De novo designed aliphatic and aromatic peptides assemble into amyloid-like cytotoxic supramolecular nanofibrils.

Peptides are very interesting biomolecules that upon self-association form a variety of thermodynamically stable supramolecular structures of nanometric dimension e.g. nanotubes, nanorods, nanovesicles, nanofibrils, nanowires and many others. Herein, we report six peptide molecules having a general chemical structure, H-Gaba-X-X-OH (Gaba: γ-aminobutyric acid, X: amino acid). Out of these six peptides, three are aromatic and the others are aliphatic. Atomic force microscopic (AFM) studies reveal that except peptide 6 (H-Gaba-Trp-Trp-OH), all the reported peptides adopt nanofibrillar morphology upon aggregation in aqueous medium. These supramolecular assemblies can recognize amyloid-specific molecular probe congo red (CR) and thioflavine t (ThT) and exhibit all the characteristic properties of amyloids. The MTT cell viability assay reveals that the toxicity of both aliphatic and aromatic peptides increases with increasing concentration of the peptides to both cancer (HeLa) and non-cancer (HEK 293) cells. Of note, the aromatic peptides show a slightly higher cytotoxic effect compared to the aliphatic peptides. Overall, the studies highlight the self-assembling nature of the de novo designed aliphatic and aromatic peptides and pave the way towards elucidating the intricacies of pathogenic amyloid assemblies.

Macrophage activation highlight an important role for NER proteins in the survival, latency and multiplication of Mycobacterium tuberculosis.

Nucleotide excision repair (NER) is one of the most extensively studied DNA repair processes in both prokaryotes and eukaryotes. The NER pathway is a highly conserved, ATP-dependent multi-step process involving several proteins/enzymes that function in a concerted manner to recognize and excise a wide spectrum of helix-distorting DNA lesions and bulky adducts by nuclease cleavage on either side of the damaged bases. As such, the NER pathway of Mycobacterium tuberculosis (Mtb) is essential for its survival within the hostile environment of macrophages and disease progression. This review focuses on present published knowledge about the crucial roles of Mtb NER proteins in the survival and multiplication of the pathogen within the macrophages and as potential targets for drug discovery.

Genome-wide analysis reveals a regulatory role for G-quadruplexes during Adenovirus multiplication.

The G-quadruplex (GQ) motifs have recently been gaining prominence because of their role as gene cis-regulatory elements in a variety of organisms and as potential druggable targets for anti-cancer therapy and ageing. Several studies have demonstrated the existence of GQs in the genomes of emerging and re-emerging human pathogens, such as hepatitis virus, herpesviruses, Ebola virus, Zika virus and Nipah virus. Human Adenovirus (HAdV) exhibits a large number of clinical manifestations especially infecting the children and the immunocompromised patients. Moreover, the HAdV-based vectors have been widely used to deliver foreign DNAs to cells in gene therapy. However, the DNA secondary structural elements in AdV-based vectors could significantly determine the gene delivery efficacy of the vectors. In this study, using a combination of whole genome sequence analysis, biochemical, biophysical and interaction assays, we revealed fifteen putative GQs that are conserved across the different species of HAdV. We further showed that the GQs are embedded in the sequences of essential viral genes, namely E1B, E2B, and L3 genes (among others), which are involved in the early and late stages of the viral life cycle. Notably, Braco-19 (a well-known GQ binding ligand) interacted specifically with the HAdV GQs and increased their stability and further blocked the HAdV multiplication in human cells. Taken together, our data strongly supported the existence of G-quadruplex structures in the HAdV genome that affect the virus multiplication and posit that such structures may influence the efficacy of the gene-delivery vectors or even the HAdV virus life-cycle.

Identification and characterization of two conserved G-quadruplex forming motifs in the Nipah virus genome and their interaction with G-quadruplex specific ligands.

The G-quadruplex (GQ) motifs are considered as potential drug-target sites for several human pathogenic viruses such as Zika, Hepatitis, Ebola, and Human Herpesviruses. The recent outbreaks of Nipah virus (NiV) in India, the highly fatal emerging zoonotic virus is a potential threat to global health security as no anti-viral drug or vaccine in currently available. Therefore, here in the present study, we sought to assess the ability of the putative G-quadruplex forming sequences in the NiV genome to form G-quadruplex structures and act as targets for anti-viral compounds. Bioinformatics analysis underpinned by various biophysical and biochemical techniques (such as NMR, CD, EMSA, DMS footprinting assay) confirmed the presence of two highly conserved G-quadruplex forming sequences (HGQs) in the G and L genes of NiV. These genes encode the cell attachment glycoprotein and RNA-dependent RNA polymerase, respectively and are essential for the virus entry and replication within the host cell. It remains possible that stabilization of these HGQs by the known G-quadruplex binding ligands like TMPyP4 and Braco-19 represents a promising strategy to inhibit the expression of the HGQ harboring genes and thereby stop the viral entry and replication inside the host cell. Accordingly, we report for the first time, that HGQs in Nipah virus genome are targets for G-quadruplex specific ligands; therefore, could serve as potential targets for anti-viral therapy.

