Alexandra M. Deaconescu

Structural Basis for Bacterial Transcription-Coupled DNA Repair

Coupling of transcription and DNA repair in bacteria is mediated by transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the transcription elongation complex, provide mechanistic insights into TRCF function.

Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1

The microtubule regulator APC is now shown to also regulate actin filament dynamics through its C-terminal “Basic” domain.

Tubulin tyrosine ligase structure reveals adaptation of an ancient fold to bind and modify tubulin

Tubulin tyrosine ligase (TTL) catalyzes the post-translational C-terminal tyrosination of α-tubulin. Tyrosination regulates recruitment of microtubule-interacting proteins. TTL is essential. Its loss causes morphogenic abnormalities and is associated with cancers of poor prognosis. We present the first crystal structure of TTL (from Xenopus tropicalis), defining the structural scaffold upon which the diverse TTL-like family of tubulin-modifying enzymes is built. TTL recognizes tubulin using a bipartite strategy. It engages the tubulin tail through low-affinity, high-specificity interactions, and co-opts what is otherwise a homo-oligomerization interface in structurally related ATP grasp-fold enzymes to form a tight hetero-oligomeric complex with the tubulin body. Small-angle X-ray scattering and functional analyses reveal that TTL forms an elongated complex with the tubulin dimer and prevents its incorporation into microtubules by capping the tubulin longitudinal interface, possibly modulating the partition of tubulin between monomeric and polymeric forms.

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF–UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.

X-ray structure of Saccharomyces cerevisiae homologous mitochondrial matrix factor 1 (Hmf1).

Introduction. Homologous mitochondrial matrix factor 1 Hmf1 (also known as YEO7 and YER057c) is a cytoplasmic homolog of Saccharomyces cerevisiae mitochondrial matrix factor 1 Mmf1, which has been proposed to serve as a sensor for isoleucine deficiency and a regulator of branched-chain amino acid transaminases. Both proteins belong to the YjgF/YER057c/UK114 protein superfamily, which has been highly conserved among eubacteria, archaea, and eukaryotes. This family is characterized by a C-terminal signature sequence of [PA]-[ASTPV]-R[SACVF]-x-[LIVMFY]-x(2)-[GSAKR]-x-[LMVA]-x(5,8)[LIVM]-E-[MI] (Fig. 1), and its members are found in all three living kingdoms as independent domains having an average molecular weight of 15 kDa (Fig. 1). Superfamily members found in all three living kingdoms exist as independent domains having an average molecular weight of 15 kDa (Fig. 1). Some genomes even contain multiple paralogs (e.g., four in Escherichia coli and two in S. cerevisiae). Despite the high degree of sequence conservation, functional studies have documented that members of this protein superfamily perform a variety of biochemical functions. Mammalian YER057c family members play roles in protein translation (human UK14, rat UK14), modulation of calpain affinity for calcium (bovine UK14), and heat-shock response (mouse HR12). Chronic administration of rat protein UK14 curtails the development of diabetes and adjuvant-induced arthritis. Human UK14 (also known as UK-114 and p14.5) was characterized as a tumor antigen expressed by various malignant neoplasms and was observed to be upregulated during cell differentiation. Bacterial members of the family affect biosynthetic pathways. Bacillus subtilis YABJ regulates purine biosynthesis by binding to the purine repressor purR and possibly stabilizing its association with DNA, whereas Lactococcus lactis ALDR and Salmonella thyphimurium YJGF block an intermediate step in the isoleucine biosynthetic pathway. The two paralogs present in S. cerevisiae, mitochondrial matrix factor 1 (Mmf1 or YIL051c) and homologous mitochondrial matrix factor 1 (Hmf1), share 71% sequence identity and have also been implicated in the transamination step of isoleucine biosynthesis plus maintenance of mitochondrial DNA (mtDNA). Mmf1 localizes to the mitochondria because of the presence of a 16-residue targeting leader peptide (Fig. 1), where it associates with mtDNA structures. Hmf1 is found mainly in the cytoplasm. Yil051c mutants are isoleucine auxotrophs in which mitochondrial respiratory activity is dramatically decreased and mtDNA is eventually lost. Deletion of the yer057c gene does not produce an obvious phenotype. Nevertheless, when fused to a mitochondrial targeting leader sequence, Hmf1 complements the yil051c deletion, indicating that Hmf1 and Mmf1 can perform similar functions, but in distinct cellular compartments. In addition, the human homolog can rescue the yil051c deletion phenotype. These results strongly suggest that the function of these homologs has been conserved, at least in part, among eukaryotes. This article reports the X-ray structure of Hmf1, which represents the first structure of a eukaryotic member of the YjgF/YER057c/UK114 superfamily. Hmf1 is a homotrimer that folds into a triangular, pseudo / barrel with narrow, deep grooves located at the intermonomer surfaces. On the basis of the high structural similarity of Hmf1 to B. subtilis chorismate mutase as well as sequence conservation, it is suggested that these intermonomer grooves represent ligand-binding regions, and possibly, enzyme active sites. Materials and Methods. Hmf1 represents target P003 of the New York Structural Genomics Research Consortium.

