Biochemistry, Third Edition
Central Dogma of Molecular Biology
Information Transfer in Cells
• Information encoded in a DNA molecule is
transcribed via synthesis of an RNA
molecule
• The sequence of the RNA molecule is
"read" and is translated into the sequence
of amino acids in a protein.
Garrett and Grisham, Biochemistry, Third Edition
DNA is the repository of genetic information
(replication), RNA serves in the expression of
this information through the processes of
transcription and translation.
Garrett and Grisham, Biochemistry, Third Edition
Essential Questions
• What are the structures of the nucleotides?
• How are nucleotides joined together to form
nucleic acids?
• What is the higher-order structure of DNA
• What are the biological functions of
nucleotides and nucleic acids?
Garrett and Grisham, Biochemistry, Third Edition
Outline1
• What Is the Structure and Chemistry of
Nitrogenous Bases?
• What Are Nucleosides?
• What Is the Structure and Chemistry of
Nucleotides?
• What Are Nucleic Acids?
• What Are the Different Classes of Nucleic
Acids?
Garrett and Grisham, Biochemistry, Third Edition
Outline2
• What Sorts of Secondary Structures Can
Double-Stranded DNA Molecules Adopt?
• Can the Secondary Structure of DNA Be
Denatured and Renatured?
• What is the Tertiary Structure of DNA?
• What Is the Structure of Eukaryotic
Chromosomes?
Garrett and Grisham, Biochemistry, Third Edition
Structure and Chemistry of
Nitrogenous Bases
The bases of nucleotides and nucleic
acids are derivatives of either pyrimidine
or purine.
• Pyrimidines
– Cytosine (DNA, RNA)
– Uracil (RNA)
– Thymine (DNA)
• Purines
– Adenine (DNA, RNA)
– Guanine (DNA, RNA)
Garrett and Grisham, Biochemistry, Third Edition
The pyrimidine ring system (six-membered heterocylic aromatic ring
with two nitrogen atoms); by convention, atoms are numbered as
indicated. (b) The purine ring system consists of two rings (pyrimidine
and imidazole), nine atoms numbered as shown.
Both are relatively insoluble in water due to aromatic character.
Garrett and Grisham, Biochemistry, Third Edition
The common pyrimidine bases – cytosine (DNA and RNA), uracil
(RNA), and thymine (DNA).
Garrett and Grisham, Biochemistry, Third Edition
The common purine bases —adenine and guanine (found in both DNA
and RNA).
Garrett and Grisham, Biochemistry, Third Edition
What Are Nucleosides?
•
•
•
•
•
•
When a base is linked to a sugar it forms a Nucleoside.
The sugars are Pentoses
D-ribose (in RNA)
2-deoxy-D-ribose (in DNA)
The difference - 2'-OH vs 2'-H
This difference affects secondary structure and stability
of nucleic acids
Garrett and Grisham, Biochemistry, Third Edition
Pentose in a five membered ring is known as furanose. Furanose
structures (ribose and deoxyribose) are presented above. Presence of a
hydroxyl group at the 2-position has dramatic effect on secondary
structures available to DNA and RNA as well as their susceptibilities to
hydrolysis. DNA is more stable.
Garrett and Grisham, Biochemistry, Third Edition
continued …
• Base in nucleosides is linked to a sugar via a
glycosidic bond
• 1’C of sugar links to 9 N of purine or to the 1 N of
pyrimidine base
• Nucleosides named by adding –idine (ur-idine)
to the root name of a pyrimidine or –osine
(aden-osine) to the root name of a purine
• Sugars make nucleosides more water-soluble
than free bases
Garrett and Grisham, Biochemistry, Third Edition
b-Glycosidic bonds link nitrogenous
bases and sugars to form nucleosides.
1’C links to 9 N of purine and to the
1 N of pyrimidine base.
Garrett and Grisham, Biochemistry, Third Edition
The common ribonucleosides—cytidine, uridine, adenosine, and
guanosine. Also, inosine (uncommon nucleoside) is drawn.
