The yeast protein encoded by PUB1 binds T-rich single stranded DNA

Area of Science:

• * Molecular Biology
• * Genetics
• * Biochemistry

Background:

• * Yeast autonomously replicating sequences (ARS) are crucial for DNA replication initiation.
• * Specific DNA-binding proteins are essential for recognizing and interacting with ARS elements.
• * Characterizing these proteins provides insight into the regulation of DNA replication.

Purpose of the Study:

• * To identify and characterize proteins that bind to the T-rich strand of the yeast ARS consensus element.
• * To purify and determine the identity of these DNA-binding proteins.
• * To assess the binding affinities and specificities of the purified proteins.

Main Methods:

• * DNA affinity chromatography using the ARS consensus element.
• * Protein purification via heparin-sepharose chromatography.
• * SDS-PAGE and peptide sequencing for protein identification.

Main Results:

• * Two distinct binding activities, ACBP-60 and ACBP-67, were identified and purified.
• * ACBP-60, a 60kDa protein, binds the ARS consensus T-rich strand with high affinity (Kd 10^-9 to 10^-10 M) and was identified as a polyuridylate binding protein (PUB1/RNP1).
• * ACBP-67, a 67kDa protein, binds with lower affinity and was identified as the major polyA binding protein (PAB1).

Conclusions:

• * Yeast possesses specific proteins that recognize the ARS consensus sequence.
• * ACBP-60 (PUB1/RNP1) is a high-affinity binder to the ARS T-rich strand.
• * ACBP-67 (PAB1) also binds the ARS T-rich strand, albeit with lower affinity, suggesting potential roles in replication initiation or regulation.