81
3 Formation of DNA Lesions, its Prevention and Repair
way that the damaged base enters exactly into its active site where its repair can
be completed accurately. It is also argued that repair proteins possess an inherent
intriguing gate keeping strategy by which they deny unmodified bases access to its
active site [224].
Molecular dynamical studies have recently emerged as a valuable tool to understand DNA repair by nucleotide flipping. Analysis of distributions of DNA bending
and angle opening can indicate how nucleotide flipping occurs in DNA. Thermodynamical and kinetic factors associated with nucleotide flipping can be calculated
which may give valuable insights into the mechanism of this process. Mechanisms
of nucleotide flipping of thymine dimers by EndoV and uracil by UDG have been
studied recently by molecular dynamics simulation [224]. This study revealed that
due to base damage, DNA becomes quite flexible and the energy difference between the closed and open states decreases. This flexibility in DNA is sensed by
the protein which binds to DNA at the damaged site. Due to this binding, DNA gets
distorted which diminishes the barrier energy required for nucleotide flipping. In
another similar study, it has been found that DNA bending due to base modification and internal DNA dynamics is the initial stage of base flipping where DNA
becomes heavily distorted at the site of a mutated base pair. As a result, the mutated
base may become slightly extrahelical which will then be captured by the protein
that would subsequently push it into its active site after making it completely extrahelical [195]. As during nucleotide flipping, only a few bases close to the lesion
site are involved, DFT can be employed to understand the detailed mechanism of
nucleotide flipping. For example, using DFT, a two-step mechanism of nucleotide
flipping has recently been proposed for the repair of O6-methylgunine (O6MG) by
AGT [196]. According to this mechanism, at the first-step, AGT recruits one of its
amino acids (Arg128) to intercalate into DNA at the lesion site that perturbs base
pairing interactions of O6MG with C. In the second-step, Arg128 pushes O6MG
out of the DNA double helix into the enzyme active site for catalysis and takes the
vacant position of O6MG by making necessary hydrogen bonds with C to stabilize
the DNA [196].
3.5.3 Repair by Catalysis
After placing the nucleotide into the protein’s active site correctly, the enzyme initiates the catalytic reaction to repair the damage. Different enzymes use different catalytic processes depending on the nature of the lesion [76]. For example, alkylated
DNA base damages are repaired by removing the alkyl group attached to the base
by DNA alkyl transferases [225, 226]. However, oxidative damages are generally
removed from DNA by different DNA glycosylases [224]. Monofunctional glycosylases cleave the N-glycosidic bond of one of the bases in the affected base pair,
thereby creating an isolated base and apurinic or apyrimidinic (AP) site [208, 210,
212, 224, 225]. AP sites are then further processed by AP endonucleases forming
3’-hydroxyl and 5’-deoxyribose phosphate termini (Fig. 3.10a). In contrast, bifunctional glycosylases not only cleave the N-glycosidic bond but can also cleave the AP
3 Formation of DNA Lesions, its Prevention and Repair
way that the damaged base enters exactly into its active site where its repair can
be completed accurately. It is also argued that repair proteins possess an inherent
intriguing gate keeping strategy by which they deny unmodified bases access to its
active site [224].
Molecular dynamical studies have recently emerged as a valuable tool to understand DNA repair by nucleotide flipping. Analysis of distributions of DNA bending
and angle opening can indicate how nucleotide flipping occurs in DNA. Thermodynamical and kinetic factors associated with nucleotide flipping can be calculated
which may give valuable insights into the mechanism of this process. Mechanisms
of nucleotide flipping of thymine dimers by EndoV and uracil by UDG have been
studied recently by molecular dynamics simulation [224]. This study revealed that
due to base damage, DNA becomes quite flexible and the energy difference between the closed and open states decreases. This flexibility in DNA is sensed by
the protein which binds to DNA at the damaged site. Due to this binding, DNA gets
distorted which diminishes the barrier energy required for nucleotide flipping. In
another similar study, it has been found that DNA bending due to base modification and internal DNA dynamics is the initial stage of base flipping where DNA
becomes heavily distorted at the site of a mutated base pair. As a result, the mutated
base may become slightly extrahelical which will then be captured by the protein
that would subsequently push it into its active site after making it completely extrahelical [195]. As during nucleotide flipping, only a few bases close to the lesion
site are involved, DFT can be employed to understand the detailed mechanism of
nucleotide flipping. For example, using DFT, a two-step mechanism of nucleotide
flipping has recently been proposed for the repair of O6-methylgunine (O6MG) by
AGT [196]. According to this mechanism, at the first-step, AGT recruits one of its
amino acids (Arg128) to intercalate into DNA at the lesion site that perturbs base
pairing interactions of O6MG with C. In the second-step, Arg128 pushes O6MG
out of the DNA double helix into the enzyme active site for catalysis and takes the
vacant position of O6MG by making necessary hydrogen bonds with C to stabilize
the DNA [196].
3.5.3 Repair by Catalysis
After placing the nucleotide into the protein’s active site correctly, the enzyme initiates the catalytic reaction to repair the damage. Different enzymes use different catalytic processes depending on the nature of the lesion [76]. For example, alkylated
DNA base damages are repaired by removing the alkyl group attached to the base
by DNA alkyl transferases [225, 226]. However, oxidative damages are generally
removed from DNA by different DNA glycosylases [224]. Monofunctional glycosylases cleave the N-glycosidic bond of one of the bases in the affected base pair,
thereby creating an isolated base and apurinic or apyrimidinic (AP) site [208, 210,
212, 224, 225]. AP sites are then further processed by AP endonucleases forming
3’-hydroxyl and 5’-deoxyribose phosphate termini (Fig. 3.10a). In contrast, bifunctional glycosylases not only cleave the N-glycosidic bond but can also cleave the AP