Molecular basis for processing of topoisomerase 1-triggered DNA damage by Apn2/APE2

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Topoisomerase 1 (Top1) incises DNA at ribonucleotides, creating complex DNA lesions.
• APE2 (Apn2 in yeast) is known to resolve these Top1-induced DNA damages.
• The precise mechanism by which Apn2 processes these lesions remains largely unknown.

Purpose of the Study:

• To elucidate the molecular mechanism of Apn2 in processing Top1-incised DNA lesions.
• To understand how Apn2 engages and cleaves DNA termini containing ribonucleotides.
• To investigate the in vivo relevance of Apn2's function in maintaining genome stability.

Main Methods:

• X-ray crystallography of Apn2-DNA complexes.
• Biochemical assays to analyze Apn2 activity.
• Genetic analysis of Apn2 deletion and mutant strains in Saccharomyces cerevisiae.

Main Results:

• Structural and biochemical data reveal Apn2 employs a wedging mechanism to fray and cleave 3' DNA termini.
• Apn2 performs endonucleolytic cleavages of 1-6 nucleotides.
• Apn2 deletion or wedge-mutant strains exhibit mutator phenotypes, growth defects, and genotoxic stress sensitivity, particularly in RER-defective backgrounds.

Conclusions:

• Apn2's broad-specificity nuclease activity is characterized by a wedge-and-cut mechanism.
• This mechanism is essential for mitigating mutagenic and genome instability phenotypes arising from Top1 activity at genomic ribonucleotides.
• Apn2 plays a critical role in DNA repair pathways involving ribonucleotide incorporation.