Garrett and Grisham, Biochemistry, Third Edition
Structure and Chemistry of
Nucleotides
Nucleotides or Nucleoside phosphates result
when phosphoric acid is esterified to a sugarOH group of nucleoside (at C-5)
•Most nucleotides are ribonucleotides
•Nucleotides have acidic properties
•Nucleic acids, which are polymers of
nucleosides derive their names from the acidity
of phosphate groups.
Garrett and Grisham, Biochemistry, Third Edition
Structures of the four common ribonucleotides —AMP, GMP,
CMP, and UMP—together with their two sets of full names, for
example, adenosine 5'-monophosphate and adenylic acid. Also
shown is the nucleoside 3'-AMP (uncommon, product of
hydrolysis).
Garrett and Grisham, Biochemistry, Third Edition
Functions of Nucleotides
• NTPs and dNTPs are substrates for nucleic acids
• Nucleoside 5'-triphosphates are carriers of energy.
Energy is stored in phosphoric bonds.
• Bases serve as recognition units or information symbol
but not involved in the biochemistry of metabolism
• ATP is central to energy metabolism (energy currency)
• GTP drives protein synthesis
• CTP drives lipid synthesis
• UTP drives carbohydrate metabolism
• Cyclic nucleotides are signal molecules and regulators
of cellular metabolism and reproduction
Garrett and Grisham, Biochemistry, Third Edition
Phosphoryl and pyrophosphoryl group transfer, the major biochemical
reactions of nucleotides. Phosphoric bonds are prime source of chemical
energy to do biological work (ATP, GTP, CTP and UTP, also deoxycounterparts).
Garrett and Grisham, Biochemistry, Third Edition
Cyclic nucleotides are cyclic
phosphodiesters.
Structures of the cyclic nucleotides
cAMP and cGMP. Phosphoric acid
is esterified to two of the available
ribose hydroxyl groups.
Important! They are regulators of
cellular metabolism and are found
in all cells.
Garrett and Grisham, Biochemistry, Third Edition
What are Nucleic Acids?
Polynucleotides!
•Facts to remember:
•Linear polymers of nucleotides linked 3‘C to 5‘C
by phosphodiester bridges
•Ribonucleic acid and Deoxyribonucleic acid
•Know the shorthand notations
•Sequence is always read 5' to 3'
•In terms of genetic information, this corresponds
to "N to C" in proteins
Garrett and Grisham, Biochemistry, Third Edition
3'-5' phosphodiester bridges link nucleotides together to form polynucleotide
chains.
Garrett and Grisham, Biochemistry, Third Edition
Shorthand notations for polynucleotide structures.
Furanoses are represented by vertical lines; phosphodiesters are represented
by diagonal slashes in this shorthand notation for nucleic acid structures.
Bases serve as distinctive side chains and give the polymer it’s unique
identity.
Garrett and Grisham, Biochemistry, Third Edition
What Are the Different Classes of
Nucleic Acids?
• DNA - one type, one purpose
• RNA - 3 (or 4) types, 3 (or 4) purposes
– ribosomal RNA - the basis of structure and
function of ribosomes
– messenger RNA - carries the message
– transfer RNA - carries the amino acids
– Small nuclear RNA
– Small non-coding RNAs
Garrett and Grisham, Biochemistry, Third Edition
DNA & RNA Differences?
Why does DNA contain thymine?
• Cytosine spontaneously deaminates to
form uracil (C-G pair could result in U-A)
• Repair enzymes recognize these
"mutations" and replace these Us with Cs
• But how would the repair enzymes
distinguish natural U from mutant U
• Nature solves this dilemma by using
thymine (5-methyl-U) in place of uracil
Garrett and Grisham, Biochemistry, Third Edition
Deamination of cytosine forms uracil.
Garrett and Grisham, Biochemistry, Third Edition
The 5-methyl
group on
thymine labels it
as a special kind
of uracil.
