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]
                                          DNA SynthesisDNA Synthesis
                                                                   5'       3'

 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
 

 

 

 
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 A
     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.
 
                                                      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
 
                   Consider misincorporation due to a rare tautomer of A
 
2nd  replication    -A-
 --
1st replication -A(imino)- ­
 -C-
5’-A- -----> -A(imino)  ­
3’--  --> --              ||
-A-
--  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: alpha, beta, delta, gama  -
                                                                        a and  delta  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 3° structure
Differences
 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
    DNA nucleare <== Reverse  transcription of telomeres
 
                                    Notable exception: retroviruses
 RNA ||--------->>  DNA ---   --->  RNA  ----------------->   Proteins --------->>>>>  Cellular action
Reverse ||­---> transcription --> ­ transcription       || ­translation ||                         --------->>>> ­­­­­­­­­­­­­
    DNA cytosolic      nucleare
 
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
 
CCCAACCCAACCCAA    RNA
||<-- RT
CCCAACCCAACCCAA    RNA
 AGGGAGGGAGGG - DNA   hybrid
||<-- Rnase H  ---------------------------------------> CCCAACCCAACCCAA         RNA
||<--  RT
AGGGAGGGAGGG     ss -DNA
||<--  RT
 CCCAACCCAA CCCAA      ds   DNA
 AGGGAGGGAGGG     ds -DNA
 
Termination of Polymerization: The Key to Nucleoside Drugs
 
  
AZT                                        Ziagen                                                                   Acyclovir

 

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

                                                                                     MonoPhosphate                                            ||

       TriPhosphate                                                                          DiPhosphate

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
DNA Damage: oxidative stress
                                                           Reactive oxygen species: HO•, H2O2, 1O2, LOOH
 
  glycol
  Guanine 8-oxo-Guanine
  DNA Damage: UV light
 
 
Chemical Mutagens
 
Mutations can occur when the normal bases that are incorporated are changed.
  1. Base analogs or bases that have altered hydrogen bonding capabilities can cause transitions.
          Ex. Bromouracil and Guanine or 2-aminopurine and cytosine
2. Bases can be modified on the DNA by mutagens.
•adenine is oxidatively deaminated to hypoxanthine, Cytosine to , Guanine to Xanthine
3. Intercalating Agents          
                                             • insertion and deletion mutants
 
           Cytosine Deamination
                        

 

Normal base pairing in DNA and
an example of mispairing via chemically modified nucleobase
 
     
                   Adenine         A=               O6-Alkyl-Guuanine   
 
                              
            Guanine              G = C Cytosine

 

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.
 
Excision Repair
Takes advantage of the double-stranded (double information) nature of the DNA molecule.
 * Mismatch repair
* Base excision repair

* Nucleotide excision repair

 

Nucleotide Excision Repair
• Extremely flexible
 • Corrects any damage that both distorts the DNA molecule and
                                                                        alters the chemistry of the DNA molecule.
 
Nucleotide Excision Repair
 
• In all organisms, NER involves the following steps:
 
1. Damage recognition
 2. Binding of a multi-protein complex at the damaged site
 3. Double incision of the damaged strand several nucleotides away
                                                   from the damaged site, on both the 5' and 3' sides
 4. Removal of the damage-containing oligonucleotide from between the two 2 nicks
 5. Filling in of the resulting gap by a DNA polymerase
 6. Ligation
 
DNA Synthesis: Take Home Message
 
1) DNA synthesis is carried out by DNA polymerases with high fidelity.
 2) DNA synthesis is characterized by initiation,
                          priming and processive synthesis steps and proceeds in 5’--> 3’ direction.
 3) Modifications of DNA base pairs, if not repaired, can lead to
                                                                                          mutations of the DNA sequence.