The Topoisomerase II (topo II) DNA incision and ligation cycle can be poisoned (e.g following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5′-phosphotyrosyl (5′-Y) linked topo II-DNA adducts. Here, X-ray structures of mouse Tdp2-DNA complexes reveal that a Tdp2 β-2-helix-β DNA damage binding “grasp”, helical “cap”, and DNA lesion binding elements fuse to form an elongated protein-DNA conjugate substrate interaction groove. The Tdp2 DNA binding surface is highly tailored for engagement of 5′-adducted ssDNA ends, and restricts non-specific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analyses support a single-metal ion catalytic mechanism for the endonuclease–exonuclease–phosphatase (EEP) nuclease superfamily, and establish a molecular framework for targeted small molecule blockade of Tdp2-mediated resistance to anti-cancer topoisomerase drugs.

Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.

is an absolute requirement for the downstream activities of the major base excision repair enzymes, it may act as a regulator for the base excision repair pathway for efficient and balanced repair of damaged bases, which are often less toxic and/or mutagenic than their subsequent repair product intermediates.Cellular DNA is continuously exposed to endogenous or exogenous chemical or physical agents that induce DNA lesions. DNA base damage threatens genomic stability and cellular viability. Multiple DNA repair pathways exist in all organisms, from bacteria to humans, to preserve the integrity of the genome (1). If not repaired, damaged bases could be mutagenic (2) and/or cause cell death by blocking DNA replication (3).In all organisms, repair of DNA-containing small adducts, as well as altered and abnormal bases, occurs primarily via the base excision repair (BER) 2 pathway, beginning with cleavage of the base by a DNA glycosylase (1, 2). Mechanistically, DNA glycosylases are categorized into two classes: mono-and bifunctional DNA glycosylases. Monofunctional DNA glycosylases, such as N-methylpurine-DNA glycosylase (MPG) and uracil-DNA glycosylase, use an activated water molecule as a nucleophile to generate an apurinic or apyrimidinic (AP) site in DNA. Bifunctional DNA glycosylases/AP lyases, such as NTH1 and OGG1, use an activated amino group (Lys) or imino group (Pro) as the nucleophile to create a Schiff base intermediate that coordinates base removal and subsequent strand incision (AP lyase) 3Ј to the AP site (4, 5). The mammalian MPG is known to excise at least 17 structurally diverse modified bases from DNA (6). These lesions include 3-alkylpurines, 7-alkylguanine, 1,N 6 -ethenoadenine (⑀A), N 2 ,3-ethenoguanine, and hypoxanthine (Hx), all of which are purine derivatives (7-12). Moreover, the base alterations are located in both the major and minor grooves of duplex DNA. Its orthologs in Escherichia coli (AlkA) and yeast (MAG) have an overlapping although not identical substrate range. Nonetheless mammalian MPG and E. coli AlkA do not share significant sequence similarity or structural homology (13,14), despite this functional similarity and the fact that 3-methyladenine is a preferred substrate for both. MPG excises ⑀A and Hx more efficiently than AlkA and MAG (11), but unlike AlkA, it cannot excise O 2 -alkylpyrimidines (15, 16) and oxidized bases such as 5-formyluracil and 5-hydroxymethyluracil (17) (22); however, the reduction was more pronounced for the AP-lyase activity. The Schiff base formation between hOGG1-and 8-oxoG-containing DNA was abrogated in the presence of Mg 2ϩ . These results suggest that hOGG1 operates mainly as a monofunctional glycosylase under physiologic concentrations of Mg 2ϩ (22). There

Background: DNA glycosylase NEIL1 initiates prereplicative repair of oxidized DNA. Results: NEIL1 forms a multiprotein complex with DNA replication proteins via its C-terminal domain (CTD), allowing recruitment at the replication fork. Isolated CTD inhibits this interaction and repair in vitro. Conclusion:The interactions of NEIL1 are necessary for prereplicative repair. Significance: The NEIL1 CTD could serve as a target for adjuvant cancer therapy.