Garrett and Grisham, Biochemistry, Third Edition
DNA & RNA Differences?
Why is DNA 2'-deoxy and RNA is not?
• Vicinal -OH groups (2' and 3') in RNA
make it more susceptible to hydrolysis
• DNA, lacking 2'-OH is more stable
• This makes sense - the genetic material
must be more stable
• RNA is designed to be used and then
broken down
Garrett and Grisham, Biochemistry, Third Edition
How Do Scientist Determine the
Primary Structure of Nucleic Acids?
Sequencing Nucleic Acids
• Chain termination method (dideoxy method),
developed by F. Sanger
• Base-specific chemical cleavage, developed
by Maxam and Gilbert
• Both use autoradiography - X-ray film
develops in response to presence of
radioactive isotopes in nucleic acid
molecules
Garrett and Grisham, Biochemistry, Third Edition
DNA Replication
• Chain termination method is based on biochemistry
of DNA replication
• Each strand of the double-helical DNA molecule
must be copied in complementary fashion by DNA
polymerase
• Each strand can serve as a template for copying
• DNA polymerase requires template and primer
• Primer: an oligonucleotide that pairs with the end of
the template molecule to form dsDNA
• DNA polymerases add nucleotides in 5'-3' direction
Garrett and Grisham, Biochemistry, Third Edition
DNA polymerase copies ssDNA in vitro in the presence of the four
deoxynucleotide monomers. A double-stranded region of DNA must be
artificially generated by adding a primer, an oligonucleotide capable of
forming a short stretch of dsDNA by base pairing with the ssDNA. The
primer must have a free 3'-OH end from which the new polynucleotide
chain can grow as the first residue is added in the initial step of the
polymerization process.
Garrett and Grisham, Biochemistry, Third Edition
Chain Termination Method
Based on DNA polymerase reaction
• Run four separate reactions
• Each reaction mixture contains dATP,
dGTP, dCTP and dTTP, one of which is P32-labelled
• Each reaction also contains a small
amount of one dideoxynucleotide: either
ddATP, ddGTP, ddCTP or ddTTP
Garrett and Grisham, Biochemistry, Third Edition
Chain Termination Method
• Most of the time, the polymerase uses
normal nucleotides and DNA molecules
grow normally
• Occasionally, the polymerase uses a
dideoxynucleotide, which adds to the chain
and then prevents further growth in that
molecule
• Random insertion of dd-nucleotides leaves
(optimally) at least a few chains terminated
at every occurrence of a given nucleotide
Garrett and Grisham, Biochemistry, Third Edition
Chain Termination Method
• Run each reaction mixture on electrophoresis gel
• Short fragments go to bottom, long fragments
on top
• Read the "sequence" from bottom of gel to top
• Convert this "sequence" to the complementary
sequence
Garrett and Grisham, Biochemistry, Third Edition
The chain termination
or dideoxy method of
DNA sequencing. (a)
DNA polymerase
reaction. (b) Structure
of dideoxynucleotide.
(c) Four reaction
mixtures with
nucleoside
triphosphates plus one
dideoxynucleoside
triphosphate. (d)
Electrophoretogram.
Note that the
nucleotide sequence as
read from the bottom
to the top of the gel is
the order of nucleotide
addition carried out by
DNA polymerase.
Garrett and Grisham, Biochemistry, Third Edition
Chemical Cleavage Method
•
•
•
•
Not used as frequently as Sanger's
Start with ssDNA labelled with P-32 at one end
Strand is cleaved by chemical reagents
Assumption is that strands of all possible
lengths will be produced, each cleaved at just
one of the occurrences of a given base.
Fragments are electrophoresed and sequence is
read
Garrett and Grisham, Biochemistry, Third Edition
A photograph of the
autoradiogram from
an actual
sequencing gel. A
portion of the DNA
sequence of nit-6,
the Neurospora gene
encoding the
enzyme nitrite
reductase.
Garrett and Grisham, Biochemistry, Third Edition
Structure of DNA
summary
The fundamental structure of DNA is a Double
Helix stabilized by hydrogen bonds!