Deciphering the essentiality and function of SxSx motif in Mycobacterium tuberculosis UvrB.

The UvrB subunit is a central component of the UvrABC incision complex and plays a pivotal role in damage recognition, strand excision and repair synthesis. A conserved structural motif (the SxSx motif) present in UvrB is analogous to a similar motif (TxGx) in the helicases of superfamily 2, whose function is not fully understood. To elucidate the significance of the SxSx (Ser143-Val144-Ser145-Cys146) motif in Mycobacterium tuberculosis UvrB (MtUvrB), different variants of MtUvrB subunit were constructed and characterized. The SxSx motif indeed was found to be essential for MtUvrB function: while Ser143 and Cys146 residues within this motif were crucial for MtUvrB function, Ser145 plays an important but less essential role. The SxSx motif-deleted mutant was drastically attenuated and three single (S143A, S145A and C146A) mutants and a double (S143A/S145A) mutant exhibited various degrees of severity in their DNA-binding, DNA helicase and ATPase activities. Taken together, these results highlight a hitherto unrecognized role for SxSx motif in the catalytic activities of UvrB.

Targeting G-quadruplex DNA with synthetic dendritic peptide: modulation of the proliferation of human cancer cells.

Telomerase, a reverse transcriptase enzyme, is found to over express in most cancer cells. It elongates the telomere region by repeated adding of TTAGGG in the 3'-end and leads to excess cell proliferation which causes cancer. G-quadruplex (G4) formation can inhibit such telomere lengthening. So, stabilization of G4 structure as well as inhibition of telomerase activity is very promising approach in targeted cancer therapy. Herein, the aptitude of a synthetic dendritic peptide, C δ2-(YEE)-E (peptide 1), to target specifically the human telomeric G4 DNA, dAGGG(TTAGGG)3, has been evaluated. Both biochemical and biophysical techniques including gel mobility shift assay, isothermal titration calorimetry and fluorescence spectroscopy have been employed for the purpose. Circular dichroism study reveals that the targeting results an increase in thermal stability of G4 DNA. Interestingly, replacement of N-terminal tyrosine residue of peptide 1 by valine, C δ2-(VEE)-E, (peptide 2) consequences in loss of its G4 DNA targeting ability, although both the peptides exhibit comparable affinity toward double-stranded DNA. Of note, peptide 1 causes cessation of growth of human cancer cells (HeLa and U2OS) and induces apoptosis in vitro. But it has no significant inhibitory effect on the growth of normal human embryonic kidney 293 cells. Mechanistically, Telomeric Repeat Amplification Protocol (TRAP) assay indicates that peptide 1 effectively inhibits the telomerase activity in human cell extracts. Overall, this study demonstrates the usefulness of a synthetic dendritic peptide as an inhibitor of tumor cell growth by inducing apoptosis upon targeting the telomeric G4 DNA.

Elucidating the functional role of Mycobacterium smegmatis recX in stress response.

The RecX protein has attracted considerable interest because the recX mutants exhibit multiple phenotypes associated with RecA functions. To further our understanding of the functional relationship between recA and recX, the effect of different stress treatments on their expression profiles, cell yield and viability were investigated. A significant correlation was found between the expression of Mycobacterium smegmatis recA and recX genes at different stages of growth, and in response to different stress treatments albeit recX exhibiting lower transcript and protein abundance at the mid-log and stationary phases of the bacterial growth cycle. To ascertain their roles in vivo, a targeted deletion of the recX and recArecX was performed in M. smegmatis. The growth kinetics of these mutant strains and their sensitivity patterns to different stress treatments were assessed relative to the wild-type strain. The deletion of recA affected normal cell growth and survival, while recX deletion showed no significant effect. Interestingly, deletion of both recX and recA genes results in a phenotype that is intermediate between the phenotypes of the ΔrecA mutant and the wild-type strain. Collectively, these results reveal a previously unrecognized role for M. smegmatis recX and support the notion that it may regulate a subset of the yet unknown genes involved in normal cell growth and DNA-damage repair.

