Biochemistry of Medicinals I Phar 6151 Chapter One
Instructor: Dr. Natalia Tretyakova, Ph.D.
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Professor
                                                                                       1.    Introduction   
||<----------Nucleic Acids------------------->||
DNA                                         RNA
Central Dogma of Molecular Biology:
Genetic information is stored in the form of DNA,
                                                                                      but gene action is expressed in the form of proteins:

                                                         Central Dogma of Biology
 =========Þ = DNA  =========Þ  RNA  =========Þ   Proteins   =========Þ   Cellular Action
  Replication              transcription             translation                 =========Þ Signal transduction
         How is DNA replicated?
         How is DNA synthesis initiated?
         How is DNA fidelity maintained?
         What kinds of DNA defects can occur?
During the past 40 years there has been an explosion of information concerning the synthesis and manipulation of DNA in biological systems.  The knowledge gained from the study of nucleic acids has given rise to everything from the field of biotechnology to potential remedies for genetic diseases.
           Cancer: ex.,  Xeroderma pigmentosum
           Genetic diseases: ex., cystic fibrosis, sickle-cell anemia, inborn errors of metabolism
           Genetic typing: ex., drug metabolism (pharmaco-genetics)
           Rational drug design: ex., antitumor drugs
           Genetic engineering, e.g insulin, growth hormone
II.   Building Blocks of Nucleic Acids
DNA is a bio-polymer of repeating nucleotide units. Each nucleotide contains a nitrogen base
(purine or pyrimidine), a pentose sugar, and a phosphate:

               Nitrogen bases present in nucleic acids: 
           Purine                    Guanine (G)                Adenine (A)
Pyrimidine                  Cytosine (C)            Thymine (T)                       Uracyl (U)
Nomenclature of nucleobases, nucleosides, and nucleotides
nucleo-base nucleoside mono-nucleotide  
  Adenine   (A)
Guanine  (G)
Thymine (T)
Cytosine  (C)
Uracyl     (U)
Deoxyadenosine (dA)
Deoxyguanosine (dG)
Deoxythymidine (dT)
Deoxycytidine     (dC)
Uridine                 (U)
Deoxyadenosine 5’-phosphate (5’-dAMP)
Deoxyguanosine 5’-phosphate (5’-dGMP)
Deoxythymidine 5’-phosphate (5’-dTMP)
Deoxycytidine     5’-phosphate (5’-dCMP)
            Uridine     5’-phosphate (5’- UMP)

Anty conformation             Syn conformation                  D-de-oxy-ribose               D-ribose

Nucleosides are converted intracellularly to mono-, di- and tri-phosphates by nucleoside kinases:

         Nucleoside                                     Mono phosphate                                  Di-phosphate ¯¬ATP 4- 
                              Tri-phosphate NTP 4-
                                             III. DNA Structure
A. Linking in DNA biopolymer.
Nucleotide units are linked to each other through phospho-di-ester bonds:

       B. Model of DNA Structure
Based on biochemical data for the frequency of purine and pyrimidine bases appearing with one another in DNA and X-ray diffraction data from DNA fibers, Watson and Crick proposed the following model for DNA structure.
1. Two 2 polynucleotide chains running in opposite directions wind about a common axis to form
                                                                                                                                               a right-handed helix.
2. The bases are in the interior while the phosphates and the ribose sugar moieties are on the outside of the helix.
The planes of the bases are perpendicular to the helix axis, and each base is rotated 36° relative to its neighbor base.
3. The two 2 chains are held together by stacking interactions, hydrogen bonds, and electrostatic  forces.
4. Base pairing is in the order A=T , which contains two 2 hydrogen bonds, and GºC , which contains three
                                                                         3 hydrogen bonds.
             DNA                                                RNA                             Anti-parallel Strands of DNA
C. Types of DNA Structure.
DNA is very flexible and capable of a great deal of structural complexity.
                                                                                                      There are three 3 general types of structures.
1.         B-DNA = most prevalent form in chromosomes (the form discovered by Watson and Crick)
• Planes of bases are nearly perpendicular to the helix axis. 3.4 Å helix rise between adjacent base pair.
Sugars are in 2’-endo conformation and are nearly perpendicular to the planes of the bases.
Bases have a helical twist of 36°  it takes 10.4 bases per helix turn (360°)
• Right handed helix
• Contains a minor and a major groove that wind about the outside of the helix
Wide and deep major groove
Narrow and deep minor groove
2.               A-DNA = dehydrated DNA; high ionic strength; ds RNA; RNA-DNA hybrids
Bases are tilted 23° with respect to helical axis. 2.56 Å distance between adjacent base pairs.
                                                                                                     Like in B-DNA, glycosidic bonds are anti.
• Differs from B-DNA due to changes in ribose puckering (3’-endo favored over 2’-endo)
• Wider and flatter than B-DNA, contains an axial hole in the middle
11 bases per helix turn
Right handed helix, 20 Å helix diameter
Narrow and deep major groove
Very broad and shallow minor groove

