DNA expansions generated by human Polμ on iterative sequences

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA polymerase mu (Polµ) possesses unique template-directed and terminal transferase activities.
• Polµ tolerates primer and template strand distortions, leading to misinsertions and extension of mismatched termini.
• Dinucleotide repeat expansions are implicated in various human genetic disorders.

Purpose of the Study:

• To propose a model for human Polµ-mediated dinucleotide expansion based on sequence context.
• To elucidate the mechanistic role of specific Polµ residues in regulating nucleotide expansions.
• To understand the implications of Polµ activity in DNA repair and mutagenesis.

Main Methods:

• In vitro biochemical assays to study Polµ activity on various DNA substrates.
• Site-directed mutagenesis to investigate the function of key Polµ residues (His329, Arg387).
• Analysis of DNA substrate requirements for Polµ-mediated expansions.

Main Results:

• A model was developed where Polµ-mediated dinucleotide expansion requires initial DNA dislocation and stabilization.
• Human Polµ residues His329 and Arg387 were identified as critical regulators of iterative nucleotide polymerization.
• These residues modulate nucleotide expansions similarly to their role in terminal transferase activity.
• Polµ can generate large sequence expansions on substrates mimicking non-homologous end-joining (NHEJ) intermediates.

Conclusions:

• Human Polµ plays a significant role in dinucleotide repeat expansions through a dislocation-stabilization mechanism.
• Specific amino acid residues within Polµ actively regulate the extent of nucleotide polymerization.
• Polµ's iterative synthesis activity can lead to frameshift mutations, DNA damage, and instability.
• Dysregulated Polµ activity may contribute to human diseases such as cancer and congenital abnormalities.