The Anionic Phospholipids in the Plasma Membrane Play an Important Role in Regulating the Biochemical Properties and Biological Functions of RecA Proteins.

Escherichia coli RecA (EcRecA) forms discrete foci that cluster at cell poles during normal growth, which are redistributed along the filamented cell axis upon induction of the SOS response. The plasma membrane is thought to act as a scaffold for EcRecA foci, thereby playing an important role in RecA-dependent homologous recombination. In addition, in vivo and in vitro studies demonstrate that EcRecA binds strongly to the anionic phospholipids. However, there have been almost no data on the association of mycobacterial RecA proteins with the plasma membrane and the effects of membrane components on their function. Here, we show that mycobacterial RecA proteins specifically interact with phosphatidylinositol and cardiolipin among other anionic phospholipids; however, they had no effect on the ability of RecA proteins to bind single-stranded DNA. Interestingly, phosphatidylinositol and cardiolipin impede the DNA-dependent ATPase activity of RecA proteins, although ATP binding is not affected. Furthermore, the ability of RecA proteins to promote DNA strand exchange is not affected by anionic phospholipids. Strikingly, anionic phospholipids suppress the RecA-stimulated autocatalytic cleavage of the LexA repressor. The Mycobacterium smegmatis RecA foci localize to the cell poles during normal growth, and these structures disassemble and reassemble into several foci along the cell after the induction of DNA damage. Taken together, these data support the notion that the interaction of RecA with cardiolipin and phosphatidylinositol, the major anionic phospholipids of the mycobacterial plasma membrane, may be physiologically relevant, as they provide a scaffold for RecA storage and may regulate recombinational DNA repair and the SOS response.

TRPV4 activation in rat carotid artery in DOCA hypertension involves eNOS and endothelium-derived contractile factor (EDCF).

Aim: Role of TRPV4 channel in regulation of endothelial function in the carotid artery in deoxycorticosterone acetate (DOCA) model of hypertension in rat was studied. Methods: 8-10 weeks old albino Wistar rats divided into three groups namely Control, UNX and hypertensive animals. Vascular smooth muscle response was studied in isolated carotid artery of rat with acetylcholine, sodium nitroprusside, GSK1016790A (GSK) in presence and absence of L-NAME and indomethacin. Results: At the end of the 6th week, the mean systolic blood pressure was increased in DOCA-treated hypertensive rats (166 ± 8 mm Hg) compared to Control and UNX (125 ± 5 mm Hg). ACh (10-9 to 10-5 M) produced almost 100% relaxation in Control (Emax = 97.48 ± 1.06 %) and UNX animals (Emax = 93.16 ± 2.33 %) which was attenuated in DOCA-treated hypertensive animals (Emax = 70.85 ± 1.65 %). No significant changes seen in SNP (10-12 to 10-5 M) induced relaxation. GSK1016790A (10-12 to 10-7 M)-mediated relaxation was significantly attenuated in DOCA-treated hypertensive animals (Emax = 25.58 ± 13.60%) compared to the control (Emax = 80.59 ± 6.86%) and UNX (Emax = 87.32 ± 2.01%) animals. L-NAME (10-4 M) potently blocked GSK-induced relaxation, and a contractile response to GSK was observed in presence of L-NAME in all the three groups of animals which was sensitive to indomethacin (10-5 M). Conclusion: TRPV4 may regulate the vascular tone of rat carotid artery through an attenuated NO pathway and stimulation of the release of contractile prostanoids in the DOCA hypertensive rats.

The second messenger cyclic di-AMP negatively regulates the expression of Mycobacterium smegmatis recA and attenuates DNA strand exchange through binding to the C-terminal motif of mycobacterial RecA proteins.

