Physical interactions between Mcm10, DNA, and DNA polymerase alpha.

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Mcm10 is crucial for eukaryotic DNA replication initiation and elongation.
• Mcm10 facilitates the recruitment of replication proteins, including DNA polymerase alpha (pol alpha), to chromatin.
• Previous studies indicated Mcm10's internal (ID) and C-terminal (CTD) domains bind single-stranded DNA (ssDNA) and pol alpha's p180 subunit.

Purpose of the Study:

• To elucidate the structural basis of Mcm10 interactions with ssDNA and pol alpha.
• To understand the mechanism of pol alpha recruitment and stabilization at the replication fork.

Main Methods:

• X-ray crystallography to determine the structure of Mcm10-ID complexed with ssDNA.
• NMR spectroscopy and fluorescence spectroscopy to map binding interfaces and competition.
• Site-directed mutagenesis to identify Mcm10 binding regions on p180.

Main Results:

• ssDNA binds to the oligonucleotide/oligosaccharide binding (OB)-fold cleft of Mcm10-ID.
• The p180 subunit of pol alpha also binds to the Mcm10-ID OB-fold.
• ssDNA and p180 compete for binding to the Mcm10-ID OB-fold, with a minimal Mcm10 binding site identified on p180 (residues 286-310).

Conclusions:

• Mcm10 utilizes a handoff mechanism involving competition for its OB-fold to load and stabilize pol alpha at the replication fork.
• These findings provide mechanistic insights into Mcm10's role in coordinating DNA replication.
• Structural data clarifies critical intermolecular interactions essential for replication fork progression.