Single-stranded DNA (ssDNA)-binding (SSB) proteins are uniformly required to bind and protect single-stranded intermediates in DNA metabolic pathways. All bacterial and eukaryotic SSB proteins studied to date oligomerize to assemble four copies of a conserved domain, called an oligonucleotide͞oligosaccharide-binding (OB) fold, that cooperate in nonspecific ssDNA binding. The vast majority of bacterial SSB family members function as homotetramers, with each monomer contributing a single OB fold. However, SSB proteins from the Deinococcus-Thermus genera are exceptions to this rule, because they contain two OB folds per monomer. To investigate the structural consequences of this unusual arrangement, we have determined a 1.8-Å-resolution x-ray structure of Deinococcus radiodurans SSB. The structure shows that D. radiodurans SSB comprises two OB domains linked by a ␤-hairpin motif. The protein assembles a four-OB-fold arrangement by means of symmetric dimerization. In contrast to homotetrameric SSB proteins, asymmetry exists between the two OB folds of D. radiodurans SSB because of sequence differences between the domains. These differences appear to reflect specialized roles that have evolved for each domain. Extensive crystallographic contacts link D. radiodurans SSB dimers in an arrangement that has important implications for higher-order structures of the protein bound to ssDNA. This assembly utilizes the N-terminal OB domain and the ␤-hairpin structure that is unique to Deinococcus and Thermus species SSB proteins. We hypothesize that differences between D. radiodurans SSB and homotetrameric bacterial SSB proteins may confer a selective advantage to D. radiodurans cells that aids viability in environments that challenge genomic stability.I n all organisms, single-stranded DNA (ssDNA)-binding (SSB) proteins sequester and protect ssDNA intermediates that arise during DNA replication, recombination, and repair (1). Their prominent roles in genome maintenance reactions make SSB proteins a requirement for cellular life (2). Although the sequences of SSB family members are highly variable, two common functional themes have emerged that link this class of proteins across evolution. The first is that SSB proteins use a conserved domain called an oligonucleotide͞oligosaccharide-binding (OB) fold to bind ssDNA (3, 4). OB domains bind ssDNA in a cleft formed primarily by ␤-strands, by using aromatic residues that stack against nucleotide bases and positively charged residues that form ionic interactions with the DNA backbone (5-8). The second common feature of cellular SSB proteins is obligate oligomerization that brings together four DNA-binding OB folds. For example, Escherichia coli SSB contains a single OB fold per monomer, but the active form of the protein is a homotetramer with four OB folds (1). This general arrangement appears to define a structural paradigm for bacterial SSB family proteins because all but three of the Ͼ250 currently identifiable bacterial ssb genes encode proteins with a single OB fold.Different...

Helicases are specialized molecular motors that separate duplex nucleic acids into single strands. The RecQ family of helicases functions at the interface of DNA replication, recombination and repair in bacterial and eukaryotic cells. They are key, multifunctional enzymes that have been linked to three human diseases: Bloom's, Werner's and Rothmund-Thomson's syndromes. This review summarizes recent studies that relate the structures of RecQ proteins to their biochemical activities.

RecQ helicases are key genome maintenance enzymes that function in DNA replication, recombination, and repair. In contrast to nearly every other identified RecQ family member, the RecQ helicase from the radioresistant bacterium Deinococcus radiodurans encodes three "Helicase and RNase D C-terminal" (HRDC) domains at its C terminus. HRDC domains have been implicated in structure-specific nucleic acid binding with roles in targeting RecQ proteins to particular DNA structures; however, only RecQ proteins with single HRDC domains have been examined to date. We demonstrate that the HRDC domains can be proteolytically removed from the D. radiodurans RecQ (DrRecQ) C terminus, consistent with each forming a structural domain. Using this observation as a guide, we produced a panel of recombinant DrRecQ variants lacking combinations of its HRDC domains to investigate their biochemical functions. The N-terminal-most HRDC domain is shown to be critical for high affinity DNA binding and for efficient unwinding of DNA in some contexts. In contrast, the more C-terminal HRDC domains attenuate the DNA binding affinity and DNAdependent ATP hydrolysis rate of the enzyme and play more complex roles in structure-specific DNA unwinding. Our results indicate that the multiple DrRecQ HRDC domains have evolved to encode DNA binding and regulatory functions in the enzyme.

Protein thermal shift assays (TSAs) provide a means for characterizing target engagement through ligand-induced thermal stabilization. Although these assays are widely utilized for screening libraries and validating hits in drug discovery programs, they can impose encumbering operational requirements, such as the availability of purified proteins or selective antibodies. Appending the target protein with a small luciferase (NanoLuc) allows coupling of thermal denaturation with luminescent output, providing a rapid and sensitive means for assessing target engagement in compositionally complex environments such as permeabilized cells. The intrinsic thermal stability of NanoLuc is greater than mammalian proteins, and our results indicate that the appended luciferase does not alter thermal denaturation of the target protein. We have successfully applied the NanoLuc luciferase thermal shift assay (NaLTSA) to several clinically relevant protein families, including kinases, bromodomains, and histone deacetylases. We have also demonstrated the suitability of this assay method for library screening and compound profiling.

RecQ helicases are critical for maintaining genome integrity in organisms ranging from bacteria to humans by participating in a complex network of DNA metabolic pathways. Their diverse cellular functions require specialization and coordination of multiple protein domains that integrate catalytic functions with DNA–protein and protein–protein interactions. The RecQ helicase from Deinococcus radiodurans (DrRecQ) is unusual among RecQ family members in that it has evolved to utilize three ‘Helicase and RNaseD C-terminal’ (HRDC) domains to regulate its activity. In this report, we describe the high-resolution structure of the C-terminal-most HRDC domain of DrRecQ. The structure reveals unusual electrostatic surface features that distinguish it from other HRDC domains. Mutation of individual residues in these regions affects the DNA binding affinity of DrRecQ and its ability to unwind a partial duplex DNA substrate. Taken together, the results suggest the unusual electrostatic surface features of the DrRecQ HRDC domain may be important for inter-domain interactions that regulate structure-specific DNA binding and help direct DrRecQ to specific recombination/repair sites.