Binding properties of replication protein A from human and yeast cells

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Replication protein A (RP-A), also known as replication factor A or human SSB, is a crucial single-stranded DNA-binding protein complex.
• RP-A is essential for DNA replication, including that of simian virus 40 (SV40).
• RP-A exists as a stable complex of three subunits (70, 32, and 14 kDa) in both human and yeast cells.

Purpose of the Study:

• To investigate the DNA-binding properties of human and yeast RP-A.
• To elucidate the role of RP-A in DNA replication initiation.
• To understand the sequence-specific binding preferences of RP-A.

Main Methods:

• Analysis of DNA-binding properties of purified human and yeast RP-A.
• Determination of binding affinities and site sizes for single-stranded DNA (ssDNA).
• Comparison of binding to ssDNA, RNA, and double-stranded DNA (dsDNA).

Main Results:

• Human RP-A exhibits high affinity (10^9 M-1) for ssDNA, with low affinity for RNA and dsDNA.
• RP-A binds to ssDNA with a site size of approximately 30 nucleotides, without cooperative binding.
• Binding is partially sequence-dependent, with a 50-fold higher affinity for pyrimidines over purines.

Conclusions:

• Yeast and human RP-A share similar DNA-binding properties.
• Both proteins preferentially bind to pyrimidine-rich strands at origins of replication (ARS307 and SV40 origin).
• This asymmetric binding strongly suggests a direct role for RP-A in initiating DNA replication.