Cyclic di-GMP and cyclic di-AMP are second messengers produced by a wide variety of bacteria. They influence bacterial cell survival, biofilm formation, virulence and bacteria-host interactions. However, many of their cellular targets and biological effects are yet to be determined. A chemical proteomics approach revealed that Mycobacterium smegmatis RecA (MsRecA) possesses a high-affinity cyclic di-AMP binding activity. We further demonstrate that both cyclic di-AMP and cyclic di-GMP bind specifically to the C-terminal motif of MsRecA and Mycobacterium tuberculosis RecA (MtRecA). Escherichia coli RecA (EcRecA) was devoid of cyclic di-AMP binding but have cyclic di-GMP binding activity. Notably, cyclic di-AMP attenuates the DNA strand exchange promoted by MsRecA as well as MtRecA through the disassembly of RecA nucleoprotein filaments. However, the structure and DNA strand exchange activity of EcRecA nucleoprotein filaments remain largely unaffected. Furthermore, M. smegmatis ΔdisA cells were found to have undetectable RecA levels due to the translational repression of recA mRNA. Consequently, the ΔdisA mutant exhibited enhanced sensitivity to DNA-damaging agents. Altogether, this study points out the importance of sequence diversity among recA genes, the role(s) of cyclic di-AMP and reveals a new mode of negative regulation of recA gene expression, DNA repair and homologous recombination in mycobacteria.

Structural Characterization of i-Motif Structure in the Human Acetyl-CoA Carboxylase†1 Gene Promoters and Their Role in the Regulation of Gene Expression.

The polypurine/polypyrimidine-rich sequences within the promoters (PI and PII) of human acetyl coenzyme A (CoA) carboxylase 1 (ACC1) gene play a vital role in determining hormone- or diet-inducible expression of ACC1. PI and PII contain consecutive runs of three and three to five G/C base pairs, respectively. In a previous study, G-rich DNA sequences of human ACC1 PI and PII were found to fold into G-quadruplex structures; these consequently acted as strong barriers to transcription and DNA replication. Typically, stretches of C-rich sequences that coexist with stretches of guanines have the capacity to form another four-stranded secondary structure known as an i-motif. However, studies on the i-motif structure are limited and its functional significance is unclear. In the current study, through the use of a combination of different techniques, it is demonstrated that C-rich single-stranded DNA derived from ACC1 PI and PII form intramolecular i-motif structures and affect normal DNA metabolic processes. Additionally, the C-rich strands of PI and PII in duplex DNA adopt the i-motif conformation in crowded solution environments at neutral pH. Notably, the i-motif-forming sequences of PI and PII suppressed luciferase gene transcription in HeLa cells. Furthermore, substitution of a nucleotide sequence that has no potential to form the i-motif structure increases luciferase gene expression in HeLa cells. These results support the idea that C-rich sequences within ACC1 PI and PII can form intramolecular i-motif structures, cause suppression of transcription, and thus reveal the functional significance of C-rich sequences in the regulation of ACC1 gene expression.

Targeting G-quadruplex DNA structures in the telomere and oncogene promoter regions by benzimidazole‒carbazole ligands.

Recent studies support the idea that G-quadruplex structures in the promoter regions of oncogenes and telomere DNA can serve as potential therapeutic targets in the treatment of cancer. Accordingly, several different types of organic small molecules that stabilize G-quadruplex structures and inhibit telomerase activity have been discerned. Here, we describe the binding of benzimidazole-carbazole ligands to G-quadruplex structures formed in G-rich DNA sequences containing the promoter regions of human c-MYC, c-KIT1, c-KIT2, VEGF and BCL2 proto-oncogenes. The fluorescence spectroscopic data indicate that benzimidazole-carbazole ligands bind and stabilize the G-quadruplexes in the promoter region of oncogenes. The molecular docking studies provide insights into the mode and extent of binding of this class of ligands to the G-quadruplexes formed in oncogene promoters. The high stability of these G-quadruplex structures was validated by thermal denaturation and telomerase-catalyzed extension of the 3' end. Notably, benzimidazole-carbazole ligands suppress the expression of oncogenes in cancer cells in a dose-dependent manner. We anticipate that benzimidazole-carbazole ligands, by virtue of their ability to stabilize G-quadruplex structures in the promoter regions of oncogenes, might reduce the risk of cancer through the loss of function in the proteins encoded by these genes.

Novel ruthenium azo-quinoline complexes with enhanced photonuclease activity in human cancer cells.

Coordinatively saturated ruthenium complexes with a variable net charge are currently under intense investigation for their anticancer potential. These complexes, possessing long wavelength metal-to-ligand charge transfer with DNA photonuclease activity, have shown promising cytotoxic profiles. Although most of the ruthenium complexes exhibit significant photochemotherapeutic activity, their poor entry into cells hinder their development as potential drug molecules. Here, we report the synthesis and characterization of four new ruthenium (II) azo-8-hydroxyquinoline complexes, their mode of in vitro DNA binding and antiproliferative properties against cultured human cancer cell lines. The activity of these compounds prior to photoirradiation is minimal. However, they could induce DNA photonuclease activity through the generation of reactive oxygen species upon exposure to light. The activities exhibited by these complexes were found to be more efficient (>5-fold) than cisplatin, emphasizing their therapeutic potential. Collectively, these results support the idea that ruthenium (II) azo-8-hydroxyquinoline complexes can serve as potential agents in photodynamic anticancer therapy.

