James L. Keck

SSB as an organizer/mobilizer of genome maintenance complexes

When duplex DNA is altered in almost any way (replicated, recombined, or repaired), single strands of DNA are usually intermediates, and single-stranded DNA binding (SSB) proteins are present. These proteins have often been described as inert, protective DNA coatings. Continuing research is demonstrating a far more complex role of SSB that includes the organization and/or mobilization of all aspects of DNA metabolism. Escherichia coli SSB is now known to interact with at least 14 other proteins that include key components of the elaborate systems involved in every aspect of DNA metabolism. Most, if not all, of these interactions are mediated by the amphipathic C-terminus of SSB. In this review, we summarize the extent of the eubacterial SSB interaction network, describe the energetics of interactions with SSB, and highlight the roles of SSB in the process of recombination. Similar themes to those highlighted in this review are evident in all biological systems. Keywords

High‐resolution structure of the E.coli RecQ helicase catalytic core

RecQ family helicases catalyze critical genome maintenance reactions in bacterial and eukaryotic cells, playing key roles in several DNA metabolic processes. Mutations in recQ genes are linked to genome instability and human disease. To define the physical basis of RecQ enzyme function, we have determined a 1.8 Å resolution crystal structure of the catalytic core of Escherichia coli RecQ in its unbound form and a 2.5 Å resolution structure of the core bound to the ATP analog ATPγS. The RecQ core comprises four conserved subdomains; two of these combine to form its helicase region, while the others form unexpected Zn2+‐binding and winged‐helix motifs. The structures reveal the molecular basis of missense mutations that cause Blooms syndrome, a human RecQ‐associated disease. Finally, based on findings from the structures, we propose a mechanism for RecQ activity that could explain its functional coordination with topoisomerase III.

Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11

In all organisms, type II DNA topoisomerases are essential for untangling chromosomal DNA. We have determined the structure of the DNA‐binding core of the Methanococcus jannaschii DNA topoisomerase VI A subunit at 2.0 Å resolution. The overall structure of this subunit is unique, demonstrating that archaeal type II enzymes are distinct from other type II topoisomerases. However, the core structure contains a pair of domains that are also found in type IA and classic type II topoisomerases. Together, these regions may form the basis of a DNA cleavage mechanism shared among these enzymes. The core A subunit is a dimer that contains a deep groove that spans both protomers. The dimer architecture suggests that DNA is bound in the groove, across the A subunit interface, and that the two monomers separate during DNA transport. The A subunit of topoisomerase VI is homologous to the meiotic recombination factor, Spo11, and this structure can serve as a template for probing Spo11 function in eukaryotes.

The HRDC domain of BLM is required for the dissolution of double Holliday junctions.

Blooms syndrome is a hereditary cancer‐predisposition disorder resulting from mutations in the BLM gene. In humans, BLM encodes one of five members of the RecQ helicase family. One function of BLM is to act in concert with topoisomerase IIIα (TOPO IIIα) to resolve recombination intermediates containing double Holliday junctions by a process called double Holliday junction dissolution, herein termed dissolution. Here, we show that dissolution is highly specific for BLM among human RecQ helicases and critically depends upon a functional HRDC domain in BLM. We show that the HRDC domain confers DNA structure specificity, and is required for the efficient binding to and unwinding of double Holliday junctions, but not for the unwinding of a simple partial duplex substrate. Furthermore, we show that lysine‐1270 of BLM, which resides in the HRDC domain and is predicted to play a role in mediating interactions with DNA, is required for efficient dissolution.

A Central Role for SSB in Escherichia coli RecQ DNA Helicase Function

RecQ DNA helicases are critical components of DNA replication, recombination, and repair machinery in all eukaryotes and bacteria. Eukaryotic RecQ helicases are known to associate with numerous genome maintenance proteins that modulate their cellular functions, but there is little information regarding protein complexes involving the prototypical bacterial RecQ proteins. Here we use an affinity purification scheme to identify three heterologous proteins that associate with Escherichia coli RecQ: SSB (single-stranded DNA-binding protein), exonuclease I, and RecJ exonuclease. The RecQ-SSB interaction is direct and is mediated by the RecQ winged helix subdomain and the C terminus of SSB. Interaction with SSB has important functional consequences for RecQ. SSB stimulates RecQ-mediated DNA unwinding, whereas deletion of the C-terminal RecQ-binding site from SSB produces a variant that blocks RecQ DNA binding and unwinding activities, suggesting that RecQ recognizes both the SSB C terminus and DNA in SSB·DNA nucleoprotein complexes. These findings, together with the noted interactions between human RecQ proteins and Replication Protein A, identify SSB as a broadly conserved RecQ-binding protein. These results also provide a simple model that explains RecQ integration into genome maintenance processes in E. coli through its association with SSB.

Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75

Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109–Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109–Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by ∼100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT–chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

Structure and function of RecQ DNA helicases.

RecQ family helicases play important roles in coordinating genome maintenance pathways in living cells. In the absence of functional RecQ proteins, cells exhibit a variety of phenotypes, including increased mitotic recombination, elevated chromosome missegregation, hypersensitivity to DNA-damaging agents, and defects in meiosis. Mutations in three of the five human RecQ family members give rise to genetic disorders associated with a predisposition to cancer and premature aging, highlighting the importance of RecQ proteins and their cellular activities for human health. Current evidence suggests that RecQ proteins act at multiple steps in DNA replication, including stabilization of replication forks and removal of DNA recombination intermediates, in order to maintain genome integrity. The cellular basis of RecQ helicase function may be explained through interactions with multiple components of the DNA replication and recombination machinery. This review focuses on biochemical and structural aspects of the RecQ helicases and how these features relate to their known cellular function, specifically in preventing excessive recombination.

Structure of the calcium-rich signature domain of human thrombospondin-2

Thrombospondins (THBSs) are secreted glycoproteins that have key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6-Å-resolution crystal structure of the glycosylated signature domain of human THBS2, which includes three epidermal growth factor–like modules, 13 aspartate-rich repeats and a lectin-like module. These elements interact extensively to form three structural regions termed the stalk, wire and globe. The THBS2 signature domain is stabilized by these interactions and by a network of 30 bound Ca2+ ions and 18 disulfide bonds. The structure suggests how genetic alterations of THBSs result in disease.

Structural basis of Escherichia coli single-stranded DNA-binding protein stimulation of exonuclease I

Bacterial single-stranded DNA (ssDNA)-binding proteins (SSBs) play essential protective roles in genome biology by shielding ssDNA from damage and preventing spurious DNA annealing. Far from being inert, ssDNA/SSB complexes are dynamic DNA processing centers where many different enzymes gain access to genomic substrates by exploiting direct interactions with SSB. In all cases examined to date, the C terminus of SSB (SSB-Ct) forms the docking site for heterologous proteins. We describe the 2.7-Å-resolution crystal structure of a complex formed between a peptide comprising the SSB-Ct element and exonuclease I (ExoI) from Escherichia coli. Two SSB-Ct peptides bind to adjacent sites on ExoI. Mutagenesis studies indicate that one of these sites is important for association with the SSB-Ct peptide in solution and for SSB stimulation of ExoI activity, whereas the second has no discernable function. These studies identify a correlation between the stability of the ExoI/SSB-Ct complex and SSB-stimulation of ExoI activity. Furthermore, mutations within SSBs C terminus produce variants that fail to stimulate ExoI activity, whereas the SSB-Ct peptide alone has no effect. Together, our findings indicate that SSB stimulates ExoI by recruiting the enzyme to its substrate and provide a structural paradigm for understanding SSBs organizational role in genome maintenance.

The Origin Recognition Complex and Sir4 Protein Recruit Sir1p to Yeast Silent Chromatin through Independent Interactions Requiring a Common Sir1p Domain

ABSTRACT Sir1p is one of four SIR (silent information regulator) proteins required for silencing the cryptic mating-type locus HMR a in the budding yeast Saccharomyces cerevisiae. A Sir1p interaction with Orc1p, the largest subunit of the origin recognition complex (ORC), is critical for Sir1ps ability to bind HMR a and function in the formation of silent chromatin. Here we show that a discrete domain within Sir1p, the ORC interaction region (OIR), was necessary and sufficient for a Sir1p-ORC interaction. The OIR contains the originally defined silencer recognition-defective region as well as additional amino acids. In addition, a Sir1p-Sir4p interaction required a larger region of Sir1p that included the OIR. Amino acid substitutions causing defects in either a Sir1p-Orc1p or a Sir1p-Sir4p interaction reduced HMR a silencing and Sir1p binding to HMR a in chromatin. These data support a model in which Sir1ps association with HMR a is mediated by separable Sir1p-ORC and Sir1p-Sir4p interactions requiring a common Sir1p domain, and they indicate that a Sir1p-ORC interaction is restricted to silencers, at least in part, through interactions with Sir4p.