Domain 1B Research Articles

ARID1B, a member of the human SWI/SNF chromatin remodeling complex, exhibits tumour-suppressor activities in pancreatic cancer cell lines

Background:The human ATP-dependent SWItch/sucrose nonfermentable (SWI/SNF) complex functions as a primary chromatin remodeler during ontogeny, as well as in adult life. Several components of the complex have been suggested to function as important regulators of tumorigenesis in various cancers. In the current study, we have characterised a possible tumour suppressor role for the largest subunit of the complex, namely the AT-rich interaction domain 1B (ARID1B).Methods:We performed Azacytidine and Trichostatin A treatments, followed by bisulphite sequencing to determine the possible DNA methylation-induced transcription repression of the gene in pancreatic cancer (PaCa) cell lines. Functional characterisation of effect of ARID1B ectopic expression in MiaPaCa2 PaCa cell line, which harboured ARID1B homozygous deletion, was carried out. Finally, we evaluated ARID1B protein expression in pancreatic tumour samples using immunohistochemistry on a tissue microarray.Results:ARID1B was transcriptionally repressed due to promoter hypermethylation, and ectopic expression severely compromised the ability of MiaPaCa2 cells to form colonies in liquid culture and soft agar. In addition, ARID1B exhibited significantly reduced/loss of expression in PaCa tissue, especially in samples from advanced-stage tumours, when compared with normal pancreas.Conclusion:The results therefore suggest a possible tumour-suppressor function for ARID1B in PaCa, thus adding to the growing list of SWI/SNF components with a similar function. Given the urgent need to design efficient targeted therapies for PaCa, our study assumes significance.

Abstract 4625: An impact of epigenetic approaches targeting JARID1B to modulate malignant behaviors of gastrointestinal cancer.

Abstract Background. Although there are emerging epigenetic factors that contribute to the occurrence, development and metastasis of Gastrointestinal (GI) cancer, the biological roles of epigenetic molecular modifications in different subpopulations, such as cancer stem cells, remains to be understood fully. Here we aimed to clarify the functional roles of the H3K4 demethylase, Jumonji, AT Rich Interactive Domain 1B (JARID1B), an epigenetic factor required for continuous cell growth of melanomas, in GI cancer. Materials and Methods. Based on our ptrevious expression studies showing a high expression level of JARID1B transcript, we focused on four GI cancer cell lines for the analysis: colorectal cancer Colo201 and HCT116; esophageal cancer TE4 and TE8. To study the underlined mechanism, the chromatin immunoprecipitation (ChIP) assay was performed. We performed in vitro experiments, including cell growth, invasion, sphere formation and immunocytochemistry; and in vivo ones, such as chemo-sensitivity and tumorigenesis. For specific knockdown of target genes, lentiviral mediated shRNAs and synthesize siRNA were used. To track biological behaviors of GI cancer cells, a lineage tracing technology was performed to visualize to the mode of tumor cell growth in vitro and in vivo. Result. CD44+/ALDH+ slowly proliferating immature GI cancer populations expressed relatively low levels of JARID1B and the differentiation marker CD20, with a relatively high level of the tumor suppressor p16/INK4A. Fluorescent ubiquitination-based cell cycle indicator transfections to GI cancer cells indicated that endogenous JARID1B increased in the late G1, showing a critical role in G1 to S transition checkpoint. The ChIP assay demonstrated clearly that JARID1B knockdown status leads to the methylation of p16/INK4A gene promoter. Importantly, continuous knockdown of JARID1B resulted in the loss of epithelial differentiation and suppressed cell growth, which associated with phosphorylation induction by Jnk/Sapk, and the irreversible induction of senescence (detected by SA β-galactosidase activity). Finally, green fluorescent-labeled cell tracking indicated that GI cancer cells, which reduced JARID1B expression, had lesser tumorigenicity than JARID1B-positive cells do in vivo. Conclusion. JARID1B plays a critical role in cell cycle regulation at G1/S transition of GI cancer cells. The continuous inhibition of JARID1B would contribute for tumor eradication. Taken together with melanoma study (Cell, 2011), the present study indicated that JARID1B would be a bona fide target for the eradication of chemotherapy-resistant, dormant GI cancer cells, by the induction of endogenous mechanism of cellular senescence. Learning Objective. We identified the novel function and transcriptional targets of epigenetics and verified the important biological behaviors of epigenetics in GI cancer. Citation Format: Katsuya Ohta, Yoshihiro Kano, Masamitsu Konno, Yoshinori Kagawa, Shimpei Nishikawa, Shinichiro Hasegawa, Takahito Fukusumi, Hisataka Ogawa, Atsushi Hamabe, Taroh Satoh, Naotsugu Haraguchi, Hirofumi Yamamoto, Yuichiro Doki, Masaki Mori, Hideshi Ishii. An impact of epigenetic approaches targeting JARID1B to modulate malignant behaviors of gastrointestinal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4625. doi:10.1158/1538-7445.AM2013-4625

Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC50 of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.

Depletion of JARID1B induces cellular senescence in human colorectal cancer

The global incidence of colorectal cancer (CRC) is increasing. Although there are emerging epigenetic factors that contribute to the occurrence, development and metastasis of CRC, the biological significance of epigenetic molecular regulation in different subpopulations such as cancer stem cells remains to be elucidated. In this study, we investigated the functional roles of the H3K4 demethylase, jumonji, AT rich interactive domain 1B (JARID1B), an epigenetic factor required for the continuous cell growth of melanomas, in CRC. We found that CD44(+)/aldehyde dehydrogenase (ALDH)(+) slowly proliferating immature CRC stem cell populations expressed relatively low levels of JARID1B and the differentiation marker, CD20, as well as relatively high levels of the tumor suppressor, p16/INK4A. Of note, lentiviral‑mediated continuous JARID1B depletion resulted in the loss of epithelial differentiation and suppressed CRC cell growth, which was associated with the induction of phosphorylation by the c‑Jun N‑terminal kinase (Jnk/Sapk) and senescence‑associated β‑galactosidase activity. Moreover, green fluorescent‑labeled cell tracking indicated that JARID1B‑positive CRC cells had greater tumorigenicity than JARID1B‑negative CRC cells after their subcutaneous inoculation into immunodeficient mice, although JARID1B‑negative CRC cells resumed normal growth after a month, suggesting that continuous JARID1B inhibition is necessary for tumor eradication. Thus, JARID1B plays a role in CRC maintenance. JARID1B may be a novel molecular target for therapy‑resistant cancer cells by the induction of cellular senescence.

Genome-wide association study of sensory disturbances in the inferior alveolar nerve after bilateral sagittal split ramus osteotomy.

BackgroundBilateral sagittal split ramus osteotomy (BSSRO) is a common orthognatic surgical procedure. Sensory disturbances in the inferior alveolar nerve, including hypoesthesia and dysesthesia, are frequently observed after BSSRO, even without distinct nerve injury. The mechanisms that underlie individual differences in the vulnerability to sensory disturbances have not yet been elucidated.MethodsThe present study investigated the relationships between genetic polymorphisms and the vulnerability to sensory disturbances after BSSRO in a genome-wide association study (GWAS). A total of 304 and 303 patients who underwent BSSRO were included in the analyses of hypoesthesia and dysesthesia, respectively. Hypoesthesia was evaluated using the tactile test 1 week after surgery. Dysesthesia was evaluated by interview 4 weeks after surgery. Whole-genome genotyping was conducted using Illumina BeadChips including approximately 300,000 polymorphism markers.ResultsHypoesthesia and dysesthesia occurred in 51 (16.8%) and 149 (49.2%) subjects, respectively. Significant associations were not observed between the clinical data (i.e., age, sex, body weight, body height, loss of blood volume, migration length of bone fragments, nerve exposure, duration of anesthesia, and duration of surgery) and the frequencies of hypoesthesia and dysesthesia. Significant associations were found between hypoesthesia and the rs502281 polymorphism (recessive model: combined χ2 = 24.72, nominal P = 6.633 × 10-7), between hypoesthesia and the rs2063640 polymorphism (recessive model: combined χ2 = 23.07, nominal P = 1.563 × 10-6), and between dysesthesia and the nonsynonymous rs2677879 polymorphism (trend model: combined χ2 = 16.56, nominal P = 4.722 × 10-5; dominant model: combined χ2 = 16.31, nominal P = 5.369 × 10-5). The rs502281 and rs2063640 polymorphisms were located in the flanking region of the ARID1B and ZPLD1 genes on chromosomes 6 and 3, whose official names are “AT rich interactive domain 1B (SWI1-like)” and “zona pellucida-like domain containing 1”, respectively. The rs2677879 polymorphism is located in the METTL4 gene on chromosome 18, whose official name is “methyltransferase like 4”.ConclusionsThe GWAS of sensory disturbances after BSSRO revealed associations between genetic polymorphisms located in the flanking region of the ARID1B and ZPLD1 genes and hypoesthesia and between a nonsynonymous genetic polymorphism in the METTL4 gene and dysesthesia.

