Transcription Translation
 
Tentative Lecture schedule    Biochemistry of Medicinals I                 Phar 6151     Part 2 – Nucleic Acids
 
Instructor: Dr. Natalia Tretyakova, Ph.D. «hyperlink "mailto:[email protected]"»   -   6-3432
 
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Professor  e-mail: [email protected]
 
                                                                  DNA Topology
 
1. DNA has to be coiled to fit inside the cell  - interior the nucleus


Average human chromosome: 2.05•108 nucleotides (8.75 cm) 24*8.75=210.00 23.529410*8.75=205.882338


Nucleus diameter: about 10 µm


DNA polymers have to be folded into to fit into the nucleus (tertiary structure 3°). 


2. Many viruses and bacteria have circular genoms – tertiary structure 3°.
 
DNA Topology:
 
             Negative supercoiling :


DNA is twisted in the direction opposite ­¯ to the direction ¯ of the double helix (underwound)


            Positive supercoiling :


DNA is twisted in the same direction ¯¯ as the direction ¯ of the double helix (overwound)
 
DNA Topology: linking number
 
Topoisomers can be quantitatively defined by the linking number (Lk).


Lk is the number of times a strand of DNA winds in the
            right handed direction around the helix axis when the axis is constrained.


Tw (twist) is the helical winding of the strands around each other.


Wr (Writh) is the number of superhelical turns


Lk = Tw + Wr, if Lk = const.,  DTw = -  DWr


DNA Topology: example
 
               ®              1               ®                     5                        ®                   10                     ®                       15¯
          ®
             25                        ¬                    20                     ¬                       15 ¯
¬¿
Relaxed B-DNA 260 bp           25             1 

                            1                   ®                 5                       ®                      10                             ®                  15¯
¯
®                                                 20             ®            23
®
                             25                          1

 
DNA Topology: chromosomal DNA
 
• Chromosomal DNA is organized in loops (no free ends)
• It is negatively supercoiled 1 (-) supercoil per 200 nucleotides

2. Cut in pieces
Exonucleases    dNMP  +
Endonucleases    +
 
3.  Ligate DNA
- DNA ligase      +    
 
DNA Gyrase • Introduces negative supercoils
• Requires energy (ATP)
• Necessary for DNA synthesis
       DNA Gyrase
  (+) supercoiled DNA            (-) supercoiled DNA           DNA chain 
DNA containing one 1 positive (+) SuperCoil


DNA containing one 1 negative (-) SuperCoil 
 
                            Drugs that inhibit DNA gyrase              chlorobiocin

DNA Gyrase-Novobiocin 1AJ6.PDB;  clorobiocin + Gyrase 1KZN.spt

 
DNA Topoisomerase   Relax supercoiled DNA: W = + 1 to ® W = 0
 
     
 
Human DNA Topoisomerase I:  1EJ9.PDB side view
 

 
 1EJ9.PDB side view     Human DNA Topoisomerase I  1EJ9.PDB top view
 

 
Drugs that inhibit DNA Topoisomerase I
 
Topotecan (Hycamtin)• Anti- tumor activity
 
• Stabilizes topoisomerase I-DNA intermediate, preventing DNA strand re-ligation
 
Enzymes that cut DNA: exonucleases
 
Exonucleases    dNMP  +
Endonucleases    +
 
• Degrade DNA in a stepwise manner by removing mononucleotides in 5' ---> 3' or 3' ---> 5' direction
• Require a free —OH
• Most exonucleases are active on both single and double-stranded DNA
• Used for degrading foreign DNA and in proofreading during DNA synthesis
 
DNA Restriction Endonucleases
 
2.         Cut in pieces • Cleave internal phosphodiester bonds resulting in 3'-OH and 5'-phosphate ends
Endonucleases    +
 
• Found in bacteria where they are used for protection against foreign DNA
 
EcoRI recognition site:     Eco - for Escherichia coli ;  Pvu - for Proteus Vulgaris  1PVI.PDB

 
* asterisks - idicates bases that are methylated (where known) ; Bam - for Bacillus amylolique-faciens
Biochemistry, 4th Edition, W.H.Freeman Company
 


                                            Pvu II                                                                     Bam H
PvuII - for 1PVI.PDB                     Proteus Vulgaris    Eco - for 1RVA.PDB                  Escherichia colli 
Bam - for 1BHM.PDB Bacillus amyloliquefaciens H
 
DNA Ligase                     Ligate DNA
 

 
• Forms phospho-di-ester bonds between 3'OH and 5' phosphate
• Requires double-stranded DNA
• Activates 5’phosphate to nucleophilic attack by trans-esterification  with activated AMP
 
--->
2. E-+NH2-AMP1- + H5'-DNA ---> E-+NH3 + AMP1--5'-DNA  
3. DNA-3'-OH + H2O + AMP1--O5'-DNA ---> DNA-3'--O5'-DNA +H2HO+ +  AMP2-
             DNA cloning