Defining the Functionally Important Domain and Amino Acid Residues in Mycobacterium tuberculosis Integration Host Factor for Genome Stability, DNA Binding, and Integrative Recombination.

The integration host factor of Mycobacterium tuberculosis (mIHF) consists of a single polypeptide chain, the product of the ihf gene. We previously revealed that mIHF is a novel member of a new class of nucleoid-associated proteins that have important roles in DNA damage response, nucleoid compaction, and integrative recombination. The mIHF contains a region of 86 amino acids at its N terminus, absent from both α- and ß-subunits of Escherichia coli IHF. However, the functional significance of an extra 86-amino-acid region in the full-length protein remains unknown. Here, we report the structure/function relationship of the DNA-binding and integrative recombination-stimulating activity of mIHF. Deletion mutagenesis showed that an extra 86-amino-acid region at the N terminus is dispensable; the C-terminal region possesses the sequences essential for its known biological functions, including the ability to suppress the sensitivity of E. coli ΔihfA and ΔihfB cells to DNA-damaging agents, DNA binding, DNA multimerization-circularization, and stimulation of phage L5 integrase-catalyzed integrative recombination. Single and double alanine substitutions at positions Arg170 and Arg171, located at the mIHF DNA-binding site, abrogated its capacity to suppress the sensitivity of E. coli ΔihfA and ΔihfB cells to DNA-damaging agents. The variants encoded by these mutant alleles failed to bind DNA and stimulate integrative recombination. Interestingly, the DNA-binding activity of the mIHF-R173A variant remained largely unaffected; however, it was unable to stimulate integrative recombination, thus revealing a separation-of-function allele of mIHF. The functional and structural characterization of this separation-of-function allele of mIHF could reveal previously unknown functions of IHF.IMPORTANCE The integration host factor of Mycobacterium tuberculosis is a novel nucleoid-associated protein. mIHF plays a vital role in DNA damage response, nucleoid compaction, and integrative recombination. Intriguingly, mIHF contains an extra 86-amino-acid region at its N terminus, absent from both α- and ß-subunits of Escherichia coli IHF, whose functional significance is unknown. Furthermore, a triad of arginine residues located at the mIHF-DNA interface have been implicated in a range of its functions. Here, we reveal the roles of N- and C-terminal regions of mIHF and the individual residues in the Arg triad for their ability to provide protection in vivo against DNA damage, bind DNA, and stimulate integrase-catalyzed site-specific recombination.

Saccharomyces cerevisiae Red1 protein exhibits nonhomologous DNA end-joining activity and potentiates Hop1-promoted pairing of double-stranded DNA.

Elucidation of the function of synaptonemal complex (SC) in Saccharomyces cerevisiae has mainly focused on in vivo analysis of recombination-defective meiotic mutants. Consequently, significant gaps remain in the mechanistic understanding of the activities of various SC proteins and the functional relationships among them. S. cerevisiae Hop1 and Red1 are essential structural components of the SC axial/lateral elements. Previous studies have demonstrated that Hop1 is a structure-selective DNA-binding protein exhibiting high affinity for the Holliday junction and promoting DNA bridging, condensation, and pairing between double-stranded DNA molecules. However, the exact mode of action of Red1 remains unclear, although it is known to interact with Hop1 and to suppress the spore viability defects of hop1 mutant alleles. Here, we report the purification and functional characterization of the full-length Red1 protein. Our results revealed that Red1 forms a stable complex with Hop1 in vitro and provided quantitative insights into their physical interactions. Mechanistically, Red1 preferentially associated with the Holliday junction and 3-way junction rather than with single- or double-stranded DNA with overhangs. Although Hop1 and Red1 exhibited similar binding affinities toward several DNA substrates, the two proteins displayed some significant differences. Notably, Red1, by itself, lacked DNA-pairing ability; however, it potentiated Hop1-promoted intermolecular pairing between double-stranded DNA molecules. Moreover, Red1 exhibited nonhomologous DNA end-joining activity, thus revealing an unexpected role for Red1 in recombination-based DNA repair. Collectively, this study presents the first direct insights into Red1's mode of action and into the mechanism underlying its role in chromosome synapsis and recombination.

