Nucleic Acids

James Watson, Francis Crick, Rosalind Franklin

Modern molecular biology really started with ___________, __________, and ________ ‘s 1953 discovery of DNA’s structure

Gregor Mendel

• discovered the laws of heredity in 1865

• Studied well-defined characteristics (unigenic trait) rather than gross morphology like size (multigenic trait – led others nowhere)

• Developed concepts of genes as units of heredity that can be dominant or recessive

  
  

Phenotype

Physical appearance

Genotype

gene complement of phenotype

Friedrich Miescher

• discovered nucleic acid in 1869

• Obtained pus from soldier’s wounds - good white blood cell source

• Digested alcohol-washed pus with pepsin (a protease to free DNA), extracted with salts and acid, obtained cell nuclei (1st subcellular fractionation!)

Walter Sutton, Theodor Boveri

• made connection between genetics and cell biology in 1900

• recognized chromosomes’ behavior during meiosis similar to behavior of Mendel's gametes during meiosis –diploid chromosomes segregate  each gamete gets 1 chromosome

• Led to realization that genes were located on chromosomes

Frederick Griffith (1928)

• found that mixing heat-killed crude pathogenic bacteria could change non-pathogenic bacteria into killers

• Hypothesis: Material in dead bacterial cells can genetically transform living bacterial cells

• Conclusion: A chemical substance from one cell is capable of genetically transforming another cell

Protein

Most biochemists thought genes were _________ until 1944

Oswald Avery

• An ex-Haligonian, ____________, used DNA purified from dead pathogenic cells to transform non-pathogens into pathogens

• Purified deoxyribonuclease enzyme destroyed transforming factor (DNase has no effect on RNA or protein)

• Purified RNA, lipids, and protein failed to transform bacteria, RNase or protease degradative enzymes had no effect on transformation

• First demonstration that DNA was the active genetic particle

Alfred Hershey, Martha Chase

• ____________ and ____________ (1952) demonstrated DNA to be the genetic material in a more biochemical way (measure it!)

• Only phage DNA needs to enter the cell for virus replication to occur

1950

• X-ray diffraction first used in 1938, but good pictures of DNA weren’t available until _______ (William Ashbury, Rosalind Franklin)

• These suggested helix of not 1, but 2 or 3 polynucleotide chains

• Organic chemists found DNA to have polarity (5'  3') (Alexander Todd)

• Watson & Crick’s 1st model was embarrassingly wrong, (chemically impossible), worked on new model for 1.5yrs

Clone

a molecule, cell, or organism that was produced from another single entity

Restriction Enzymes

DNA cutting enzymes (molecular scissors)

Plasmid DNA Vectors

• circular form of self-replicating DNA

• Can be manipulated to carry and clone other pieces of DNA

Polymerase Chain Reaction (PCR)

• 1983

• Specific amplification of gene sequences using custom DNA ‘primers

Watson-Crick

Original _________ model required that while one strand would be copied in a 5′ to 3′ direction, the other strand would have to be copied in a 3′ to 5’ direction (Anti-parallel!)

Antiparallel

DNA must be made in __________ fashion

Arthur Kornberg

• Demonstrated DNA synthesis in cell-free extracts of bacteria

• Identified DNA polymerase I, won Nobel prize for it (obtained mg quantities from thousands of litres of bacterial culture…)

Purines

Adenine, Guanine

Pyrimidines

Cytosine, Urasil, Thymine

H₂O

Joining of PO₄^3- to deoxyribose, or base will yield ________ (Condensation reactions in nucleotide formation)

AsO₄^3-

NASA’s Astrobiology Institute held news conference in 2010 with a major claim from published research that  GFAJ bacterium could use ________ in place of PO₄^3-

Xeno Nucleic Acids, XNAs

• Artificial nucleic acids that use same bases and phosphodiester bonds as DNA but use very different nucleoside sugars

• Still form Watson-Crick double helixes

• can be replicated from DNA with mutant polymerases, and copied back to DNA using another mutant polymerase – accurately!

• invulnerable to acids, nucleases (enzymes that cleave up DNA, RNA)

  Can perhaps be used to target viral DNA

  
  

Matthew Meselson, Frank Stahl

• Demonstrated that the two strands of the double helix

separate during DNA replication

• Used stable heavy isotope 15N to differentially label the

parental and daughter DNA strands

• Separated heavy from light DNA in cesium chloride density

gradients that exploits minor mass difference of 15N and 14N

• Led to discovery of ‘semi-conservative’ DNA replication

Stable Isotope Labelling

Semi-conservative DNA replication discovered by ______________

Distributive, Semi-Conservative, Conservative

3 possible models of DNA replication

Conservative Replication

Parental strands stay together

Semi-Conservative Replication

Mix, 1 strand each

Distributive Replication

Chopped up

1

RNA can also assume double helix shape, but with only ____, not 2 polynucleotide chains

Non-Classical Base-Pairing

RNA can assume lots of diverse tertiary structures, also _______________

Right-Handed

DNA structure is a _________ double-helix (5′ curls & points around like right

index finger grasping a cup of water)

35.6

In a DNA double-helix, on average each base pair is rotated _______° from the adjacent base pair.