JARID1B Protein Expression and Prognostic Implications in Uveal Melanoma

It has been suggested recently that stem cell marker expression of jumonji AT-rich interactive domain 1B (JARID1B) is required for continuous tumor growth and maintenance in human cutaneous melanoma cells. The aim of this study is to determine whether JARID1B is also expressed in uveal melanoma (UM) and whether JARID1B marks an expanded cancer stem cell pool in poor prognosis UM. Based on the available data, this is the first time JARID1B expression in UM has been studied. A total of 121 consecutive patients diagnosed with UM and who underwent enucleation were included in the study. Immunohistochemical staining with JARID1B antibodies was performed and immunoreactivity was assessed. Correlations of JARID1B expression with established clinicopathological parameters and overall survival (OS) were evaluated in univariate and multivariate analyses. JARID1B positive expression, as defined by >0% staining, was present in 55% of UMs and invariably in ciliary body epithelium. The correlation between JARID1B negative expression and JARID1B expression >5% inside the tumor tissue and OS was borderline statistically significant based on LogRank test at 5% significance level (P = 0.06). There were significantly more JARID1B positive cells in tumors with extrascleral extension than in tumors with no or minimal intrascleral invasion (P < 0.01, Mann-Whitney test). This study demonstrates that JARID1B is expressed by UM cells. Despite that JARID1B was highly expressed in UM, a statistically significant association (P < 0.05) between JARID1B expression and OS could not be obtained. However, a P-value of 0.06 could suggest that high JARID1B expression is correlated with lower survival; thus, a follow up study with a greater patient sample is recommended. In addition, samples of tumors characterized by high invasiveness showed a higher JARID1B expression. Furthermore, this study substantiates the presence of progenitor cells in the ciliary body epithelium.

Overexpression of Jumonji AT-rich interactive domain 1B and PHD finger protein 2 is involved in the progression of esophageal squamous cell carcinoma

Jumonji AT-rich interactive domain 1B (JARID1B) and PHD finger protein 2 (PHF2), members of the histone demethylases, have been found to be involved in many types of tumors. However, the expression and prognostic significance of JARID1B and PHF2 in esophageal squamous cell carcinoma (ESCC) still remains unclear. In this study, JARID1B and PHF2 expression were detected on tissue microarrays of ESCC samples in 120 cases using immunohistochemical staining. Our results showed that JARID1B and PHF2 were overexpressed in ESCCs. In addition, a significant correlation was observed between JARID1B nuclear expression level and histological grade (P=0.003). Kaplan–Meier survival analysis showed a tendency that high cytoplasmic expression of JARID1B and PHF2 was associated with decreased overall survival of ESCC patients, whereas JARID1B high expression in the nucleus was associated with high overall survival, although there was no statistical significance. Overall, our data suggest that JARID1B and PHF2 are overexpressed in ESCC and that they may play crucial roles in the course of ESCC initiation and/or progression.

MiR-125b Targets ARID3B in Breast Cancer Cells

Mounting evidence suggests involvement of deregulated microRNA (miRNA) expression during the complex events of tumorigenesis. Among such deregulated miRNAs in cancer, miR-125b expression is reported to be consistently low in breast cancers. In this study, we screened a panel of breast cancer cell lines (BCCLs) for miR-125b expression and detected decreased expression in 14 of 19 BCCLs. Due to the heterogeneity of breast cancers, MCF7 cells were chosen as a model system for ERBB2 independent breast cancers to restore miR-125b expression (MCF7-125b) to investigate the phenotypical and related functional changes. Earlier, miR-125b was shown to regulate cell motility by targeting ERBB2 in ERBB2 overexpressing breast cancer cells. Here we showed decreased motility and migration in miR-125b expressing MCF7 cells, independent of ERBB2. MCF7-125b cells demonstrated profoundly decreased cytoplasmic protrusions detected by phalloidin staining of filamentous actin along with decreased motility and migration behaviors detected by in vitro wound closure and transwell migration assays compared to empty vector transfected cells (MCF7-EV). Among possible numerous targets of miR-125b, we showed ARID3B (AT-rich interactive domain 3B) to be a novel target with roles in cell motility in breast cancer cells. When ARID3B was transiently silenced, the decreased cell migration was also observed. In light of these findings, miR-125b continues to emerge as an interesting regulator of cancer related phenotypes.