•DNA consists of two polynucleotide strands
wound together to form DNA double helix
•Strands run in opposite direction (antiparallel)
•Two strands are held together through interchain hydrogen bonds
•These H bonds pair the bases of nucleotides in
one chain to complementary bases in the other –
base pairing.
Garrett and Grisham, Biochemistry, Third Edition
The DNA Double Helix
•Erwin Chargaff had the pairing data, but didn't
understand its implications. Chargaff rule – the
number of purine residues equals the number of
pyrimidine residues in all organisms (A=T, G=C).
•Rosalind Franklin's X-ray fiber diffraction data
was crucial (Helix!)
•Watson-Crick model of the DNA double helix.
Garrett and Grisham, Biochemistry, Third Edition
(a) Double-stranded
DNA as an
imaginary
ladderlike structure.
(b) A simple righthanded twist
converts the ladder
to a helix.
Garrett and Grisham, Biochemistry, Third Edition
A model of DNA double helix.
The nucleotides are linked covalently
by phoshodiester bonds through the
3’-hydroxil (-OH) group of one sugar
and the 5’-phosphate (P) of the next.
Two DNA strands are held together
by hydrogen bonds between the
paired bases. Two hydrogen bonds
form between A and T, while three
form between G and C. The bases can
pair in this way only if the two
polynucleotide chains that contain
them are antiparallel to each other.
The coiling of the two strands around
each other creates two groves in the
double helix.
Consequences – each strand of DNA
contains a sequence of nucleotides
that are exactly complementary to
the sequence of its partner strand.
Garrett and Grisham, Biochemistry, Third Edition
The bases in a base
pair are not directly
across the helix axis
from one another
but rather are
slightly displaced.
This displacement,
and the relative
orientation of the
glycosidic bonds
linking the bases to
the sugar–phosphate
backbone, leads to
differently sized
grooves in the
cylindrical column
created by the
double helix, the
major groove and
the minor groove,
each coursing along
its length.
How grooves are formed?
Garrett and Grisham, Biochemistry, Third Edition
Comparison of A, B, Z DNA
ABZs of DNA Secondary Structure
• A: right-handed, short and broad, 2.3 Å, 11
bp per turn (dehydrated DNA, probably
does not exist in vivo)
• B: right-handed, longer, thinner, 3.32 Å, 10
bp per turn, (most common)
• Z: left-handed, longest, thinnest, 3.8 Å, 12
bp per turn (G-C rich regions)
Garrett and Grisham, Biochemistry, Third Edition
Comparison of the A-, B-, and Z-forms of the DNA double helix. The distance
required to complete one helical turn is shorter in A-DNA than it is in B-DNA.
The alternating pyrimidine–purine sequence of Z-DNA is the key to the “lefthandedness” of this helix.
Garrett and Grisham, Biochemistry, Third Edition
Can the Secondary Structure of DNA
Be Denatured and Renatured?
•
•
•
•
Important for Study of Genome complexity
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 temperature is lowered, the absorbance
drops, reflecting the re-establishment of stacking
Garrett and Grisham, Biochemistry, Third Edition
Steps in the thermal denaturation and renaturation of DNA. Renaturation
(re-annealing) depends on DNA concentration and time. The nucleation
phase of the reaction is depending on sequence alignment of the two strands.
This process takes place slowly because it takes time for complementary
sequences to encounter one another in solution and then align themselves in
register. Once the sequences are aligned, the strands zipper up quickly.
