Dissecting endonuclease and exonuclease activities in endonuclease V from Thermotoga maritima

Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 3′-side 1 nt from a deaminated base lesion. DNA cleavage analysis using mutants defective in DNA binding and Mn2+ as a metal cofactor reveals a novel 3′-exonuclease activity in endonuclease V [Feng,H., Dong,L., Klutz,A.M., Aghaebrahim,N. and Cao,W. (2005) Defining amino acid residues involved in DNA-protein interactions and revelation of 3′-exonuclease activity in endonuclease V. Biochemistry, 44, 11486–11495.]. This study defines the enzymatic nature of the endonuclease and exonuclease activity in endonuclease V from Thermotoga maritima. In addition to its well-known inosine-dependent endonuclease, Tma endonuclease V also exhibits inosine-dependent 3′-exonuclease activity. The dependence on an inosine site and the exonuclease nature of the 3′-exonuclease activity was demonstrated using 5′-labeled and internally-labeled inosine-containing DNA and a H214D mutant that is defective in non-specific nuclease activity. Detailed kinetic analysis using 3′-labeled DNA indicates that Tma endonuclease V also possesses non-specific 5′-exonuclease activity. The multiplicity of the endonuclease and exonuclease activity is discussed with respect to deaminated base repair

DNA DEAMINATION REPAIR ENZYMES IN BACTERIAL AND HUMAN SYSTEMS

DNA repair enzymes and pathways are diverse and critical for living cells to maintain correct genetic information. Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the uracil DNA glycosylase superfamily. We report that a bacterial SMUG1 ortholog in Geobacter metallireducens (Gme) and the human SMUG1 enzyme are not only uracil DNA glycosylases (UDG), but also xanthine DNA glycosylases (XDG). Mutations at M57 (M57L) and H210 (H210G, H210M, H210N) can cause substantial reductions in XDG and UDG activities. Increased selectivity is achieved in the A214R mutant of Gme SMUG1 and G60Y completely abolishes XDG and UDG activity. Most interestingly, a proline substitution at the G63 position switches the Gme SMUG1 enzyme to an exclusive uracil DNA glycosylase. Mutational analysis and molecular dynamics (MD) simulations of Gme SMUG1 identify important structural determinants in conserved motifs 1 and 2. Our study offers insights on the important role that modulation of conformational flexibility may play in defining specificity and catalytic efficiency. Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 3\u27 side one nucleotide from a deaminated base lesion. This study defines the endonuclease and exonuclease activity in endonuclease V from Thermotoga maritima (Tma) in an assay condition with Mn2+ as a metal cofactor. Tma endonuclease V exhibits inosine-dependent 3\u27-exonuclease activity. Detailed kinetic analysis using 3\u27-labeled DNA indicates that Tma endonuclease V also possesses nonspecific 5\u27-exonuclease activity. The multiplicity of the endonuclease and exonuclease activity is discussed with respect to deaminated base repair. Biochemical properties of human endonuclease V with respect to repair of deaminated base lesions were reported. We determined repair activities of human endonuclease V on inosine (I)-, xanthosine (X)-, oxanosine (O)- and uridine (U)-containing DNA. Human endonuclease V is most active with inosine-containing DNA; however, with minor activity on xanthosine-containing DNA. Mg2+ and to a much less extent, Mn2+, Ni2+, Co2+ can support the endonuclease activity. Introduction of human endonuclease V into Escherichia coli cells caused two-fold reduction in mutation frequency. This is the first report of deaminated base repair activity from human endonuclease V

BIOCHEMICAL STUDY OF ENDONUCLEASE V AND ITS APPLICATION IN MUTATION SCANNING

The integrity of the genetic information encoded by DNA is essential to all living organisms, yet the reactive bases of DNA are constantly attacked by endogenous and exogenous agents resulting in as many as one million individual molecular lesions per cell per day. Excessive DNA damage or deficiency in DNA repair enzymes may cause cancer, premature aging, and neurodegenerative diseases. Endonuclease V (Endo V) is a DNA repair enzyme which can recognize all four types of DNA deamination products, specifically, uracil, hypoxanthine, xanthine and oxanine. It was also shown that endo V can recognize mismatches. We screened about 60 mutants of endo V from Thermotoga maritima and found some mutants had altered base preferences for mismatches. Tma endo V Y80A was shown to become a C-specific mismatch endonuclease. G13D mutation in K-ras oncogene which was not recognized by wild type Tma endo V was successfully cleaved by Tma endo V Y80A. This study provides valuable information on base recognition and active site organization of Tma endo V. Tma endo V mutants can be used for cancer mutation scanning and mutation recognition. In order to further understand the role of Y80 of endo V in base recognition, we substituted the Y80 with sixteen amino acids. Together with three Y80 mutants isolated before, we characterized all nineteen mutants of Tma endo V Y80 using deaminated base-containing DNA substrates and mismatch-containing DNA substrates. This comprehensive amino acid substitution at a single site (Y80) underlines the importance of aromatic ring and hydrogen bond donor capacity in base recognition by endo V, reveals additional Y80 mutants with altered base preferences in mismatch cleavage, and offers new insight on the role of Y80 in base recognition. Though endo V was shown to be important for repair of deaminated lesions in vivo, its DNA repair pathway remains unknown. In order to understand the DNA repair pathway mediated by endo V, we have developed a cell-free system from Escherichia coli. The preliminary results indicated that the repair patch of endo V mediated DNA repair pathways may consist of a long patch and a short patch repair pathway