3. Z-DNA
= prevalent form for short oligonucleotides for sequences of alternating pyrimidines (T , C) and             purines (G, C) in the presence of high ­ salt concentration C
      Glycosidic angle alternates between Syn (purines) and Anti (pyrimidines).
      Backbone zig-zags, sugar pucker alternates between 2’ endo and 2’ exo
      12 bases per helix turn
      Left-handed helix, 18 Å helix diameter
      Flat major groove
      Narrow and deep minor groove
D. DNA Properties facilitating its recognition by proteins
Major and minor groove of the double helix are lined by potential hydrogen bond donors and acceptors: oxygen and nitrogen atoms displayed on different sides of the AT and GC base pairs. The key sequence recognition features are governed by the hydrogen bonding patterns:
       N       H                              O        H
                        Major groove

                                 Minor groove
Major Groove of B-DNA
       12 Å wide, 8.5 Å deep
Minor Groove of B-DNA
       6Å wide, 7.5Å deep
The major groove is bigger and has more opportunities for H-bond formation that facilitate its ability to interact with proteins. DNA binding molecules 
IV. Biophysical properties of DNA
Biophysical properties of DNA allow it to fulfill its biological role and also make it possible to manipulate
                                                                                                                                                              DNA in vitro.
1.                DNA melting and renaturation
The non-covalent bonds that stabilize double helix can be disrupted by heating  ­T or
        by reducing ¯ salt concentration C..The process of strand separation is called denaturation or melting.

2.                DNA precipitation
Most techniques for isolation and purification of cellular DNA use the ability of DNA to precipitate out ¯ of an aqueous solution in the presence of high ­ salt and organic solvent (ethanol, diethyl ether, acetone).
The DNA pellet can then be collected free of other cellular constituents.
V. Topology of DNA
DNA double helix does not exist in a cell as a long straight rod: it is coiled in space to fit the dimensions of the cell. If the ends of DNA polymer are fixed in place, such as in circular DNA (plasmid DNA), it can be supercoiled (twisted around its own axis ­).
Negative supercoiling: double helix is twisted in the direction opposite ­¯ to the direction of the helix (underwound DNA). Negative supercoiling generates a torsional force that helps unwind DNA when required for replication and transcription.
Positive supercoiling: double helix is twisted in the same direction ­­ as the winding of the helix (overwound DNA).
1.       The Role of Linking
DNA conformations differing in their degree of supercoiling (topoisomers) are defined by their
linking number. Linking number (Lk) is the number of times Lk one strand winds around the other in the right-handed direction.
Tw (twist) is the number of turns of the helix. Tw = # of base pairs per turn ; Tw = # bp / turn 
Wr (Writh) is number of super-helical turns
Lk = Tw + Wr
For a closed circle, Lk = const is constant and cannot be changed without breaking DNA stand. Therefore, if Wr is changed due to positive (+) or negative (—) supercoiling, Tw has to change in the opposite direction:
If Lk  = const ,         DTw = - DWr
If Lk  is changed, it is more energetically favorable to change Wr, not Tw.

