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Structural alterations of pathologically or physiologically modified DNA
Abstract:
We have studied the alterations of DNA conformation in in vitro depurinated or methylated topological isomers of the plasmid pAT 153. Depurination by heat/acid treatment or alkylation by methyl methanesulfonate (pathological modifications) result in DNA unwinding detected as a reduction in the degree of supercoiling of DNA topoisomers as measured by the alteration of electrophoretic mobility on agarose gel. On the contrary, in vitro enzymic methylation at the C-5 position of cytosine (physiological modification) does not measurably alter the tertiary structure of the circular substrates. From the average number of modified sites needed to remove one superhelical twist from each single topoisomer of a population of partially relaxed DNA molecules, we have calculated an unwinding angle smaller than -3.4 degree per methylated purine and of approximately -12.0 degree per apurinic site. These results, together with previously reported values of unwinding by pyrimidine dimers, suggest a possible mechanism of recognition of damaged sites by repair mechanisms that are not single-damage specific.
Insights
Pathological DNA damage, like depurination, unwinds DNA, altering supercoiling. Physiological methylation does not affect DNA structure, suggesting distinct repair pathway recognition for DNA damage.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA conformation and supercoiling are crucial for DNA replication and repair.
- Pathological DNA modifications can alter DNA structure, potentially impacting cellular processes.
- Understanding how DNA damage affects DNA topology is key to deciphering DNA repair mechanisms.
Purpose of the Study:
- To investigate the impact of depurination and methylation on DNA conformation.
- To quantify the unwinding angle associated with specific DNA modifications.
- To explore the implications for DNA damage recognition by repair enzymes.
Main Methods:
- In vitro analysis of topological isomers of plasmid pAT 153.
- Induction of depurination (heat/acid) and methylation (methyl methanesulfonate, enzymatic).
- Assessment of DNA unwinding via changes in supercoiling and electrophoretic mobility on agarose gel.
Main Results:
- Depurination and methyl methanesulfonate alkylation caused significant DNA unwinding, reducing supercoiling.
- Enzymatic methylation at cytosine C-5 did not alter DNA tertiary structure.
- Calculated unwinding angles: < -3.4° per methylated purine and ~ -12.0° per apurinic site.
Conclusions:
- Pathological DNA modifications induce structural changes (unwinding) detectable by supercoiling alterations.
- Physiological methylation does not impact DNA tertiary structure.
- DNA unwinding associated with various damage types may facilitate recognition by non-specific repair mechanisms.