Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• The base excision repair (BER) pathway is crucial for repairing oxidative DNA damage, preventing disruptions in cellular processes like replication.
• NEIL1 is a human DNA glycosylase initiating BER by recognizing and excising DNA lesions.
• NEIL1 interacts with key replication proteins, including proliferating cellular nuclear antigen (PCNA).

Purpose of the Study:

• To investigate the structural and mechanistic details of the NEIL1-PCNA interaction in the presence of DNA.
• To elucidate how NEIL1 binding affects the quaternary structure of PCNA.
• To understand the potential functional implications of this complex for DNA repair and replication.

Main Methods:

• Size exclusion chromatography (SEC) to isolate and characterize the NEIL1-PCNA-DNA complex.
• Native gel electrophoresis and mass spectrometry to confirm the interaction.
• Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) to analyze structural changes in PCNA.
• Ab initio shape reconstruction for solution structure determination.

Main Results:

• A stable complex of NEIL1, PCNA, and DNA was isolated.
• SEC and AFM indicated dissociation of the PCNA homotrimer upon complex formation with NEIL1 and DNA.
• SAXS confirmed NEIL1-mediated PCNA trimer dissociation and the formation of a 1:1:1 NEIL1-DNA-PCNA(monomer) complex.
• Ab initio modeling provided the solution structure of this novel complex.

Conclusions:

• NEIL1 binding to DNA induces the dissociation of the PCNA trimer into monomers.
• This structural transition suggests a regulatory mechanism, potentially switching cellular machinery between DNA replication and base excision repair.
• The findings offer insights into the coordination of DNA repair and replication processes.