Instructor: Dr. Natalia Tretyakova, Ph.D. «hyperlink "mailto:[email protected]"»    -   6-3432

 

PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Prof. mailto:[email protected]

                                                                   5'       3'                 DNA SynthesisDNA Synthesis

 3'       5'                                                                                                                       3'         5'

Primer Synthesis and removal

 

3'  5'
|| Primase
3'  5'
5'  3'
RNA primer +  || DNA Pol III
3'  5'
5' -->       3'
 ¯ DNA Pol I
DNA strand                           3'  5'
5'  3'
degraded               <- primer + || DNA Ligase
3'  5'
5'  3'

 

Replication Fork Garland

 

 

 

 

Antitumor drug examples

 

Cyclophosphamide      Cytoxan®           
Melphalan                   Alkeran®
Busulfan                      Myleran®             form crosslinks
Chlorambucil              Leukeran®
Mitomycin                   Mutamycin®           
Cisplatin                      Platinol®
Bleomycin                    Blenoxane®         cuts DNA strands between A= or G º C
Dactinomycin              Cosmegen ® inserts into the double helix preventing its unwinding

 

Chemical modification of DNA

 

                      Carcinogen (X) ---------------> detoxification --> excretion
              metabolic activation ||  
         reactive metabolite (X-) + DNA
              ||        DNA adducts                  
||repair          ||                            || replication
                          intact DNA                 cell death       mutations

 

DNA cross-linking agents as anti-tumor drugs

 

Nitrogen mustards
         
Mechlorethamine       Chlorambucil                    Cycloamide
Mechanism:
>->

 

Interstrand cross-links block strand separation

                                                                   5'       3'              required for DNA Synthesis

 

                                  N7-alkylguanines can be stabilized by 2 pathways

 

-->||
                                      Depurination          |                                     |Ring cleavage
                                Abasic site    

 

Importance of DNA Repair

 

• DNA is the only biological macromolecule that is repaired. All others are replaced.
 
• More than 100 genes are required for DNA repair,
                                                                    even in organisms with very small genomes.
 
• Cancer is a consequence of inadequate DNA repair.

 

DNA Repair Strategy

 

• Take advantage of the double-stranded (double information) nature of the DNA molecule.
 
• Remove damaged nucleobase (nucleotide) and fill the gap
                                                                   using the complementary strand as a template

DNA Repair Types

 

¤  Base excision repair
¤  Direct repair
¤  Nucleotide excision repair
¤  Mismatch repair
¤  Recombination repair

                                          Base excision repair

 

¤ Used for repair of small damaged bases in DNA (AP sites, 8-oxo-G, _)
¤ Human gene hogg1 is often deleted in lung cancer
¤ Several steps are involved: 
       a) modified base is excised by N-glycosylase 
       b) the abasic site is cleaved with endonuclease
       c) the resulting gap is filled by Polymerase b?
       d) DNA Ligase seals the strands

 

Base excision repair mechanism

 

                      

 

                                Direct repair

                           ¤ || DNA photolyase -->
-->
¤O6-alkyltransferase
                   O6-alkylguanine DNA alkyltransferase

 

¤ Directly repaires alkylation damage (O6-Me-dG, O6-POB-dG)
¤ stoichiometric reaction, enzyme inactivated via alkylation of Cys in the binding site
+ AGT-CH2--H --> + AGT-CH2--CH3
 O6-methylguanine                                             
© AGT enzyme is highly conserved:
     a) helix-turn-helix motif
     b) hydrophobic side-chains form alkyl-binding pocket

 

Nucleotide excision repair (NER)

 

¨ Corrects any damage that both distorts the DNA molecule and alters
                                                                the chemistry of the DNA molecule
(pyrimidine dimers, benzo[a]pyrene-dG adducts, cisplatin-cross-links).
¨ Xeroderma pigmentosum is a genetic disorder resulting in defective NER
¨ Special excision nuclease (exinuclease) hydrolyzes phosphodiester bond to release a short oligonucleotide fragment (about 12-20 nucleotides,  3-5th 3’ from the lesion and 7-12 nucleotides 5’ from the lesion)
4.    Pol d/e replaces the removed strand
5.    DNA ligase seals the ends
 

                                                                                  Arginine Flipper

 

       Nucleotide excision repair (NER)

DNA with dimer                          Mammalian Enzyme
Dimer reconized and DNA cut    exinuclease
Dimer excised
Gap filled by DNA polymerase  Pol d/e
Nick sealed by DNA ligase         DNA ligase

 

Recombination repair

 

               

 

Genetic diseases associated with defective DNA repair

 

Xeroderma Pigmentosum                                             NER
Hereditary nonpolyposis colorectal cancer                MMR
Cockrayne’s syndrome                                                  NER
Falconi’s anemia                                                            DNA ligase
Bloom’s syndrome                                                          BER, ligase
Lung cancer (?)                                                              BER

 

Predict repair mechanisms for the following types of DNA damage

 site                                           DNA Synthesis: Take Home Message
 
double strand break                  1) DNA synthesis is carried out by DNA polymerases
                                                                              glycol with high fidelity.
UV photo products                    2) DNA synthesis is characterized by initiation,
:G mismatch                                          priming and processive synthesis steps and
polyclic aromatic                                                           proceeds in 5’ ® 3’ direction.
|       hydrocarbon adducts         3) Modifications of DNA base pairs, if not repaired,
exocyclic adducts (propano)                                          can lead to mutations of the
N7-methylguanine                                                                            DNA sequence.
O6-   ethylguanine