Structures of the mitochondrial single-stranded DNA binding protein with DNA and DNA polymerase γ

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• Mitochondrial single-stranded DNA binding protein (mtSSB/SSBP1) is crucial for maintaining mitochondrial DNA (mtDNA) integrity.
• Clinical mutations in SSBP1 are associated with severe mitochondrial disorders.
• The molecular mechanisms of mtSSB-ssDNA interaction and its interplay with other replication factors are poorly understood.

Purpose of the Study:

• To elucidate the structural basis of mtSSB binding to single-stranded DNA (ssDNA).
• To investigate the interaction between mtSSB and mitochondrial DNA polymerase (Polγ).
• To provide structural insights into SSBP1-related mitochondrial diseases.

Main Methods:

• X-ray crystallography to determine the structure of human mtSSB bound to ssDNA at 1.9-Å resolution.
• Site-directed mutagenesis to confirm DNA binding sites.
• Cryo-electron microscopy (cryo-EM) to analyze the tetrameric assembly and interaction with Polγ.

Main Results:

• A high-resolution crystal structure revealed two distinct ssDNA binding sites on mtSSB: a low-affinity and a high-affinity site.
• The high-affinity site involves key residues R38 and W84, important for DNA binding and stability.
• Cryo-EM confirmed mtSSB tetramer formation and its interaction interface with Polγ.

Conclusions:

• The study reveals detailed molecular interactions of mtSSB with ssDNA, highlighting critical residues and binding modes.
• Structural insights into mtSSB-Polγ interaction provide a foundation for understanding mitochondrial DNA replication.
• This work offers a molecular basis for SSBP1 mutations linked to mitochondrial diseases.