A rapid and efficient method to purify proteins at replication forks under native conditions.

Area of Science:

• Molecular Biology
• Epigenetics
• Biochemistry

Background:

• Studying replication fork dynamics is crucial for understanding DNA replication, repair, histone deposition, and epigenetic memory.
• Isolation of protein on nascent DNA (iPOND) is a key method for purifying replication fork proteins.

Purpose of the Study:

• To present accelerated native iPOND (aniPOND), a simplified and improved method for isolating replication fork proteins.
• To enhance protein yield and speed compared to the original iPOND procedure.

Main Methods:

• Combined cell membrane lysis and nuclei harvesting into a single step to minimize sample loss.
• Employed a mild nuclei lysis protocol to preserve DNA-protein complexes.
• Developed aniPOND, a faster method that avoids formaldehyde cross-linking.

Main Results:

• aniPOND significantly improved protein yield (5-fold for CAF1-complex, 20-fold for PCNA) compared to iPOND.
• Detected polycomb repressive complex 2 (PRC2) components (SUZ12, EZH2, RBBP4) at replication forks using aniPOND, but not iPOND.
• aniPOND is faster and yields more proteins than standard iPOND.

Conclusions:

• aniPOND is a more efficient method for isolating replication fork proteins.
• This improved technique facilitates Mass Spectrometry (MS) analysis of replication fork complexes.
• aniPOND enables detection of previously unobserved protein complexes at replication forks.