Structure-Function Study of Mammalian Munc18-1 and C. elegans UNC-18 Implicates Domain 3b in the Regulation of Exocytosis

Munc18-1 is an essential synaptic protein functioning during multiple stages of the exocytotic process including vesicle recruitment, docking and fusion. These functions require a number of distinct syntaxin-dependent interactions; however, Munc18-1 also regulates vesicle fusion via syntaxin-independent interactions with other exocytotic proteins. Although the structural regions of the Munc18-1 protein involved in closed-conformation syntaxin binding have been thoroughly examined, regions of the protein involved in other interactions are poorly characterised. To investigate this we performed a random transposon mutagenesis, identifying domain 3b of Munc18-1 as a functionally important region of the protein. Transposon insertion in an exposed loop within this domain specifically disrupted Mint1 binding despite leaving affinity for closed conformation syntaxin and binding to the SNARE complex unaffected. The insertion mutation significantly reduced total amounts of exocytosis as measured by carbon fiber amperometry in chromaffin cells. Introduction of the equivalent mutation in UNC-18 in Caenorhabditis elegans also reduced neurotransmitter release as assessed by aldicarb sensitivity. Correlation between the two experimental methods for recording changes in the number of exocytotic events was verified using a previously identified gain of function Munc18-1 mutation E466K (increased exocytosis in chromaffin cells and aldicarb hypersensitivity of C. elegans). These data implicate a novel role for an exposed loop in domain 3b of Munc18-1 in transducing regulation of vesicle fusion independent of closed-conformation syntaxin binding.

Identification of circulating placental mRNA in maternal blood of pregnancies affected with fetal congenital heart diseases at the second trimester of pregnancy: implications for early molecular screening

To investigate whether a significantly aberrant expression of circulating placental mRNA genes related with cardiogenesis can be detected at the second trimester of pregnancy. The study was performed in two stages. First stage (development model group): match of 14 placental tissues at delivery of fetuses with congenital heart disease versus 20 controls. Second stage (validation model group): mRNA amplification of abnormal expressed genes in maternal blood samples from 26 women bearing a fetus with a congenital heart disease matched with 28 controls. We identified four functional categories of genes possibly involved in abnormal heart development: cardiac morphogenesis: tenascin, thioredoxin, salvador homolog 1 protein; extracellular matrix (ECM) and valvular tissue biosynthesis; placental-associated plasma protein, collagen, type I, alpha 2, fibulin-1, heparanase, procollagen-proline, 2-oxoglutarate 4-dioxygenase, alpha polypeptide II, Jumonji, AT rich interactive domain 1B RBP2-like; normal contractile activity: actinin, alpha 4, fascin homolog 1, actin-bundling protein; and congestive heart failure. Altered placental genetic expression was found at term delivery in affected fetuses. The aberration was also confirmed in maternal blood at the second trimester of women bearing a fetus with congenital heart disease. Sensitivity for the most aberrant genes ranged between 42% and 95% at a false positive rate (FPR) of 10%.

The Epidermal Growth Factor Receptor Responsive miR-125a Represses Mesenchymal Morphology in Ovarian Cancer Cells

The epithelial-to-mesenchymal transition (EMT) that occurs during embryonic development is recapitulated during tumor metastasis. Important regulators of this process include growth factors, transcription factors, and adhesion molecules. New evidence suggests that microRNA (miRNA) activity contributes to metastatic progression and EMT; however, the mechanisms leading to altered miRNA expression during cancer progression remain poorly understood. Importantly, overexpression of the epidermal growth factor receptor (EGFR) in ovarian cancer correlates with poor disease outcome and induces EMT in ovarian cancer cells. We report that EGFR signaling leads to transcriptional repression of the miRNA miR-125a through the ETS family transcription factor PEA3. Overexpression of miR-125a induces conversion of highly invasive ovarian cancer cells from a mesenchymal to an epithelial morphology, suggesting miR-125a is a negative regulator of EMT. We identify AT-rich interactive domain 3B (ARID3B) as a target of miR-125a and demonstrate that ARID3B is overexpressed in human ovarian cancer. Repression of miR-125a through growth factor signaling represents a novel mechanism for regulating ovarian cancer invasive behavior.