T

A bonds with _______ (2 H-Bonds)

G

C bonds with ______ (3 H-Bonds)

Purines

larger, double‑ring nitrogenous bases

Pyrimidines

smaller, single‑ring nitrogenous bases

Tautomeric

Normally, each nitrogenous base has a common form that overwhelmingly predominates. But occasionally, a proton (H⁺) shifts position, and the electrons/double bonds rearrange along with it. This produces a rare _______ form.

Wrong Base

When a base changes into its rare tautomer, the positions where it can donate or accept hydrogen bonds change.

That means the rare form may temporarily pair with the __________ during DNA replication.

Nucleotide

If DNA polymerase happens to copy the DNA while the base is in that rare form, it can insert the wrong __________

Bacterial Virus

A _________ (phage) utilizes alternative base in place of A, called 2-aminoadenine

Base forms a triple bond with T, whereas A forms only a double bond (temp. stability increases)

Why? Bacteria use enzymes to destroy invading phage DNA but can’t degrade this base as easily

Biosynthesis pathway only found in 2021 (50 yrs after discovery!)

Z, P

• Synthetic biologists created 2 more bases, ‘__’ and ‘___’

• Z & P base pair, but do not pair with any other base (like C-G, A-T), are replicated faithfully

• DNA replicating enzymes (polymerases) can replicate Z & P both in vitro and in vivo

• Sequencing services, cloning all work with Z and P

A-DNA, B-DNA, Z-DNA

DNA can exist in 3 conformations:

A-DNA

• occurs in crystalline structures where the water concentration is reduced

• the structure is distorted and the bases are no longer co-planar

• Right-handed

B-DNA

• right-handed

• is the standard form in biological systems

Z-DNA

• occurs occasionally in the middle of a B-form molecule.

• Left-handed.

• Bases are co-planar in both the B and Z forms.

Propeller Twist

• The 2 members of each base pair don’t always lie in same plane

• This is called “_________”- normal in (a), twisted in (b)

• This causes alteration in width of major and minor grooves, so DNA is never a perfect helix (we just draw it like one!)

• this is dictated by local DNA sequence (like 2o structure in proteins)

Topoisomerase

• DNA replication induces positively supercoiled regions ahead of replication fork

• Must remove these “over-wound” regions otherwise replication stalls

• an enzyme needed: __________

Topology

study of geometric properties and spatial relations unaffected by the continuous change of shape or size of figures

surrounding ionic environment (water, salt), protein bound with the DNA

DNA is a flexible molecule but….

..flexibility constrained by;

1) __________________

2) __________________

10

DNA rotates once per _____ bases (36o turn per base)

Rotate

linear DNA should freely ________ to accommodate any changes

Length

long linear DNA can’t rotate due to ________, DNA-binding proteins

covalently closed circular DNA (cccDNA)

two polynucleotide backbones of (circular) DNA too wound up to be separated unless covalent bond broken

Linking Number (Lk)

number of times 2 DNA strands need to be passed over each other to be entirely separate from the other

L(k)

= T(w) + W(r)

T(w)

Twist (# of helical crossing)

W( r )

Writhe (# of Super-helical crossing)

Macromolecules

DNA is unique among biological ____________ in the simple and direct way in which its structure is related to its biological function

Double-Stranded

In the cell, DNA molecules are ____________

Complementary

Cytosine on one strand makes three hydrogen bonds with the __________ surface of guanine on the other

Watson-Crick Base Pairing

• the specific way nitrogenous bases link together in DNA and RNA through hydrogen bonds.

• Adenine (A) always pairs with Thymine (T) in DNA (or Uracil (U) in RNA) using two hydrogen bonds.

• Guanine (G) always pairs with Cytosine (C) using three hydrogen bonds.

