Single-stranded telomere-binding protein employs a dual rheostat for binding affinity and specificity that drives...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Single-stranded DNA (ssDNA) binding proteins are essential for chromosome biology.
• Most ssDNA-binding proteins lack sequence specificity.
• The Saccharomyces cerevisiae Cdc13 protein is vital for telomere maintenance and exhibits high affinity and specificity for G-rich sequences.

Purpose of the Study:

• To investigate the roles of binding affinity and specificity in Cdc13 function.
• To understand how mutations at the DNA-binding interface affect Cdc13's biochemical properties and in vivo function.

Main Methods:

• Created an unbiased panel of alanine mutations in the Cdc13 DNA-binding interface.
• Assessed mutant protein affinity and sequence specificity in vitro.
• Evaluated the viability of yeast strains expressing mutant Cdc13 proteins in vivo.

Main Results:

• Mutations causing loss of affinity led to reduced yeast viability.
• Mutations enhancing specificity (reducing sequence tolerance) resulted in a gradient of yeast viability.
• Increased specificity correlated with decreased sequence tolerance in vitro.

Conclusions:

• DNA binding specificity is a critical, tunable factor for Cdc13 function in telomere maintenance.
• Cdc13 utilizes a cooperative binding interface allowing for plastic binding modes and sequence tolerance.
• Sequence specificity in nucleic acid-binding proteins may be more of a continuum than a binary trait, with sequence tolerance being a broader phenomenon.