Specificity and Catalytic Mechanism of DNA Glycosylases in UDG Superfamily

DNA can be damaged by several kinds of endogenous and exogenous reactive nitrogen species. Under nitosative stress, uracil (U), hypoxanthine (I), xanthine (X) and oxanine (O) are four major deaminated DNA bases derived from cytosine (C), adenine (A) and guanine (G) respectively. To repair this type of DNA damage, several different repair pathways are involved. My dissertation work mainly focused on the uracil-DNA glycosylase (UDG) superfamily, which includes several groups of enzymes that recognize the damaged DNA bases and initiate the base excision repair (BER) pathway, one of the most important repair pathways to deal with deaminated DNA bases. Chapter 1 is a general introduction of different kinds of DNA damage and their corresponding repair pathways. Chapter 2 presents a detailed functional and structural analysis of family 5 UDGb from Thermus thermophilus HB8 in order to understand the specificity and catalytic mechanism of family 5 UDGb. Chapter 3 describes the biochemical properties and catalytic mechanism of family 4 UDGa from Thermus thermophilus HB8. A special double mutant has been identified with increased enzyme activity compared to single mutants. Chapter 4 is about a potential new group of enzymes within the UDG superfamily. Members from this new group of enzymes showed robust xanthine DNA glycosylase activities with unique catalytic mechanism and protein sequences. In summary, these functional and structural analyses provide new insights into substrate specificity and catalytic mechanism of UDG superfamily

Structural Characterization of the DNA Repair Protein Complex SbcC-SbcD of Thermotoga maritima

DNA damage poses a considerable threat to genomic integrity and cell survival. One of the most harmful forms of DNA damage are double-strand breaks that arise spontaneously during regular DNA processing like replication or meiosis. In addition, they can also be induced by a variety of DNA damaging agents like UV light, cell toxins or anti-cancer drugs. Failure of the rapid repair of these breaks can lead to chromosomal rearrangements and ultimately tumorigenesis in humans. In response to these genomic threats, a highly developed DNA repair network of protein factors has evolved, where the Mre11/Rad50/Nbs1 (MRN) complex is sought to play a key role in sensing, processing and repair of DNA double-strand breaks. Orthologs of Mre11 and Rad50, but not Nbs1, are found in all taxonomic kingdoms of life, suggesting that Mre11 and Rad50 form the core of this complex. In this work structural studies were performed to decipher the overall architecture and the interaction of SbcC and SbcD, the bacterial orthologs of Rad50 and Mre11. Using X-ray crystallographic and small angle X-ray scattering techniques the crystal as well as the in solution structures of the Thermotoga maritima SbcC ATPase domain in complex with full-length SbcD were solved. The crystal and in solution structure match well fortifying the calculated models that reveal an open, elongated complex with dimensions of approximately 210 Å * 75 Å * 65 Å. The heterotetrameric protein assembly consists of two SbcD molecules that homodimerize at domains I to form the central portion of the complex. Located at the outer areas of this homodimer domains II are arranged close to lobe II of SbcC building a small protein-protein interface. The C-terminal domains III of SbcD are connected to domains II via a flexible linker and associate through hydrophobic interactions with the coiled-coils of SbcC. These arrangements in combination with earlier findings lead to a model where upon ATP-binding the complex performs a conformational switch resulting in a ring-shaped structure. This conformation would bear a central cavity to harbor DNA strands that can be processed by the inwards oriented nuclease active sites of SbcD

Fragment- and structure-based drug discovery for developing therapeutic agents targeting the DNA Damage Response

Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven “undruggable”, Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with ∼50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or “undruggable” targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of ∼2–8% and crystal structures from ∼1.8 to 3.2 Å. We consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1)

Comprehensive classification of the PIN domain-like superfamily

PIN-like domains constitute a widespread superfamily of nucleases, diverse in terms of the reaction mechanism, substrate specificity, biological function and taxonomic distribution. Proteins with PIN-like domains are involved in central cellular processes, such as DNA replication and repair, mRNA degradation, transcription regulation and ncRNA maturation. In this work, we identify and classify the most complete set of PIN-like domains to provide the first comprehensive analysis of sequence–structure–function relationships within the whole PIN domain-like superfamily. Transitive sequence searches using highly sensitive methods for remote homology detection led to the identification of several new families, including representatives of Pfam (DUF1308, DUF4935) and CDD (COG2454), and 23 other families not classified in the public domain databases. Further sequence clustering revealed relationships between individual sequence clusters and showed heterogeneity within some families, suggesting a possible functional divergence. With five structural groups, 70 defined clusters, over 100,000 proteins, and broad biological functions, the PIN domain-like superfamily constitutes one of the largest and most diverse nuclease superfamilies. Detailed analyses of sequences and structures, domain architectures, and genomic contexts allowed us to predict biological function of several new families, including new toxin-antitoxin components, proteins involved in tRNA/rRNA maturation and transcription/translation regulation