Complementary

Since the sequence of bases in one strand dictates the sequence of bases in the other, we say that the two strands are ______________ to one another

Double Helix

The most energetically favourable confirmation for double-stranded DNA is one in which the two strands wind around one another, forming a __________

Propeller

The B-DNA crystal structure confirmed all of the essential features of the Watson and Crick model, with some interesting deviations, The DNA base pairs were not perfectly planar but rather contained various degrees of __________ twist

Nucleosome

Another key feature was that the helix axis was not perfectly straight but rather was bent by about 19 degrees. The authors duly noted that this degree of bending, when extended over longer DNA fragments, would be sufficient to allow DNA to wrap around a histone core to form a _____________, the fundamental unit of eukaryotic chromatin

Base Stacking

The vertical alignment and tight physical attraction of neighboring chemical bases in a DNA strand that holds the double helix together

20

Since both A-T and G-C base pairs have similar widths, the helix has a fairly uniform diameter of _______ Å along its length

B-DNA, 10.5

Double-stranded DNA in cells generally adopts a conformation called ________, whose helical structure repeats itself approximately every ____ bp as one looks along the double helix.

3.4

In B-DNA, each successive base pair is separated by ____ Å

Major and Minor Grooves

• Two spiral depressions that run along the outside of the DNA double helix, created because the strands' sugar-phosphate backbones are not attached to the base pairs at opposite positions (13 Å vs 9 Å width)

• The edges of the base pairs are exposed in these depressions

A-DNA

• has 11 bp per turn

• base pairs tilted relative to the DNA axis, as well as being displaced farther from the DNA axis than they are in B-DNA

• Has a narrower, deeper major groove and a shallower, wider minor groove than B-DNA

• right-handed

A-DNA

• can be induced by some proteins that form a complex with DNA

• is particularly important in RNA structure, since this is the only type of double helix that double-stranded RNA can form

Bend

Regions of the DNA double helix containing successive A-T followed by T-A base pairs tend to ______ more readily than helices with G-C steps

Z-DNA

• An unusual type of DNA helix that spirals in the opposite direction, and is therefore left-handed

• can form under special conditions in DNA strands with alternating G and C bases

• favoured by a chemical modification of cytosine, methylation, and high salt concentrations

• promoted by torsional stress on the DNA

Negative Supercoils

An initial unwinding of the DNA - which is in the clockwise direction and is equivalent to separating the DNA strands - leads to accumulation of ____________

Negative, Positive

__________ supercoiling involves underwinding the DNA double helix, while __________ supercoiling involves overwinding it

Positive Supercoils

An initial twisting of the DNA in the counterclockwise direction leads to formation of _____________

Immobilized

Supercoiling can also occur in linear fragments of DNA in which the ends are _____________, and therefore not free to rotate and release superhelical tension

Linking Number (Lk)

the number of times one strand of duplex DNA wraps around the other

Twist (Tw)

• the number of turns in the DNA helix in a given fragment of DNA

• positive sign is or a right-handed helix, a negative sign is for a left-handed helix

• e.g. a 105 bp fragment of relaxed B-DNA has a _____ of 10 (=105 bp / 10.5 bp per turn)

0

A relaxed plasmid has a writhe of ____

Positive

_______ supercoiled DNA has a value of writhe greater than 0

Negative

____________ supercoiled DNA has a value of writhe that is less than 0

Topoisomerase

• Introduce or remove supercoils from DNA in the cell

• Change the supercoiling by introducing a transient break in either one of both DNA strands in the double-stranded DNA, depending on the type of enzyme

DNA

It is the ability of RNA to form base pairs with ______, as well as with RNA, that allows cells to synthesize RNA copies of genes from a DNA or RNA template

2’ OH

The presence of the ______ on ribose and the distinct chemical structure of uracil, as compared with thymine, allow RNA to adopt diverse structures that are not readily formed by DNA

Chemical Modification

Many RNA molecules require ___________ of some of their nucleotides in order to become fully functional. These modifications, which alter the chemical structure of the bases, are introduced after the RNA is synthesized

2’-deoxyribose

The nucleotide consists of a phosphate joined to a sugar, known as __________, to which a base is attached

Nucleoside

The sugar and base alone are called a ____________

Nucleotide

Adding a phosphate (or more than one phosphate) to a nucleoside creates a __________.

Phosphodiester Linkage

Nucleotides are joined to each other through _____________

5’ to 3’

DNA is always synthesized in the __________ direction

Template

DNA polymerase is unusual enzyme since it needs a _______ to work from

Xeno Nucleic Acids, XNAs

use different sugars than DNA & has practical applications

Tautomeric

Nucleic acid bases are not static but can take alternative ____________ forms

B-DNA

is the major biological conformation of DNA (but Z occasionally)

Positive

As helicases and polymerases move along DNA, unwinding and copying it as they go, they generate _______ supercoils in the double-stranded helical DNA in front of them. These _________ supercoils are a consequence of the fact that the DNA template unwinds as the two strands separate

10

During DNA replication, one full turn of the double helix is unwound for every ____ bp of DNA that is replicated

-1

Twist (Tw) changes by ____ for each turn of the helix that is unwound