TOPIC 3: DNA REPLICATION
Information flow
Central dogma of biology: genetic information flows
unidirectionally from DNA → RNA → PROTEINS
- DNA replicationDNA:copy.
- DNA transcription: DNA to RNA passage.
- RNA translation: transition from RNA to protein.
- RNA can stay in RNA and not translate.
- Reverse transcription (in virus): passage from RNA to
DNA.
- RNA replication (in virus): copy of RNA.
Thus, DNA replication is semi-conservative . Stem cells synthesize a new strand. Thus, in
each daughter cell, there is a strand of the stem cell and a new strand.
,TYPES OF DNA / RNA POLYMERASES
- Synthesize DNA or RNA, ie 5 '→ 3' phosphodiester bonds always.
- They do not work alternately, they need a reference (mold) to transcribe.
DNA POLYMERASE REACTION MECHANISM In
order for it to act, a DNA polymerase needs:
- Mold: simple DNA strand. (To know which base to incorporate)
- Primer (anchor point and start): free 3'-OH end. (Initiator)
- Molecules required for the reaction to occur: 4 dNTPs (dATP, dGTP, dCTP and dTTP) and
Mg2 + (it is the substrate and synchronizes with the previous ones).
1. Polymerase catalyzes the nucleophilic attack of free hiroxyl (OH-3 ') on phosphate.
2. The phosphate is then converted to orthophosphate and a pyrophosphate (PPi) is
released, which releases energy.
3. With the above energy, the phosphodiester bond is created between complementary
nucleotides.
, The free 3 'end must be a nucleotide paired with the complementary base. When the correct
situation is given, nucleotides are incorporated at this end.
Each time a nucleotide is incorporated, a phosphate is released.
DNA polymerase OfE.coli
Thewas first identified polymerases were bacterial (prokaryotic) and first studied were those
ofE. coli: DNA Pol. I (much more abundant), II and III.
DNA polymerase andE.coli
P presentactivity:
- DNA polymerase 5 '→ 3' (sequentially)
- DNA exonuclease 5 '→ 3' (sequential or in blocks)
DNA is eaten when repair mechanisms are put into operation of the damage and when
the primers should be removed.
- DNA exonuclease 3 '→ 5' (sequential) or corrective activity
In addition, this polymerase has proof reading → ensures that each nucleotide it
incorporates is correct and, if not, removes it by cutting the phosphodiester bond
(endonuclease) and replace it with the appropriate one.
Mechanism that corrects polymerase errors to prevent cell mutations.
Error detection: if the bases are properly paired, their OH-3 'end is free and therefore the
enzyme can continue its activity; however, if these are unpaired, the OH-3 'end is not free
and the enzyme cannot detect it to continue functioning.
This mechanism increases the replication fidelity by about 1000 times.
KLENOW FRAGMENT
Information flow
Central dogma of biology: genetic information flows
unidirectionally from DNA → RNA → PROTEINS
- DNA replicationDNA:copy.
- DNA transcription: DNA to RNA passage.
- RNA translation: transition from RNA to protein.
- RNA can stay in RNA and not translate.
- Reverse transcription (in virus): passage from RNA to
DNA.
- RNA replication (in virus): copy of RNA.
Thus, DNA replication is semi-conservative . Stem cells synthesize a new strand. Thus, in
each daughter cell, there is a strand of the stem cell and a new strand.
,TYPES OF DNA / RNA POLYMERASES
- Synthesize DNA or RNA, ie 5 '→ 3' phosphodiester bonds always.
- They do not work alternately, they need a reference (mold) to transcribe.
DNA POLYMERASE REACTION MECHANISM In
order for it to act, a DNA polymerase needs:
- Mold: simple DNA strand. (To know which base to incorporate)
- Primer (anchor point and start): free 3'-OH end. (Initiator)
- Molecules required for the reaction to occur: 4 dNTPs (dATP, dGTP, dCTP and dTTP) and
Mg2 + (it is the substrate and synchronizes with the previous ones).
1. Polymerase catalyzes the nucleophilic attack of free hiroxyl (OH-3 ') on phosphate.
2. The phosphate is then converted to orthophosphate and a pyrophosphate (PPi) is
released, which releases energy.
3. With the above energy, the phosphodiester bond is created between complementary
nucleotides.
, The free 3 'end must be a nucleotide paired with the complementary base. When the correct
situation is given, nucleotides are incorporated at this end.
Each time a nucleotide is incorporated, a phosphate is released.
DNA polymerase OfE.coli
Thewas first identified polymerases were bacterial (prokaryotic) and first studied were those
ofE. coli: DNA Pol. I (much more abundant), II and III.
DNA polymerase andE.coli
P presentactivity:
- DNA polymerase 5 '→ 3' (sequentially)
- DNA exonuclease 5 '→ 3' (sequential or in blocks)
DNA is eaten when repair mechanisms are put into operation of the damage and when
the primers should be removed.
- DNA exonuclease 3 '→ 5' (sequential) or corrective activity
In addition, this polymerase has proof reading → ensures that each nucleotide it
incorporates is correct and, if not, removes it by cutting the phosphodiester bond
(endonuclease) and replace it with the appropriate one.
Mechanism that corrects polymerase errors to prevent cell mutations.
Error detection: if the bases are properly paired, their OH-3 'end is free and therefore the
enzyme can continue its activity; however, if these are unpaired, the OH-3 'end is not free
and the enzyme cannot detect it to continue functioning.
This mechanism increases the replication fidelity by about 1000 times.
KLENOW FRAGMENT
