Elena S. Bochkareva

Single-stranded DNA mimicry in the p53 transactivation domain interaction with replication protein A

One of many protein–protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1–120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37–57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast, the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37–57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide/oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic p53N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage.

Structural Basis for the Recognition of DNA Repair Proteins UNG2, XPA, and RAD52 by Replication Factor RPA

Replication protein A (RPA), the nuclear ssDNA-binding protein in eukaryotes, is essential to DNA replication, recombination, and repair. We have shown that a globular domain at the C terminus of subunit RPA32 contains a specific surface that interacts in a similar manner with the DNA repair enzyme UNG2 and repair factors XPA and RAD52, each of which functions in a different repair pathway. NMR structures of the RPA32 domain, free and in complex with the minimal interaction domain of UNG2, were determined, defining a common structural basis for linking RPA to the nucleotide excision, base excision, and recombinational pathways of repairing damaged DNA. Our findings support a hand-off model for the assembly and coordination of different components of the DNA repair machinery.

The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding.

Replication protein A (RPA), the eukaryote single‐stranded DNA‐binding protein (SSB), is a heterotrimer. The largest subunit, RPA70, which harbours the major DNA‐binding activity, has two DNA‐binding domains that each adopt an OB‐fold. The complex of the two smaller subunits, RPA32 and RPA14, has weak DNA‐binding activity but the mechanism of DNA binding is unknown. We have determined the crystal structure of the proteolytic core of RPA32 and RPA14, which consists of the central two‐thirds of RPA32 and the entire RPA14 subunit. The structure revealed that RPA14 and the central part of RPA32 are structural homologues. Each subunit contains a central OB‐fold domain, which also resembles the DNA‐binding domains in RPA70; an N‐terminal extension that interacts with the central OB‐fold domain; and a C‐terminal helix that mediate heterodimerization via a helix–helix interaction. The OB‐fold of RPA32, but not RPA14, possesses additional similarity to the RPA70 DNA‐binding domains, supporting a DNA‐binding role for RPA32. The discovery of a third and fourth OB‐fold in RPA suggests that the quaternary structure of SSBs, which in Bacteria and Archaea are also tetramers of OB‐folds, is conserved in evolution. The structure also suggests a mechanism for RPA trimer formation.

Structure of the major single‐stranded DNA‐binding domain of replication protein A suggests a dynamic mechanism for DNA binding

Although structures of single‐stranded (ss)DNA‐binding proteins (SSBs) have been reported with and without ssDNA, the mechanism of ssDNA binding in eukarya remains speculative. Here we report a 2.5 Å structure of the ssDNA‐binding domain of human replication protein A (RPA) (eukaryotic SSB), for which we previously reported a structure in complex with ssDNA. A comparison of free and bound forms of RPA revealed that ssDNA binding is associated with a major reorientation between, and significant conformational changes within, the structural modules—OB‐folds—which comprise the DNA‐binding domain. Two OB‐folds, whose tandem orientation was stabilized by the presence of DNA, adopted multiple orientations in its absence. Within the OB‐folds, extended loops implicated in DNA binding significantly changed conformation in the absence of DNA. Analysis of intermolecular contacts suggested the possibility that other RPA molecules and/or other proteins could compete with DNA for the same binding site. Using this mechanism, protein–protein interactions can regulate, and/or be regulated by DNA binding. Combined with available biochemical data, this structure also suggested a dynamic model for the DNA‐binding mechanism.

New prospects in studying the bacterial signal recognition particle pathway

In vivo and in vitro studies have suggested that the bacterial version of the mammalian signal recognition particle (SRP) system plays an essential and selective role in protein biogenesis. The bacterial SRP system consists of at least two proteins and an RNA molecule (termed Ffh, FtsY and 4.5S RNA, respectively, in Escherichia coli). Recent evidence suggests that other putative bacterial‐specific SRP components may also exist. In vitro experiments confirmed the expected basic features of the bacterial SRP system by demonstrating interactions among the SRP components themselves, between them and ribosomes, ribosome‐linked hydrophobic nascent polypeptides or inner membranes. The availability of a conserved (and essential) bacterial SRP version has facilitated the implementation of powerful genetic and biochemical approaches for studying the cascade of events during the SRP‐mediated targeting process in vivo and in vitro as well as the three‐dimensional structures and the properties of each SRP component and complex.

