A protein complex known as Replication Protein A, or RPA, serves as a guardian for our genetic material. It is the primary protein in eukaryotes that attaches to single-stranded DNA (ssDNA). This form of DNA arises when the double helix unwinds during processes like copying or repairing the genetic code. RPA’s responsibility is to find these exposed single strands and shield them from harm, preventing breakage or degradation by cellular enzymes.
The Building Blocks of RPA
RPA is a heterotrimer, meaning it is constructed from three distinct protein subunits. These components are named according to their size: RPA1 (also called RPA70), RPA2 (RPA32), and RPA3 (RPA14). Each subunit has a specific job, and their collaboration gives the RPA complex its capabilities. They are connected by flexible linkers, allowing the structure to be dynamic in its binding.
The largest subunit, RPA70, is the primary workhorse for engaging with DNA. It contains several specialized regions known as oligonucleotide/oligosaccharide-binding folds, or OB-folds. These domains are responsible for making physical contact with and holding onto the single-stranded DNA, forming the main binding platform of the complex.
The middle subunit, RPA32, functions as a communications hub for the complex. While it contains one DNA-binding domain (DBD-D), its more prominent role involves interacting with other proteins involved in DNA metabolism. This subunit sends and receives signals, helping to coordinate DNA replication and repair machinery and recruit other necessary factors to the site.
The smallest component, RPA14, provides structural integrity to the assembly. It helps form the stable core of the complex. Its OB-fold interacts with domains on RPA70 and RPA32, locking the three pieces together. This stabilizing function ensures the RPA complex remains intact and functional as it carries out its duties.
Forming the Functional RPA Complex
The assembly of the three RPA subunits into a functional unit is a precise and orderly molecular event. The process ensures that the final heterotrimer is correctly structured to perform its duties. This construction is foundational to its ability to engage with DNA. The stability of the final complex relies on the specific interactions between the OB-folds of each subunit.
Formation begins with the two smaller subunits, RPA32 and RPA14. These two proteins bind together, forming a stable sub-complex. This initial pairing creates a foundational structure for the rest of the assembly.
Once the RPA32-RPA14 sub-complex is formed, it acts as a platform to recruit the large RPA70 subunit. The trimerization core is completed when specific domains on RPA70 and RPA32 interact with RPA14. These interactions are primarily hydrophobic, meaning they are driven by the chemical tendency of certain parts of the proteins to avoid water. This final binding event completes the assembly, yielding the fully functional RPA heterotrimer.
RPA’s Role in DNA Maintenance
With the RPA complex fully assembled, its primary function is to manage and protect single-stranded DNA (ssDNA) that appears during normal cellular activities. During DNA replication, for instance, the double helix must be unwound and separated to serve as templates for new DNA. This action exposes long stretches of ssDNA, which are unstable and susceptible to damage.
The assembled RPA complex quickly coats these exposed single strands. This coating serves two main purposes. First, it shields the ssDNA from cellular enzymes called endonucleases, which would otherwise degrade the unprotected strand. Second, it prevents the strand from forming hairpins or other secondary structures that would obstruct the replication machinery. By keeping the template strand accessible, RPA ensures that DNA polymerase can move along it efficiently.
This protective function is not limited to replication. Similar ssDNA intermediates are generated during DNA repair pathways, such as nucleotide excision repair and homologous recombination. In these contexts, RPA binds to the ssDNA, stabilizing it and preventing further damage. It also acts as a platform to recruit other repair proteins, like RAD51, to the site of the damage, facilitating corrections.