Interplay of DNA repair with transcription: from structures to mechanisms

Many DNA transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. An example is the interplay between nucleotide excision repair (NER) and transcription, also known as transcription-coupled DNA repair (TCR). Discovered decades ago, the mechanisms for TCR have remained elusive, not in small part due to the scarcity of structural studies of key players. Here we summarize recent structural information on NER/TCR factors, focusing on bacterial systems, and integrate it with existing genetic, biochemical, and biophysical data to delineate the mechanisms at play. We also review emerging, alternative modalities for recruitment of NER proteins to DNA lesions.

Crystallization and preliminary structure determination of Escherichia coli Mfd, the transcription-repair coupling factor

Transcription-repair coupling factors (TRCFs) are SF2 ATPases that couple transcription to DNA-damage repair by recognizing and removing RNA polymerase-elongation complexes stalled at DNA lesions and recruiting the nucleotide excision-repair machinery to the damaged sites. As a first step towards understanding the TRCF mechanism, the 130 kDa Escherichia coli TRCF (the product of the mfd gene) has been overexpressed, purified and crystallized using an unusual precipitant, pentaerythritol ethoxylate. Initial phases were obtained using single-wavelength anomalous dispersion with a highly redundant 4 A resolution data set collected from selenomethionyl-substituted crystals and dramatically improved by density modification and phase extension to 3.2 A resolution. Model building and refinement, which are in progress, will provide insight into transcription-coupled DNA-repair pathways, as this represents the first TRCF to be crystallized to date.

Regulation of Microtubule Assembly by Tau and not by Pin1.

The molecular mechanism by which the microtubule-associated protein (MAP) tau regulates the formation of microtubules (MTs) is poorly understood. The activity of tau is controlled via phosphorylation at specific Ser/Thr sites. Of those phosphorylation sites, 17 precede a proline, making them potential recognition sites for the peptidyl-prolyl isomerase Pin1. Pin1 binding and catalysis of phosphorylated tau at the AT180 epitope, which was implicated in Alzheimers disease, has been reported to be crucial for restoring taus ability to promote MT polymerization in vitro and in vivo [1]. Surprisingly, we discover that Pin1 does not promote phosphorylated tau-induced MT formation in vitro, refuting the commonly accepted model in which Pin1 binding and catalysis on the A180 epitope restores the function of the Alzheimers associated phosphorylated tau in tubulin assembly [1, 2]. Using turbidity assays, time-resolved small angle X-ray scattering (SAXS), and time-resolved negative stain electron microscopy (EM), we investigate the mechanism of tau-mediated MT assembly and the role of the Thr231 and Ser235 phosphorylation on this process. We discover novel GTP-tubulin ring-shaped species, which are detectable in the earliest stage of tau-induced polymerization and may play a crucial role in the early nucleation phase of MT assembly. Finally, by NMR and SAXS experiments, we show that the tau molecules must be located on the surface of MTs and tubulin rings during the polymerization reaction. The interaction between tau and tubulin is multipartite, with a high affinity interaction of the four tubulin-binding repeats, and a weaker interaction with the proline-rich sequence and the termini of tau.

RNA polymerase between lesion bypass and DNA repair

DNA damage leads to heritable changes in the genome via DNA replication. However, as the DNA helix is the site of numerous other transactions, notably transcription, DNA damage can have diverse repercussions on cellular physiology. In particular, DNA lesions have distinct effects on the passage of transcribing RNA polymerases, from easy bypass to almost complete block of transcription elongation. The fate of the RNA polymerase positioned at a lesion is largely determined by whether the lesion is structurally subtle and can be accommodated and eventually bypassed, or bulky, structurally distorting and requiring remodeling/complete dissociation of the transcription elongation complex, excision, and repair. Here we review cellular responses to DNA damage that involve RNA polymerases with a focus on bacterial transcription-coupled nucleotide excision repair and lesion bypass via transcriptional mutagenesis. Emphasis is placed on the explosion of new structural information on RNA polymerases and relevant DNA repair factors and the mechanistic models derived from it.

From Mfd to TRCF and Back Again—A Perspective on Bacterial Transcription-coupled Nucleotide Excision Repair†

Photochemical and other reactions on DNA cause damage and corrupt genetic information. To counteract this damage, organisms have evolved intricate repair mechanisms that often crosstalk with other DNA‐based processes such as transcription. Intriguing observations in the late 1980s and early 1990s led to the discovery of transcription‐coupled repair (TCR), a subpathway of nucleotide excision repair. TCR, found in all domains of life, prioritizes for repair lesions located in the transcribed DNA strand, directly read by RNA polymerase. Here, we give a historical overview of developments in the field of bacterial TCR, starting from the pioneering work of Evelyn Witkin and Aziz Sancar, which led to the identification of the first transcription‐repair coupling factor (the Mfd protein), to recent studies that have uncovered alternative TCR pathways and regulators.