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' ¾®       3'

 ¯ DNA Pol I

DNA strand                           3'  5'

5'  3'

degraded               ¬ primer + ¯ DNA Ligase

3'  5'

5'  3'


Replication Fork Garland




Fidelity of Polymerization: Absolutely Essential!!


Error Probability = Polymerization error 10-4 

                                 3' --> 5' Nuclease error 10-3 

                                              (10-4) • (10-3)  = 10-7 or 1 in 10,000,000


DNA Synthesis: addition of new dNTPs follows Watson-Crick rules



                          G = C                                                             A= 

                                                                            Polymerase errors

Very low rate of misincorporation (1 per 108)

  Errors can occur due to the presence of minor tautomers of nucleobases.


                           = A                               Cytosine    C = A Rare tautomer of Adenine

                             Normal base pairing                          Mispairing

                               Proofreading function of DNA polymerases




Figure 25-7. An example of error correction by the 3'--> 5' exonu­clease activity of DNA polymerase I. Structural analysis has located the exonuclease activity ahead of the polymerase activity as the enzyme is oriented in its movement along the DNA. A miss-matched base (here, a C=A mismatch) impedes translocation of DNA polymerase I to the next site. Sliding backward, the enzyme corrects the mistake with its 5'--> 3' exonuclease activity, then resumes its polymerase activity in the 5'--> 3' direction.


                   Consider misincorporation due to a rare tautomer of A


2nd  replication    -A-


1st replication -A(imino)- | ­


5’-A- --> -A(imino)     | ­

3’--  --> --             |


--  Normal replication

Final result: A --> G transition

                                            Mismatch Repair Enzymes

Polymerase I, III error rates:  1 per 107                          nucleotides
Observed mutation rate:       1 per 108 - 1 per 1010       nucleotides
Polymerase errors can be corrected after DNA synthesis! 
Repair of nucleotide mismatches:
• Recognize parental DNA strand (correct base) and daughter strand (incorrect base)
            Parental strand is methylated —CH3: metC      or   Amet
2. Replace a portion of the strand containing erroneous nucleotide
 (between the mismatch and a nearby methylated site –up to 1000 nt)

                                                                               DNA replication in eukaryotes

Several eukaryotic DNA polymerases are known: a, b, d, g  -
                                                                                a and  d  are thought to be the major chromosomal replicases
Similarities with E.Coli
 Always 5’ to 3’ direction -->
 Require a primer
 Similarities in active site and tertiary structure
 Eukaryotic replication is much slower (100 nt/sec)
 Several replication origins
 Polymerases are more specialized (a for lagging strand, d for leading strand)
4.   Require special processing of the chromosomal ends .

Telomerase preserves chromosomal ends


• The ends of the linear DNA strand can not be replicated due to the lack of a primer
This would lead to shortening of DNA strands after replication
3'  5'
5'        3' <--RNA primer
• Solution: the chromosomal ends are extended by DNA telomerase
This enzyme adds hundreds 200÷900 of tandem repeats of a hexa-nucleotide(AGGG in humans)
to the parental strand:
3'  5' AGGGAGGGAGGG<-- telomere
5'        3' ||||||||||||¯
3'  5' AGGGAGGGAGGG<-- telomere
5'  3' CCCAACCCAA CCCAA<-- RNA primer
Telomerase is a ribonucleoprotein that contains an RNA molecule
used as a template for elongation of the 3’ strand



Central dogma of molecular biology


 DNA ||-------------->  RNA -------------->   Proteins ----->>>>>>>  Cellular action
Replication ||    ­|| transcription         ­||||||| translation                ------>>>>|||||||||||| ­­­­­­­­­­­­­
           ||                    nucleare                                    cytosolic
    DNA nucleare <= Reverse  transcription of telomeres
                                    Notable exception: retroviruses
 RNA ||------------>  DNA -------->  RNA  ------------------>   Proteins ---------->  Cellular action
Reverse ||->||transcription ­ transcription                 ­||||||| translation                  --->>> |||||||||||| ­­­­­­­­­­­­­
    DNA cytosolic      nucleare                                    cytosolic
Reverse transcriptases (RT) are RNA directed DNA Pol
Used by RNA viruses  (HIV-I , human immunoblastosis virus, Rous sarcoma virus) :
1. Make RNA-DNA hybrid (use its own RNA as a primer)
2. Make ss DNA by exoribonuclease (RNase H) activity
3. Make ds DNA   incorporate in the host genome
||<--- RT
||<--- Rnase H  ---------------------> CCCAACCCAACCCAA         RNA
||<---  RT
||<---  RT


Termination of Polymerization: The Key to Nucleoside Drugs


AZT                                        Ziagen                                                                   Acyclovir


Inhibition of Viral DNA Polymerization by nucleoside analogs


(DNA)n bases  +  dNTZiagen  (DNA)(n+1)  bases analog ¹  
 E. coli DNA Polymerase I


Nucleosides Must Be Converted to Triphosohates to be
Part of DNA and RNA


                                                                                     MonoPhosphate                                            ¯

       TriPhosphate                                                                          DiPhosphate


Chemical modification of DNA


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


                                        Types of DNA Mutations


1.         Point mutations: substitution of one base pair for another, e.g. A for GC
• the most common form of mutation
transition;         purine to purine and             pyrimidine to pyrimidine
transversions;    purine to pyrimidine or        pyrimidine to purine
2.         Deletion of one or more base pairs 
3.         Insertion of one or more base pairs


        DNA Damage 

Sources of DNA damage: endogenous
1.        Deamination
2.        Depurination: 10,000/cell/day
3.        Oxidative stress
Sources of DNA damage: environmental
1. Alkylating agents (drugs, pollutants)
2. X-ray and UV irradiation
3. Diet
4. Smoking

Mechanisms of induced mutations


·       Altered basepairing characteristics (O6-alkyl-G)
·       Abasic sites (N7-guanine adducts)
·       Deletions/insertions due to intercalating agents (e.g acridin orange)
·       DNA strand breaks (reactive oxygen species)


Normal base pairing in DNA and
an example of mispairing via chemically modified nucleobase


                   Adenine A=                       O6-Alkyl-Guuanine   
            Guanine GºC Cytosine

                                                      DNA Damage: deamination

-->  Adenine A --> hypoXanthine
->  Guanine G --> Xanthine
 C  Cytosine -->   

           C Cytosine Deamination to ®  
      G Guanine              Depurination G or A remove by hydrolise H2O
¾®  Abasic site


DNA Damage: oxidative stress


                                                         Reactive oxygen species: HO•, H2O2, 1O2, LOOH

Guanine 8-oxo-Guanine


Guanine oxidation in DNA


LOO• HO•, HOCl, 1O2, HONO       LOO• HO•, HOCl, 1O2, HONO
   ||                     ||       ||        ||                  ||         ||       ||        ||
       dG in DNA ||                    8-oxo-dG in DNA                          
                        ||                  ||                 ||                            || 

 DNA Damage: UV light


           Also  = C , C = C dimers-neighbour           dimer-neighbour  = 

Benzo[a]pyrene- induced DNA adducts


                   benzo[a]pyrene                              (+)-trans-anti BPDE |reaction|
                                                                                                                                             N2-BPDE-dG adduct
         Structure of  N2-BPDE-dG containing DNA