The role for zinc in replication protein A.

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Replication protein A (RPA) is a heterotrimeric protein crucial for DNA replication, recombination, and repair.
• The largest subunit, RPA70, has a C-terminal domain (RPA70-CTD) with a zinc-binding motif involved in subunit interactions.

Purpose of the Study:

• To characterize the RPA70-CTD and its role in DNA binding.
• To investigate the influence of zinc on RPA complex structure and function.

Main Methods:

• Proteolysis was used to isolate the core RPA70-CTD (amino acids 432-616).
• X-ray fluorescence emission spectroscopy confirmed the presence of coordinated Zn(II) in RPA70-CTD.
• DNA binding assays were performed on a trimeric complex of RPA70-CTD, RPA32 ssDNA-binding domain, and RPA14.

Main Results:

• RPA70-CTD was found to bind Zn(II), which is essential for its tertiary structure stabilization.
• The trimeric complex exhibited significant ssDNA binding activity, comparable to other RPA domains when zinc was present.
• Chelating agents drastically reduced the trimer's DNA binding affinity, highlighting the role of zinc.

Conclusions:

• Zinc is critical for maintaining the structural integrity and DNA-binding activity of RPA70-CTD.
• RPA70-CTD itself possesses DNA-binding capabilities modulated by zinc coordination.
• ssDNA binding by the RPA trimer results from a synergistic interaction between RPA70-CTD and RPA32.