Exploring Damage Recognition Models in Prokaryotic Nucleotide Excision Repair with a Benzo[a]pyrene-Derived Lesion in UvrB

The UvrB protein is a central unit for damage recognition in the prokaryotic nucleotide excision repair system, which excises bulky DNA lesions. We have utilized molecular modeling and MD simulations based on crystal structures, mutagenesis, and fluorescence data, to model the 10R-(+)-cis-anti-B[a]P-N2-dG lesion, derived from the tumorigenic (+)-anti-B[a]PDE metabolite of benzo[a]pyrene, at different locations on the inner and outer strand in UvrB. Our results suggest that this lesion is accommodated on the inner strand where it might translocate through the tunnel created by the beta-hairpin and UvrB domain 1B and ultimately could be housed in the pocket behind the beta-hairpin prior to excision by UvrC. Lesions that vary in size and shape may be stopped at the gate to the tunnel, within the tunnel, or in the pocket when UvrC initiates excision. Common features of beta-hairpin intrusion between the two DNA strands and nucleotide flipping manifested in structures of prokaryotic and eukaryotic NER lesion recognition proteins are consistent with common recognition mechanisms, based on lesion-induced local thermodynamic distortion/destabilization and nucleotide flipping.

Mechanistic Basis of 5′-3′ Translocation in SF1B Helicases

Superfamily 1B (SF1B) helicases translocate in a 5'-3' direction and are required for a range of cellular activities across all domains of life. However, structural analyses to date have focused on how SF1A helicases achieve 3'-5' movement along nucleic acids. We present crystal structures of the complex between the SF1B helicase RecD2 from Deinococcus radiodurans and ssDNA in the presence and absence of an ATP analog. These snapshots of the reaction pathway reveal a nucleotide binding-induced conformational change of the two motor domains that is broadly reminiscent of changes observed in other SF1 and SF2 helicases. Together with biochemical data, the structures point to a step size for translocation of one base per ATP hydrolyzed. Moreover, the structures also reveal a mechanism for nucleic acid translocation in the 5'-3' direction by SF1B helicases that is surprisingly different from that of 3'-5' translocation by SF1A enzymes, and explains the molecular basis of directionality.

The PcG protein hPc2 interacts with the N-terminus of histone demethylase JARID1B and acts as a transcriptional co-repressor

JARID1B (jumonji AT rich interactive domain 1B) is a large nuclear protein that is highly expressed in breast cancers and is proposed to function as a repressor of gene expression. In this paper, a phage display screen using the N-terminus of JARID1B as bait identified one of the JARID1B interacting proteins, namely PcG protein (Polycomb group) hPc2. We demonstrated that the C-terminal region, including the COOH box, was required for the interaction with the N-terminus of JARID1B. In a reporter assay system, co-expression of JARID1B with hPc2 significantly enhanced the transcriptional repression. These results support a role for hPc2 acting as a transcriptional co-repressor.

Myeloid Transforming Protein Evi1 Interacts with Methyl-CpG Binding Domain Protein 3 and Inhibits in Vitro Histone Deacetylation by Mbd3/Mi-2/NuRD

The ecotropic viral integration site 1 ( Evi1) gene encodes a putative transcription regulator, which is aberrantly expressed in acute myeloid leukemias (AML) with chromosomal abnormalities involving the 3q26 locus. Repression and activation of transcriptional control have been reported, but it is currently unclear how Evi1 may evoke these opposing effects. Using a yeast two-hybrid screen, we identified a novel binding partner of Evi1, i.e., methyl binding domain 3b (Mbd3b) protein, a member of the Mi-2/NuRD histone deacetylase complex. Applying in vitro and in vivo assays, we found that Evi1 interacts with Mbd3b but not with other MBD family members Mbd1, -2, and -4 or MeCP2. We show that interaction of Evi1 with Mbd3 requires 40 amino acids that are adjacent and downstream of the methyl binding domain (MBD). We further demonstrate that the first three zinc fingers of Evi1 are needed for Mbd3 interaction. Evi1 acts as a transcriptional repressor when recruited to an active promoter, yet when present in the Mi-2/NuRD complex through Mbd3 interaction, it inhibits the histone deacetylation function of this multiprotein structure. Our data may in part explain how Evi1 could act as a repressor as well as an activator of transcription.

