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Interactions between DNA polymerase and aminofluorene adducts that affect the recognition and possibly the
Abstract:
Adducts of 2-aminofluorene on the C-8 position of guanine block DNA synthesis and lead to mutation. N-acetylated adducts adopt the syn conformation such that in DNA the guanine is displaced from the helix by the fluorene ring while unacetylated adducts prefer the anti conformation allowing normal base pairing of the guanine with cytosine. In vitro synthesis by both E. coli DNA polymerase I and T4 DNA polymerase terminates predominantly one nucleotide before acetylated adducts but has an increased tendency to terminate opposite the unacetylated adducts apparently reflecting the preferred conformations of the two species of reacted nucleoside. In complete contrast AMV reverse transcriptase correctly inserts cytosine opposite the acetylated adducts but prefers to terminate one nucleotide before the unacetylated adducts. We interpret these results as indicating that the specific properties of a replicating polymerase can influence the conformation of a reacted nucleoside thus altering its recognition and possibly its mutagenic activity.
Insights
DNA adducts from 2-aminofluorene can cause mutations. Different DNA polymerases interact differently with these adducts, influencing their conformation and mutagenic potential.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- 2-aminofluorene (2-AF) forms adducts at the C-8 position of guanine.
- These adducts can block DNA synthesis and lead to mutations.
- The conformation (syn vs. anti) of these adducts influences their interaction with DNA and enzymes.
Purpose of the Study:
- To investigate how different DNA polymerases interact with acetylated and unacetylated 2-aminofluorene-guanine adducts.
- To determine if polymerase properties can influence adduct conformation and mutagenic activity.
Main Methods:
- In vitro DNA synthesis assays using E. coli DNA polymerase I, T4 DNA polymerase, and AMV reverse transcriptase.
- Synthesis termination site analysis opposite modified guanine bases.
Main Results:
- E. coli and T4 DNA polymerases preferentially terminate synthesis before acetylated adducts (syn conformation) and opposite unacetylated adducts (anti conformation).
- AMV reverse transcriptase correctly inserts cytosine opposite acetylated adducts but terminates before unacetylated adducts.
- Polymerase-specific interactions appear to influence the conformational preference of the adducts.
Conclusions:
- The specific DNA polymerase can dictate the conformation of 2-aminofluorene-guanine adducts.
- This conformational influence can alter adduct recognition and potentially modulate their mutagenic activity.
- Understanding these polymerase-adduct interactions is crucial for assessing chemical mutagenicity and carcinogenicity.