Replication-dependent mutagenesis by 5-bromodeoxyuridine: identification of base change and sequence effects on...

Area of Science:

• Molecular Biology
• Genetics
• Mammalian Cell Mutagenesis

Background:

• 5-bromodeoxyuridine (BrdU) is a thymidine analog used to study DNA replication and mutagenesis.
• Replication-dependent mutagenesis involves errors occurring during DNA replication.
• The E. coli gpt gene is a commonly used reporter gene in mammalian mutagenesis studies.

Purpose of the Study:

• To elucidate the molecular mechanism of BrdU-induced reversion in mammalian cells.
• To identify factors influencing the susceptibility of specific DNA sites to BrdU mutagenesis.
• To characterize the types of base transitions induced by BrdU replication-dependent mutagenesis.

Main Methods:

• Utilizing murine cell lines with chromosomally integrated E. coli gpt genes.
• Employing BrdU for replication-dependent mutagenesis and selecting for GPT+ revertants.
• Analyzing the DNA sequence of revertant and non-revertant gpt genes to identify base transitions and flanking sequences.

Main Results:

• BrdU replication-dependent mutagenesis predominantly induces AT to GC base transitions.
• Four out of thirteen mutant cell lines were effectively reverted by BrdU.
• Non-revertible cell lines showed similar susceptibility to general mutagenesis, indicating site-specific resistance.
• DNA sequence context, particularly flanking sequences like 3'-adjacent Adenine, significantly affected BrdU mutagenesis susceptibility.

Conclusions:

• BrdU replication-dependent mutagenesis specifically causes AT to GC transitions.
• The susceptibility of a mutation site to BrdU mutagenesis is influenced by the surrounding DNA sequence.
• Specific DNA sequence motifs can confer resistance to BrdU-induced reversion.