The Trimerization Core of Human Replication Protein A
Amar Desai '08 and Michael Northcutt '08
Replication Protein A (RPA) is a eukaryotic single stranded (ss) DNA-binding protein that is vital to DNA replication, recombination, and repair. Truener et al. (1996) showed that RPA unwinds dsDNA by binding to the A+T rich internal regions of dsDNA and forcing separation of complementary DNA strands. Additional studies have shown that it undergoes protein-protein interactions with proteins involved in DNA replication and repair, and it is believed that RPA may also be involved in transcriptional regulation. Increasing evidence has revealed that RPA may have a modulatory role in cellular responses and this is shown by the fact that RPA is hyperphosphorylated upon DNA damage or replication stress, which may change the structure of RPA and the individual pathways with which it is associated (Zou et al., 2006). The protein is also essential in the initiation and elongation phases of DNA replication because it is required for loading DNA polymerase alpha and additional replication fork proteins. Once the forks are established, RPA remains associated with them throughout elongation. During DNA repair, RPA contributes to damage recognition, excision, and re-synthesis of the DNA strand. Finally RPA is important in homologous recombination of DNA because it has many interactions with a group of proteins (Rad 52) that are vital to recombination and it has been shown that the new DNA was not stable unless RPA was present (Binz et al., 2004).
II. General Structure
In humans RPA forms a heterotrimer with subunits of 70, 32, and 14 kDa.
Each subunit consists of smaller domains that play different roles in the
function of the protein, and this crystal structure shows a dimer of trimers
of the three domains that form the trimerization core. This trimerization
core is composed of DNA Binding Domain C (DBD-C) of
RPA70 , DBD-D of RPA32, and RPA14
respectively (Bochkareva et al., 2002). Each of the domains is composed
of an OB-fold (oligo-saccharide/oligo-nucleotide binding fold) domain which
is common to many single stranded DNA binding proteins and the interactions
between the trimerization core will be detailed below (Binz et al., 2004).
III. Formation of the Trimer
The formation of the heterotrimer in human RPA is mediated by three domains: DBD-C
in RPA32 and RPA 14
together form the trimerization core.
All three domains in the trimerization core
are structurally similar and are built around a central oligonucleotide/oligosaccharide binding (OB) fold and flanked by an alpha-helix at the C-terminus (See DBD-C
, and RPA14.
The three OB-folds
provide the major source of interaction between the trimerization core and these folds are packed in tandem and run half a turn around the three-helical bundle. (Bochkareva et. al 2002).
Because RPA crystallizes as a dimer of trimers, it will be shown from this point in this configuration. Trimerization is facilitated by hydrophobic interactions of three parallel C-terminal alpha-helices, and amino acids from each domain are involved in stabilizing these interactions. Tyr599, Tyr602, Leu606, Val607, Ile610, Ala614 of DBD-C
; Met152, Phe155, Ile159, Leu160, Ile163, Met167
Leu98, Ala 102, Ile105, Phe109 and Phe114 of RPA 14
An interaction believed to be of functional importance exists between the lone contact of DBD-C and DBD-D. Ile46 at the extreme N-terminus of DBD-D is only 3 Å away from the Asp522 in the loop between ß strands 2 and 3 of DBD-C (L23)
. Structural data suggests that the N-terminus of RPA32 might interact with DBD-C and/or DBD-D to regulate DNA binding of the trimer. (Bochkareva et. al 2002).
IV. Conserved DNA Binding Sites and the DBD-C Domain
Many sources of evidence indicate the conservation of the DNA binding site in all four DBD’s of the trimer. The first is the structural conservation of the OB fold in all of the DBD’s. The second key is the conservation of important aromatic amino acids (Phenylalanine, Tyrosine, Tryptophan) and analogs of these have been found in all four of the DBDs. In the trimerization core, Phe532 and Tyr 581 from DBD-C show this conservation while Trp 107 and Phe135 in DBD-D are also present.
These amino acids have been shown to stack with DNA bases in DBD-A and DBD-B (two domains not involved in trimerization and not shown in this crystal structure).
DBD-C possesses a unique characteristic with the presence of a zinc ribbon embedded in the OB-fold.
This zinc ribbon is located around amino acids 480-510 between ß strands 1 and 2. Three antiparallel ß strands form the region with four cysteine residues tetrahedrally flanking the zinc ion
The function of the zinc ribbon in RPA is thought to be cooperative binding, as particular amino acids (Asn487, Lys488, Lys489, flanking Cys486) are located close in proximity to the ssDNA-binding region and may thus interact with the sugar-phosphate backbone of bound DNA. (Bochkareva et. al 2002).
An additional feature of interest of DBD-C is a cap composed of three helices at the rear of the domain showing characteristics of the helix-turn-helix motif.
. This region may be involved in binding to double-stranded DNA. (Bochkareva et. al 2002).
V. DNA Binding
suggested multistep ssDNA-binding pathway (Bochkareva
et al 2001).
The mechanism for ssDNA binding in RPA is multistep and involves all four DBDs. The first step has DBD-A and DBD-B binding 8-10 nucleotides of ssDNA in an unstable manner. The functions of this type of binding could include anchoring RPA to ssDNA and recruiting other proteins to the complex. The next step involves a significant conformational change to a higher order binding mode of about 30 nucleotides per trimer. This new mode elongates on ssDNA in a 5’ to 3’ direction and does this to involve the trimerization core. A possible explanation for this would be so that DBD-C and DBD-D are positioned in tandem after DBD-B and thus DBD-C could contact the 3’ protruding end of ssDNA after DBD-B does. After binding to the ssDNA, the linker that connects DBD-C and DBD-B becomes resistant to proteolysis. Thus this method clearly shows how the four DBDs bind to ssDNA in a cooperative manner. (Bochkareva et. al 2001).
Binz, S.K., Sheehan, A.M., Wold, M.S. Replication Protein A Phosphorylation and the Cellular Response to DNA Damage. DNA Repair. 3, 1015-1024 (2004).
Bochkareva, E., Belegu, V., Korolev, S., Bochkareva, A. Structure of the major single-stranded DNA-binding domain of replication protein A suggests a dynamic mechanism for DNA binding. Embo. 20, 612-618 (2001).
Bochkareva, E., Korolev, S., Lees-Miller, S.P., & Bochkarev, A. Structure of the RPA Trimerization Core and its Role in the Multistep DNA-Binding Mechanism of RPA. The European Molecular Biology Organization Journal. 21, 1855-1863 (2002).
Truener, K., Ramsperger, U., & Knippers, R. Replication Protein A Induces the Unwinding of Long Double-stranded DNA Regions. J. Mol. Bio. 259, 104-112 (1996).
Zou, Y., Liu, Y., Wu, X., & Shell, S. Functions of Human Replication Protein A (RPA): From DNA Replication to DNA Damage and Stress Responses. Journal of Cellular Physiology. 208, 267-273 (2006).
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