Aris Kaksis Riga University docent - Chemistry

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' 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)- |

-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

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 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' AGGG

AGGG

<-- telomere
5'

3' ||||||||||||¯
3'

5' AGGG

AGGG

<-- telomere
5'

CCCAA

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

CCCAA

CCCAA RNA
||<--- RT

CCCAA

CCCAA RNA
AGGG

AGGG

- DNA hybrid
||<--- Rnase H --------------------->

CCCAA

CCCAA RNA
||<--- RT
AGGG

AGGG

ss -DNA
||<--- RT

CCCAA

CCCAA ds DNA
AGGG

AGGG

ds -DNA

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

glycol

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
                        ||                  ||                 ||                            ||