Backbone H, N, and C resonance assignments of the FF1 domain from P190A RhoGAP in 5 and 8 M urea.

The Rho GTPase (Ras homolog GTPases) system is a crucial signal transducer that regulates various cellular processes, including cell cycle and migration, genetic transcription, and apoptosis. In this study, we investigated the unfolded state of the first FF domain (FF1) of P190A RhoGAP, which features four tandem FF domains. For signal transduction, FF1 is phosphorylated at tyrosine 308 (Y308), which is buried in the hydrophobic core and is inaccessible to kinases in the folded domain.

Probing hot spots of protein-protein interactions mediated by the safety-belt region of REV7.

REV7 is a HORMA (Hop1, Rev7, Mad2) family adaptor protein best known as an accessory subunit of the translesion synthesis (TLS) DNA polymerase ζ (Polζ). In this role, REV7 binds REV3, the catalytic subunit of Polζ, by locking REV7-binding motifs (RBMs) in REV3 underneath the REV7 safety-belt loop. The same mechanism is used by REV7 to interact with RBMs from other proteins in DNA damage response (DDR) and mitosis.

The backbone NMR resonance assignments of the stabilized E. coli β clamp.

The 81 kDa E. coli β clamp is a ring-shaped head-to-tail homodimer that encircles DNA and plays a central role in bacterial DNA replication by serving as a processivity factor for DNA polymerases and a binding platform for other DNA replication and repair proteins. Here we report the backbone H, N, and C NMR resonance assignments of the stabilized T45R/S107R β clamp variant obtained using standard TROSY-based triple-resonance experiments (BMRB 52548).

Protein Assemblies in Translesion Synthesis.

Translesion synthesis (TLS) is a mechanism of DNA damage tolerance utilized by eukaryotic cells to replicate DNA across lesions that impede the high-fidelity replication machinery. In TLS, a series of specialized DNA polymerases are employed, which recognize specific DNA lesions, insert nucleotides across the damage, and extend the distorted primer-template. This allows cells to preserve genetic integrity at the cost of mutations.

Lead compound profiling for small molecule inhibitors of the REV1-CT/RIR Translesion synthesis Protein-Protein interaction.

Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development.

Effects of Xylanase A double mutation on substrate specificity and structural dynamics.

While protein activity is traditionally studied with a major focus on the active site, the activity of enzymes has been hypothesized to be linked to the flexibility of adjacent regions, warranting more exploration into how the dynamics in these regions affects catalytic turnover. One such enzyme is Xylanase A (XylA), which cleaves hemicellulose xylan polymers by hydrolysis at internal β-1,4-xylosidic linkages. It contains a "thumb" region whose flexibility has been suggested to affect the activity.

Backbone and ILV side-chain methyl NMR resonance assignments of human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes.

Rev7 is a versatile HORMA (Hop1, Rev7, Mad2) family adaptor protein with multiple roles in mitotic regulation and DNA damage response, and an essential accessory subunit of the translesion synthesis (TLS) DNA polymerase Polζ employed in replication of damaged DNA. Within Polζ, the two copies of Rev7 interact with the two Rev7-bonding motifs (RBM1 and RBM2) of the catalytic subunit Rev3 by a mechanism characteristic of HORMA proteins whereby the "safety-belt" loop of Rev7 closes on the top of the ligand. Here we report the nearly complete backbone and Ile, Val, Leu side-chain methyl NMR resonance assignments of the 27 kDa human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes (BMRB deposition numbers 51651 and 51652) that will facilitate future NMR studies of Rev7 dynamics and interactions.

Activation Dynamics of Ubiquitin Specific Protease 7.

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme responsible for the regulation of key human oncoproteins and tumor suppressors including Mdm2 and p53, respectively. Unlike other members of the USP family of proteases, the isolated catalytic domain of USP7 adopts an enzymatically inactive conformation that has been well characterized using X-ray crystallography. The catalytic domain also samples an active conformation, which has only been captured upon USP7 substrate-binding.

ILV methyl NMR resonance assignments of the 81 kDa E. coli β-clamp.

The ring-shaped E. coli β-clamp protein is an 81 kDa head-to-tail homodimer, which serves as a processivity factor anchoring the replicative polymerase to DNA, thereby increasing replication processivity and speed. In addition, it facilitates numerous protein transactions that take place on DNA during replication, repair, and damage response.

DNA Sequence Specificity Reveals a Role of the HLTF HIRAN Domain in the Recognition of Trinucleotide Repeats.

DNA damage tolerance (DDT) pathways enable cells to cope with a variety of replication blocks that threaten their ability to complete DNA replication. Helicase-like transcription factor (HLTF) plays a central role in the error-free DDT pathway, template switching (TS), by serving as a ubiquitin ligase to polyubiquitinate the DNA sliding clamp PCNA, which promotes TS initiation. HLTF also serves as an ATP-dependent DNA translocase facilitating replication fork remodeling.

Backbone and ILV side-chain NMR resonance assignments of the catalytic domain of human deubiquitinating enzyme USP7.

Ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme, which removes ubiquitin tag from numerous protein substrates involved in diverse cellular processes such as cell cycle regulation, apoptosis and DNA damage response. USP7 affects stability, interaction network and cellular localization of its cellular and viral substrates by controlling their ubiquitination status. The large 41 kDa catalytic domain of USP7 harbors the active site of the enzyme.

