Instructor: Dr. Natalia Tretyakova, Ph.D. «hyperlink ""»    -   6-3432


PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Prof.

                                                                   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


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-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
© 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