Independent and Coordinated Functions of Replication Protein A Tandem High Affinity Single-stranded DNA Binding Domains

The initial high affinity binding of single-stranded DNA (ssDNA) by replication protein A (RPA) is involved in the tandem domains in the central region of the RPA70 subunit (RPA70AB). However, it was not clear whether the two domains, RPA70A and RPA70B, bind DNA simultaneously or sequentially. Here, using primarily heteronuclear NMR complemented by fluorescence spectroscopy, we have analyzed the binding characteristics of the individual RPA70A and RPA70B domains and compared them with the intact RPA70AB. NMR chemical shift comparisons confirmed that RPA70A and RPA70B tumble independently in solution in the absence of ssDNA. NMR chemical shift perturbations showed that all ssDNA oligomers bind to the same sites as observed in the x-ray crystal structure of RPA70AB complexed to d(C)8. Titrations using a variety of 5′-mer ssDNA oligomers showed that RPA70A has a 5–10-fold higher affinity for ssDNA than RPA70B. Detailed analysis of ssDNA binding to RPA70A revealed that all DNA sequences interact in a similar mode. Fluorescence binding measurements with a variety of 8–10′-mer DNA sequences showed that RPA70AB interacts with DNA with ∼100-fold higher affinity than the isolated domains. Calculation of the theoretical “linkage effect” from the structure of RPA70AB suggests that the high overall affinity for ssDNA is a byproduct of the covalent attachment of the two domains via a short flexible tether, which increases the effective local concentration. Taken together, our data are consistent with a sequential model of DNA binding by RPA according to which RPA70A binds the majority of DNA first and subsequent loading of RPA70B domain is facilitated by the linkage effect.

The Crystal Structure of the SV40 T-Antigen Origin Binding Domain in Complex with DNA

DNA replication is initiated upon binding of “initiators” to origins of replication. In simian virus 40 (SV40), the core origin contains four pentanucleotide binding sites organized as pairs of inverted repeats. Here we describe the crystal structures of the origin binding domain (obd) of the SV40 large T-antigen (T-ag) both with and without a subfragment of origin-containing DNA. In the co-structure, two T-ag obds are oriented in a head-to-head fashion on the same face of the DNA, and each T-ag obd engages the major groove. Although the obds are very close to each other when bound to this DNA target, they do not contact one another. These data provide a high-resolution structural model that explains site-specific binding to the origin and suggests how these interactions help direct the oligomerization events that culminate in assembly of the helicase-active dodecameric complex of T-ag.

Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY.

Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane‐targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co‐translational targeting and assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co‐translational targeting and membrane assembly of NG. The co‐translational interaction of FtsY with the membrane was confirmed by in vitro experiments.

Membrane Targeting of Ribosomes and Their Release Require Distinct and Separable Functions of FtsY

The mechanism underlying the interaction of the Escherichia coli signal recognition particle (SRP) receptor FtsY with the cytoplasmic membrane is not fully understood. We investigated this issue by utilizing active (NG+1) and inactive (NG) mutants of FtsY. In solution, the mutants comparably bind and hydrolyze nucleotides and associate with SRP. In contrast, a major difference was observed in the cellular distribution of NG and NG+1. Unlike NG+1, which distributes almost as the wild-type receptor, the inactive NG mutant accumulates on the membrane, together with ribosomes and SRP. The results suggest that NG function is compromised only at a later stage of the targeting pathway and that despite their identical behavior in solution, the membrane-bound NG-SRP complex is less active than NG+1-SRP. This notion is strongly supported by the observation that lipids stimulate the GTPase activity of NG+1-SRP, whereas no stimulation is observed with NG-SRP. In conclusion, we propose that the SRP receptor has two distinct and separable roles in (i) mediating membrane targeting and docking of ribosomes and (ii) promoting their productive release from the docking site.

Structure of the origin-binding domain of simian virus 40 large T antigen bound to DNA

The large T antigen (T‐ag) protein binds to and activates DNA replication from the origin of DNA replication (ori) in simian virus 40 (SV40). Here, we determined the crystal structures of the T‐ag origin‐binding domain (OBD) in apo form, and bound to either a 17 bp palindrome (sites 1 and 3) or a 23 bp ori DNA palindrome comprising all four GAGGC binding sites for OBD. The T‐ag OBDs were shown to interact with the DNA through a loop comprising Ser147–Thr155 (A1 loop), a combination of a DNA‐binding helix and loop (His203–Asn210), and Asn227. The A1 loop traveled back‐and‐forth along the major groove and accounted for most of the sequence‐determining contacts with the DNA. Unexpectedly, in both T‐ag‐DNA structures, the T‐ag OBDs bound DNA independently and did not make direct protein–protein contacts. The T‐ag OBD was also captured bound to a non‐consensus site ATGGC even in the presence of its canonical site GAGGC. Our observations taken together with the known biochemical and structural features of the T‐ag–origin interaction suggest a model for origin unwinding.