2.       Types of Conformers: example of a 260 bp B-DNA

Relaxed circle, no supercoiling:
        Lk = 25, Tw = 260/10.4 = 25, Wr = 0
If we disconnect the circle and unwind DNA by two 2 turns:
        Lk = 23, Tw = 23, Wr = 0
This DNA can fold to the structure containing two 2 superhelical turns (Negative supercoiling, more energetically favorable)
       Lk = 23, Tw = 25, Wr = -2
       Densely packed and compact
Natural DNA is negatively supercoiled (underwound). Small ¯ amounts of unwinding that occurs in negatively supercoiled DNA is useful for processes requiring strand separation, such as replication, recombination and transcription.
                 VI. Enzymes that Change DNA
A.    Enzymes that cut DNA
Endo-nucleases: enzymes that cleave internal phosphodiester bonds within DNA chain
Exo-nucleases: cleave nucleotides one 1 at a time from an end of a polynucleotide chain; may be specific  for 5’ end or the 3’ end of DNA chain, thus the name 5’--->3’ and 3’--->5’ exonucleases.
Examples: snake venom phospho-di-esterase (3’--->5’), calf intestinal phospho-di-esterase (5’--->3’)
Restriction Endonucleases:
• originate from bacteria for protection against unwanted DNA (degrade foreign DNA)
• recognize unmethylated DNA (their own DNA is methylated)
Type II restriction enzymes cut DNA at palindromic (two 2 fold axis ­¯ of symmetry) recognition sites
• enzyme has same two 2 fold axis ­¯ of symmetry
• highly specific; recognition takes place in the major groove of DNA via hydrogen bond formation between amino acids AA of the enzyme (Arg, Glu) and DNA nucleo-bases
B.      Ligation of DNA
Enzymes that join (ligate) DNA fragments are called DNA Ligases
     catalyzes the formation of phospho-di-ester bond between 3'- OH and 5'- phosphate
     requires energy DG<0 : bacteria use NAD+ and animal cells use ATP
     needed for DNA repair, DNA synthesis
     only acts on double stranded DNA not single stranded
     forms phospho-amide bond with active site lycine
    Mechanism of DNA ligase
( E = enzyme, ATP
4- = adenosine triphosphate, PPi = pyrophosphate -O(HO)PO-O-OP(OH)O- ):
1)         E + ATP4- + H3O+ ---> E-AMP1- + -O(HO)PO-O-OP(OH)O- + H2O
2)         E-AMP1- + -O-P-5’-DNA---> E + AMP-O---O-P-5’-DNA
3)         DNA-3’-OH + AMP-O---O-P-5’-DNA ---> DNA-3’-O--O-P-5’-DNA + AMP2- + H3O+ 
DNA-3’-OH +    + AMP2- + H2O +
             + ATP4- +                                                                                   + -O(HO)PO-O-OP(OH)O- 
Molecular biologist
create large DNA molecules with special characteristics by joining DNA fragments with ligases.

           C. Enzymes that change DNA Supercoiling 
1.            Enzymes that introduce torsional stress (supercoiling) into DNA are called DNA gyrases.
     require energy DG<0 , ATP
     only act on double stranded DNA not single stranded
     each round introduces two 2 negative (—) supercoils (  Wr = -2 ) 
     can act repeatedly to introduce more supercoiling
Mechanism of DNA gyrase:
1.   ATP-catalyzed cleavage of both DNA strands.
2.   5’-phosphate terminus of each strand is linked to a tyrosine residue in the enzyme.
3.   Pass DNA through the gap between fixed ends.
4.   Seal (ligate) DNA strands
5.   ATP – catalyzed release of DNA

Left Handed DNA                           Right Handed DNA
Gyrases are very useful targets for antibiotics, since negative supercoiling (—) is necessary for the replication
                                                                                                                                                                   of bacteria
      - Novobiocin       = inhibits ATP binding to gyrase
      - Ciprofloxacin  = inhibits cleavage and re-ligation of DNA strands
      - Nalidixic Acid = inhibits cleavage  and re-ligation of DNA strands
2.         Enzymes that relax torsional stress (reduce supercoiling) of DNA are called topoisomerases
     use the energy DG<0 stored in supercoiled DNA
     only act on double stranded DNA
     can act repeatedly to reduce super-coiling in steps of Wr = 1
          Mechanism of DNA topoisomerase I
1)   Cut one strand of DNA generating a transient strand break.
2)   5’ phosphate is covalently linked to a Tyr residue of the enzyme.
3)   Pass the intact strand through the gap.
4) 3’ OH on the other end of DNA chain attacks the enzyme-DNA intermediate restoring
                                                                                                                                        the original DNA chain.


Negatively supercoiled DNA                                       Positively supercoiled DNA
Because topo-isomerases are necessary for cancer cells to divide, these enzymes are excellent targets for the development of antitumor drugs, such as the campthecican analog topotecan:
   Topotecan (Hycamtin)