Probing the Potential Role of Non-B DNA Structures at Yeast Meiosis-Specific DNA Double-Strand Breaks.

A plethora of evidence suggests that different types of DNA quadruplexes are widely present in the genome of all organisms. The existence of a growing number of proteins that selectively bind and/or process these structures underscores their biological relevance. Moreover, G-quadruplex DNA has been implicated in the alignment of four sister chromatids by forming parallel guanine quadruplexes during meiosis; however, the underlying mechanism is not well defined. Here we show that a G/C-rich motif associated with a meiosis-specific DNA double-strand break (DSB) in Saccharomyces cerevisiae folds into G-quadruplex, and the C-rich sequence complementary to the G-rich sequence forms an i-motif. The presence of G-quadruplex or i-motif structures upstream of the green fluorescent protein-coding sequence markedly reduces the levels of gfp mRNA expression in S. cerevisiae cells, with a concomitant decrease in green fluorescent protein abundance, and blocks primer extension by DNA polymerase, thereby demonstrating the functional significance of these structures. Surprisingly, although S. cerevisiae Hop1, a component of synaptonemal complex axial/lateral elements, exhibits strong affinity to G-quadruplex DNA, it displays a much weaker affinity for the i-motif structure. However, the Hop1 C-terminal but not the N-terminal domain possesses strong i-motif binding activity, implying that the C-terminal domain has a distinct substrate specificity. Additionally, we found that Hop1 promotes intermolecular pairing between G/C-rich DNA segments associated with a meiosis-specific DSB site. Our results support the idea that the G/C-rich motifs associated with meiosis-specific DSBs fold into intramolecular G-quadruplex and i-motif structures, both in vitro and in vivo, thus revealing an important link between non-B form DNA structures and Hop1 in meiotic chromosome synapsis and recombination.

Enhanced G-Quadruplex DNA Stabilization and Telomerase Inhibition by Novel Fluorescein Derived Salen and Salphen Based Ni(II) and Pd(II) Complexes.

Metal based salen complexes have been considered as an important scaffold toward targeting of DNA structures. In the present work, we have synthesized nickel(II) and palladium(II) salen and salphen complexes by using readily available fluorescein as the backbone to provide an extended aromatic surface. The metal complexes exhibit affinity toward the human telomeric G-quadruplex DNA with promising inhibition of telomerase activity. This has been ascertained by their efficiency in the long term cell proliferation assay which showed significant cancer cell toxicity in the presence of the metal complexes. Confocal microscopy showed cellular internalization followed by localization in the nucleus and mitochondria. Considerable population at the sub-G1 phase of the cell cycle showed cell death via apoptotic pathway.

Genetic and biochemical evidences reveal novel insights into the mechanism underlying Saccharomyces cerevisiae Sae2-mediated abrogation of DNA replication stress.

In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in double-strand break (DSB) repair, replication stress and telomere length maintenance. Another protein linked to DSB repair is Sae2, which regulates MRX persistence at DSBs. However, very little is known about its role in DNA replication stress and repair. Here, we reveal a crucial role for Sae2 in DNA replication stress. We show that different mutant alleles of SAE2 cause hypersensitivity to genotoxic agents, and when combined with Δmre11 or nuclease-defective mre11 mutant alleles, the double mutants are considerably more sensitive suggesting that the sae2 mutations synergize with mre11 mutations. Biochemical studies demonstrate that Sae2 exists as a dimer in solution, associates preferentially with single-stranded and branched DNA structures, exhibits structure-specific endonuclease activity and cleaves these substrates from the 5' end. Furthermore, we show that the nuclease activity is indeed intrinsic to Sae2. Interestingly, sae2G270D protein possesses DNA-binding activity, but lacks detectable nuclease activity. Altogether, our data suggest a direct role for Sae2 nuclease activity in processing of the DNA structures that arise during replication and DNA damage and provide insights into the mechanism underlying Mre11-Sae2-mediated abrogation of replication stressrelated defects in S. cerevisiae.

Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity.

Much is known about the Escherichia coli nucleotide excision repair (NER) pathway; however, very little is understood about the proteins involved and the molecular mechanism of NER in mycobacteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs, and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single- and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to 5' direction and is strongly modulated by a nontranslocating 5' single-stranded tail, indicating that in addition to the translocating strand it also interacts with the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length of the duplex increases: it fails to unwind duplexes longer than 70 bp. These results, on one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in M. tuberculosis.