REV1 Inhibition Enhances Radioresistance and Autophagy.

Cancer therapy resistance is a persistent clinical challenge. Recently, inhibition of the mutagenic translesion synthesis (TLS) protein REV1 was shown to enhance tumor cell response to chemotherapy by triggering senescence hallmarks. These observations suggest REV1's important role in determining cancer cell response to chemotherapy.

Targeting protein-protein interactions in the DNA damage response pathways for cancer chemotherapy.

Cellular DNA damage response (DDR) is an extensive signaling network that orchestrates DNA damage recognition, repair and avoidance, cell cycle progression and cell death. DDR alteration is a hallmark of cancer, with the deficiency in one DDR capability often compensated by a dependency on alternative pathways endowing cancer cells with survival and growth advantage. Targeting these DDR pathways has provided multiple opportunities for the development of cancer therapies.

NMR resonance assignments for the nucleotide binding domains of the E. coli clamp loader complex γ subunit.

The E. coli γ clamp loader is a pentameric complex of δ, δ' and three γ subunits that opens and loads β-clamp proteins onto DNA in an ATP-dependent process essential for efficient DNA replication. ATP binding to the γ subunits promotes conformational changes that enable the clamp loader to bind and open the ring-shaped β-clamp homodimer.

Structure-Based Drug Design of Phenazopyridine Derivatives as Inhibitors of Rev1 Interactions in Translesion Synthesis.

Rev1 is a protein scaffold of the translesion synthesis (TLS) pathway, which employs low-fidelity DNA polymerases for replication of damaged DNA. The TLS pathway helps cancers tolerate DNA damage induced by genotoxic chemotherapy, and increases mutagenesis in tumors, thus accelerating the onset of chemoresistance. TLS inhibitors have emerged as potential adjuvant drugs to enhance the efficacy of first-line chemotherapy, with the majority of reported inhibitors targeting protein-protein interactions (PPIs) of the Rev1 C-terminal domain (Rev1-CT).

The Rev1-Polζ translesion synthesis mutasome: Structure, interactions and inhibition.

DNA contains information that must be safeguarded, but also accessed for transcription and replication. To perform replication, eukaryotic cells use the B-family DNA polymerase enzymes Polδ and Polɛ, which are optimized for accuracy, speed, and processivity. The molecular basis of these high-performance characteristics causes these replicative polymerases to fail at sites of DNA damage (lesions), which would lead to genomic instability and cell death.

Virtual Pharmacophore Screening Identifies Small-Molecule Inhibitors of the Rev1-CT/RIR Protein-Protein Interaction.

Translesion synthesis (TLS) has emerged as a mechanism through which several forms of cancer develop acquired resistance to first-line genotoxic chemotherapies by allowing replication to continue in the presence of damaged DNA. Small molecules that inhibit TLS hold promise as a novel class of anticancer agents that can serve to enhance the efficacy of these front-line therapies. We previously used a structure-based rational design approach to identify the phenazopyridine scaffold as an inhibitor of TLS that functions by disrupting the protein-protein interaction (PPI) between the C-terminal domain of the TLS DNA polymerase Rev1 (Rev1-CT) and the Rev1 interacting regions (RIR) of other TLS DNA polymerases.

Dynamics of the E. coli β-Clamp Dimer Interface and Its Influence on DNA Loading.

The ring-shaped sliding clamp proteins have crucial roles in the regulation of DNA replication, recombination, and repair in all organisms. We previously showed that the Escherichia coli β-clamp is dynamic in solution, transiently visiting conformational states in which Domain 1 at the dimer interface is more flexible and prone to unfolding. This work aims to understand how the stability of the dimer interface influences clamp-opening dynamics and clamp loading by designing and characterizing stabilizing and destabilizing mutations in the clamp.

Structural Approach To Identify a Lead Scaffold That Targets the Translesion Synthesis Polymerase Rev1.

Translesion synthesis (TLS) is a mechanism of replication past damaged DNA through which multiple forms of human cancer survive and acquire resistance to first-line genotoxic chemotherapies. As such, TLS is emerging as a promising target for the development of a new class of anticancer agents. The C-terminal domain of the DNA polymerase Rev1 (Rev1-CT) mediates assembly of the functional TLS complex through protein-protein interactions (PPIs) with Rev1 interacting regions (RIRs) of several other TLS DNA polymerases.

Conformational Dynamics of a Cysteine-Stabilized Plant Defensin Reveals an Evolutionary Mechanism to Expose Hydrophobic Residues.

Sugar cane defensin 5 (Sd5) is a small antifungal protein, whose structure is held together by four conserved disulfide bridges. Sd5 and other proteins sharing a cysteine-stabilized α-β (CSαβ) fold lack a regular hydrophobic core. Instead, they are stabilized by tertiary contacts formed by surface-exposed hydrophilic and hydrophobic residues.

Rev7 dimerization is important for assembly and function of the Rev1/Polζ translesion synthesis complex.

The translesion synthesis (TLS) polymerases Polζ and Rev1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding Rev1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the "safety belt" loop.