Crystallographic and NMR Analyses of UvsW and UvsW.1 from Bacteriophage T4

The uvsWXY system is implicated in the replication and repair of the bacteriophage T4 genome. Whereas the roles of the recombinase (UvsX) and the recombination mediator protein (UvsY) are known, the precise role of UvsW is unclear. Sequence analysis identifies UvsW as a member of the monomeric SF2 helicase superfamily that translocates nucleic acid substrates via the action of two RecA-like motor domains. Functional homologies to Escherichia coli RecG and biochemical analyses have shown that UvsW interacts with branched nucleic acid substrates, suggesting roles in recombination and the rescue of stalled replication forks. A sequencing error at the 3'-end of the uvsW gene has revealed a second, short open reading frame that encodes a protein of unknown function called UvsW.1. We have determined the crystal structure of UvsW to 2.7A and the NMR solution structure of UvsW.1. UvsW has a four-domain architecture with structural homology to the eukaryotic SF2 helicase, Rad54. A model of the UvsW-ssDNA complex identifies structural elements and conserved residues that may interact with nucleic acid substrates. The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases.

UvrB Domain 4, an Autoinhibitory Gate for Regulation of DNA Binding and ATPase Activity

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.

Base Flipping in Nucleotide Excision Repair

UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A beta-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3' side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3' incision by UvrC.

Structural Basis for Transcription-coupled Repair: the N Terminus of Mfd Resembles UvrB with Degenerate ATPase Motifs

The transcription repair coupling factor Mfd removes stalled RNA polymerase from DNA lesions and links transcription to UvrABC-dependent nucleotide excision repair in prokaryotes. We report the 2.1A crystal structure of the UvrA-binding N terminus (residues 1-333) of Escherichia coli Mfd (Mfd-N). Remarkably, Mfd-N reveals a fold that resembles the three N-terminal domains of the repair enzyme UvrB. Domain 1A of Mfd adopts a typical RecA fold, domain 1B matches the damage-binding domain of the UvrB, and domain 2 highly resembles the implicated UvrA-binding domain of UvrB. However, Mfd apparently lacks a functional ATP-binding site and does not contain the DNA damage-binding motifs of UvrB. Thus, our results suggest that Mfd might form a UvrA recruitment factor at stalled transcription complexes that architecturally but not catalytically resembles UvrB.

Identification of Residues within UvrB That Are Important for Efficient DNA Binding and Damage Processing

The UvrB protein is the central recognition protein in bacterial nucleotide excision repair. We have shown previously that the highly conserved beta-hairpin motif in Bacillus caldotenax UvrB is essential for DNA binding, damage recognition, and UvrC-mediated incision, as deletion of the upper part of the beta-hairpin (residues 97-112) results in the inability of UvrB to be loaded onto damaged DNA, defective incision, and the lack of strand-destabilizing activity. In this work, we have further examined the role of the beta-hairpin motif of UvrB by a mutational analysis of 13 amino acids within or in the vicinity of the beta-hairpin. These amino acids are predicted to be important for the interaction of UvrB with both damaged and non-damaged DNA strands as well as the formation of salt bridges between the beta-hairpin and domain 1b of UvrB. The resulting mutants were characterized by standard functional assays such as oligonucleotide incision, electrophoretic mobility shift, strand-destabilizing, and ATPase assays. Our data indicated a direct role of Tyr96, Glu99, and Arg123 in damage-specific DNA binding. In addition, Tyr93 plays an important but less essential role in DNA binding by UvrB. Finally, the formation of salt bridges between the beta-hairpin and domain 1b, involving amino acids Lys111 bound to Glu307 and Glu99 bound to Arg367 or Arg289, are important but not essential for the function of UvrB.