In both bacteria and eukaryotes the alkylated, oxidized, and deaminated bases and depurinated lesions are primarily repaired via an endogenous preventive pathway, i.e. base excision repair (BER). Radiation therapy and chemotherapy are two important modes of cancer treatment. Many of those therapeutic agents used in the clinic have the ability to induce the DNA damage; however, they may also be highly cytotoxic, causing peripheral toxicity and secondary cancer as adverse side effects. In addition, the damage produced by the therapeutic agents can often be repaired by the BER proteins, which in effect confers therapeutic resistance. Efficient inhibition of a particular BER protein(s) may increase the efficacy of current chemotherapeutic regimes, which minimizes resistance and ultimately decreases the possibility of the aforementioned negative side effects. Therefore, pharmacological inhibition of DNA damage repair pathways may be explored as a useful strategy to enhance chemosensitivity. Various agents have shown excellent results in preclinical studies in combination chemotherapy. Early phase clinical trials are now being carried out using DNA repair inhibitors targeting enzymes such as PARP, DNA-PK or MGMT. In the case of BER proteins, elimination of N-Methylpurine DNA glycosylase (MPG) or inhibition of AP-endonuclease (APE) increased sensitivity of cancer cells to alkylating chemotherapeutics. MPG(-/-) embryonic stem cells and cells having MPG knock-down by siRNA are hypersensitive to alkylating agents, whereas inhibition of APE by small molecule inhibitors sensitized cancer cells to alkylating chemotherapeutics. Thus, MPG and other BER proteins could be potential targets for chemosensitization.

N-Methylpurine DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. In this study we tested the role of N-terminal extension on MPG hypoxanthine (Hx) cleavage activity. Our results showed that MPG lacking N-terminal extension excises hypoxanthine with significantly reduced efficiency, one-third of that exhibited by full-length MPG under similar conditions. Steady-state kinetics showed full-length MPG has higher V max and lower K m than N⌬100 MPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that truncation can substantially increase the equilibrium binding constant of MPG toward Hx, but under single-turnover conditions there is apparently no effect on catalytic chemistry; however, the truncation of the N-terminal tail affected the turnover of the enzyme significantly under multiple turnover conditions. Real time binding experiments by surface plasmon resonance spectroscopy further showed that N⌬100 MPG binds approximately six times more tightly toward its product apurinic/apyrimidinic site than the substrate, whereas full-length MPG similarly binds to both the substrate and the product. We thereby conclude that the N-terminal tail in MPG plays a critical role in overcoming the product inhibition, which is achieved by reducing the differences of MPG binding affinity toward Hx and apurinic/apyrimidinic sites and thus is essential for the Hx cleavage reaction of MPG. The results from this study also affirm the need for reinvestigation of full-length MPG for its enzymatic and structural properties, which are currently available mostly for the truncated protein.Cellular DNA is continuously damaged by various endogenous or exogenous chemical or physical agents. Multiple DNA repair pathways repair damaged bases and prevent cell death and mutations responsible for genomic instability, cancer, and aging (1-3).In all organisms, repair of DNA-containing small adducts, as well as altered and abnormal bases, occurs primarily via the base excision repair pathway, beginning with cleavage of the base by a DNA glycosylase. Mammalian N-methylpurine DNA glycosylase (MPG), 2 a monofunctional glycosylase, is known to excise at least 17 structurally diverse modified purine bases, including toxic and mutagenic alkylated, deaminated, and etheno adducts from both the major and minor grooves of duplex DNA (4 -12).In our previous study, we showed that MPG is organized into three distinct domains with a protease-hypersensitive ϳ100-amino acid region at the N terminus (13). We also found that truncated (N⌬100C⌬18) and full-length enzymes retained similar binding and kinetic properties toward ⑀A (7). Later, several x-ray structures of human truncated MPG in complex with ⑀A or control DNA were published with the notion that the seemingly unstructured (protease-sensitive) N-terminal extension may hinder crystallization of MPG (14 -16). But different studies by us and others showed that the N-terminal e...

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5′-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3′- and 5′-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3′- and 5′-phosphotyrosyl linkage at the 3′- and 5′- ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5′-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetics studies showed that the 5′-activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC50 = 40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.

Protein purification is still very empirical, and a unified method to purify proteins without an affinity tag is not available yet. In the post-genomic era, functional genomics, however, strongly demands such a method. In this paper we have formulated a unique method that can be applied to purify any recombinant basic protein from E. coli. Here, we have found that if the pH of the buffer is merely one pH unit below the isoelectric point (pI) of the recombinant proteins, most of the latter bind to the column. This result supports the Henderson-Hasselbalch principle. Considering that E. coli proteins are mostly acidic, and based on the pI determined theoretically, apparently all recombinant basic proteins (at least pI-1≥6.94) may be purified from E.coli in a single-step using a cation-exchanger resin, SP-sepharose, and a selected buffer pH depending on the pI of the recombinant protein. Approximately, two-fifths of human proteome, including many if not all nucleic acid interacting proteins, have a pI of 7.94 or higher; virtually all these 12,000 proteins may be purified using this method in a single-step.