STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Mammalian CST (CTC1-STN1-TEN1) is crucial for telomere replication and genome-wide recovery from replication stress.
• CST shares functional and structural similarities with Replication Protein A (RPA), binding single-stranded DNA (ssDNA).
• The precise mechanism of CST action remains largely unelucidated.

Purpose of the Study:

• To investigate the mechanism of CST action by examining the functional consequences of a DNA-binding deficient mutant (STN1 OB-fold mutant, STN1-OBM).
• To correlate in vitro DNA-binding properties of the STN1-OBM mutant with its in vivo cellular functions.
• To propose a model for CST's dynamic DNA-binding mechanism.

Main Methods:

• Utilized a STN1 OB-fold mutant (STN1-OBM) to assess CST function in vivo and in vitro.
• Evaluated the impact of STN1-OBM on endogenous replication stress resolution and telomere duplex replication.
• Performed in vitro DNA-binding studies using ssDNA oligonucleotides of varying lengths to characterize STN1-OBM binding preferences.

Main Results:

• In vivo, STN1-OBM selectively impaired resolution of endogenous replication stress and telomere duplex replication, while sparing telomeric C-strand fill-in and new origin firing.
• In vitro, STN1-OBM demonstrated a preferential destabilization of binding to short ssDNA substrates compared to long ones.
• These findings suggest that CST engages DNA substrates of diverse lengths and structures, with STN1 playing a key role in binding shorter substrates.

Conclusions:

• The selective in vivo effects of STN1-OBM indicate distinct mechanistic roles for CST in resolving different types of replication stress.
• The in vitro data support a model where CST utilizes multiple OB folds for DNA binding, similar to RPA, but with unique architectural features.
• A dynamic DNA-binding model for CST is proposed, explaining its mechanism of action across various replication stress scenarios.