Direct visualization of DNA baton pass between replication factors bound to PCNA

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Lagging strand DNA synthesis in Eukarya and Archaea involves DNA polymerase (Pol), flap endonuclease (FEN), and DNA ligase (Lig).
• These factors form complexes with proliferating cell nuclear antigen (PCNA), creating a platform for sequential action during DNA replication.
• Previous studies visualized Pol-PCNA-DNA and Lig-PCNA-DNA complexes, but the FEN-PCNA-DNA structure remained elusive.

Purpose of the Study:

• To determine the tertiary structure of the FEN-PCNA-DNA complex.
• To visualize the FEN-Lig-PCNA-DNA complex, representing an intermediate state in DNA repair.
• To provide direct structural evidence of the hand-off mechanism between replication factors on a PCNA clamp.

Main Methods:

• Single particle analysis was employed to determine the high-resolution structures of the complexes.
• Cryo-electron microscopy was used for structure determination.

Main Results:

• The FEN-PCNA-DNA complex structure was determined for the first time.
• The structure of the FEN-Lig-PCNA-DNA complex was visualized, illustrating a putative intermediate state.
• Direct visualization of the hand-off action between FEN and Lig on the PCNA clamp was achieved.
• A significant conformational change in DNA from bent to straight was observed during factor switching.
• Dynamic motions of replication factors during the switching process were detected.

Conclusions:

• This study provides the first structural insights into the FEN-PCNA-DNA complex.
• The findings elucidate the mechanism of factor hand-off during DNA replication and repair.
• The observed DNA conformational change and dynamic factor motions highlight the adaptability of the replication machinery.