Genome-Wide Profiling of Endogenous Single-Stranded DNA Using the SSiNGLe-P1 Method

Area of Science:

• Genomics
• Molecular Biology
• Biochemistry

Background:

• Endogenous single-stranded DNA (essDNA) formation occurs through various cellular processes.
• essDNA's roles and impact on genome integrity are not fully understood.
• Mammalian genomes contain essDNA in both nuclear and mitochondrial DNA.

Purpose of the Study:

• To develop a high-throughput method for genome-wide identification of essDNA regions.
• To profile essDNA distribution in human mitochondrial and nuclear genomes.
• To investigate the functional implications of essDNA localization.

Main Methods:

• Established the SSiNGLe-P1 (Single-Stranded Nucleic Acid Identification by Limited P1 digestion) approach.
• Utilized P1 endonuclease for limited digestion to isolate essDNA regions.
• Applied the method for high-throughput, genome-wide profiling of essDNA.

Main Results:

• essDNA profiles in the mitochondrial genome support the strand-displacement model of mtDNA replication.
• Nuclear essDNA regions are enriched at origins of DNA replication and R-loops.
• A significant portion of identified essDNA regions are unannotated, suggesting novel functional elements.

Conclusions:

• The SSiNGLe-P1 method enables efficient genome-wide essDNA mapping.
• essDNA plays a role in mitochondrial DNA replication and nuclear genome organization.
• Unannotated essDNA regions may represent previously undiscovered functional genomic elements.