Garrett and Grisham, Biochemistry, Third Edition
These c0t curves show
the rates of reassociation of denatured
DNA from various
sources and illustrate
how the rate of reassociation is inversely
proportional to genome
complexity. The DNA
sources are as follows:
poly A+poly U, a
synthetic DNA duplex of
poly A and poly U
polynucleotide chains;
mouse satellite DNA, a
fraction of mouse DNA
in which the same
sequence is repeated
DNA of a more complex bacteriophage; E. coli DNA,
many thousands of
bacterial DNA; calf DNA (nonrepetitive fraction),
times; MS-2 dsRNA, the
mammalian DNA (calf) from which the highly
double-stranded form of
repetitive DNA fraction (satellite DNA) has been
RNA of MS-2, a simple
removed. Arrows indicate the genome size (in bp) of
bacteriophage; T4 DNA,
the various DNAs.
the
Garrett and Grisham, Biochemistry, Third Edition
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,
underwound (-) or overwound (+).
• Enzymes called topoisomerases or gyrases
can introduce or remove supercoils
Garrett and Grisham, Biochemistry, Third Edition
There are toroidal and interwound varieties of DNA supercoiling. (a) The DNA is
coiled in a spiral fashion about an imaginary toroid. (b) The DNA interwinds and
wraps about itself. (c) Supercoils in long, linear DNA arranged into loops whose ends
are restrained—a model for chromosomal DNA.
Garrett and Grisham, Biochemistry, Third Edition
A simple model for the action of
bacterial DNA gyrase
(topoisomerase II).
The A-subunits cut the DNA
duplex (1) and then hold onto the
cut ends (2). Conformational
changes occur in the enzyme that
allow a continuous region of the
DNA duplex to pass between the
cut ends and into an internal
cavity of the protein. The cut
ends are then re-ligated (3), and
the intact DNA duplex is released
from the enzyme. The released
intact circular
DNA now contains two negative
supercoils as a consequence of
DNA gyrase action (4).
Garrett and Grisham, Biochemistry, Third Edition
Supercoiled DNA in a toroidal form wraps readily around protein “spools.”
A twisted segment of linear DNA with two negative supercoils (a) can
collapse into a toroidal conformation if its ends are brought closer together
(b) Wrapping the DNA toroid around a protein “spool” stabilizes this
conformation of supercoiled DNA (c)
Garrett and Grisham, Biochemistry, Third Edition
Structure of Eukaryotic
Chromosomes
• 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
Garrett and Grisham, Biochemistry, Third Edition
Nucleosome Structure
• Chromatin, the nucleoprotein complex,
consists of histones and non-histone
chromosomal proteins
• Histone octamer structure has been solved
(without DNA by Moudrianakis, and with
DNA by Richmond)
• Non-histone proteins are regulators of
gene expression
Garrett and Grisham, Biochemistry, Third Edition
A diagram of the histone
octamer.
Nucleosomes consist of two
turns of DNA supercoiled
about a histone “core”
octamer.
Garrett and Grisham, Biochemistry, Third Edition
Nucleosomes as seen in the electron microscope.
“Beads on a string” – partially unfolded chromatin.
Garrett and Grisham, Biochemistry, Third Edition
The 2-nm DNA helix is wound
twice around histone octamers to
form 10-nm nucleosomes.
These nucleosomes are then
wound in solenoid fashion with six
nucleosomes per turn to form a
30-nm filament.
The 30-nm filament forms long
DNA loops, each containing about
60,000 bp, which are attached at
their base to the nuclear matrix.
Eighteen of these loops form a
miniband unit of a chromosome.
Approximately 106 of these
minibands occur in each
chromatid of human chromosome
4 at mitosis.
Garrett and Grisham, Biochemistry, Third Edition
Each DNA molecule that forms a linear chromosome must contain
a Centromere, two Telomeres and Replication Origins
Sequence of events a typical chromosome follows during the cell cycle:
The DNA replicates in interphase beginning at the origins of replication and proceeding bidirectionally from the
origins across the chromosome. Many origins are required to ensure that the entire chromosome can be
replicated rapidly. In M phase, the centromere attaches the duplicated chromosome to the mitotic spindle
so that one copy is distributed to each daughter cell during mitosis. The centromere also helps to hold the
duplicated chromosomes together until they are ready to be moved apart. The telomeres form special caps
at each chromosome end.
Garrett and Grisham, Biochemistry, Third Edition