Cellular Responses to Widespread DNA Replication Stress.

Replicative DNA polymerases are blocked by nearly all types of DNA damage. The resulting DNA replication stress threatens genome stability. DNA replication stress is also caused by depletion of nucleotide pools, DNA polymerase inhibitors, and DNA sequences or structures that are difficult to replicate. Replication stress triggers complex cellular responses that include cell cycle arrest, replication fork collapse to one-ended DNA double-strand breaks, induction of DNA repair, and programmed cell death after excessive damage. Replication stress caused by specific structures (e.g., G-rich sequences that form G-quadruplexes) is localized but occurs during the S phase of every cell division. This review focuses on cellular responses to widespread stress such as that caused by random DNA damage, DNA polymerase inhibition/nucleotide pool depletion, and R-loops. Another form of global replication stress is seen in cancer cells and is termed oncogenic stress, reflecting dysregulated replication origin firing and/or replication fork progression. Replication stress responses are often dysregulated in cancer cells, and this too contributes to ongoing genome instability that can drive cancer progression. Nucleases play critical roles in replication stress responses, including MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, FEN1, and TATDN2. Several of these nucleases cleave branched DNA structures at stressed replication forks to promote repair and restart of these forks. We recently defined roles for EEPD1 in restarting stressed replication forks after oxidative DNA damage, and for TATDN2 in mitigating replication stress caused by R-loop accumulation in BRCA1-defective cells. We also discuss how insights into biological responses to genome-wide replication stress can inform novel cancer treatment strategies that exploit synthetic lethal relationships among replication stress response factors.

TATDN2 resolution of R-loops is required for survival of BRCA1-mutant cancer cells.

BRCA1-deficient cells have increased IRE1 RNase, which degrades multiple microRNAs. Reconstituting expression of one of these, miR-4638-5p, resulted in synthetic lethality in BRCA1-deficient cancer cells. We found that miR-4638-5p represses expression of TATDN2, a poorly characterized member of the TATD nuclease family. We discovered that human TATDN2 has RNA 3' exonuclease and endonuclease activity on double-stranded hairpin RNA structures. Given the cleavage of hairpin RNA by TATDN2, and that BRCA1-deficient cells have difficulty resolving R-loops, we tested whether TATDN2 could resolve R-loops. Using in vitro biochemical reconstitution assays, we found TATDN2 bound to R-loops and degraded the RNA strand but not DNA of multiple forms of R-loops in vitro in a Mg2+-dependent manner. Mutations in amino acids E593 and E705 predicted by Alphafold-2 to chelate an essential Mg2+ cation completely abrogated this R-loop resolution activity. Depleting TATDN2 increased cellular R-loops, DNA damage and chromosomal instability. Loss of TATDN2 resulted in poor replication fork progression in the presence of increased R-loops. Significantly, we found that TATDN2 is essential for survival of BRCA1-deficient cancer cells, but much less so for cognate BRCA1-repleted cancer cells. Thus, we propose that TATDN2 is a novel target for therapy of BRCA1-deficient cancers.

In Vitro Reconstitutive Base Excision Repair (BER) Assay.

The mammalian cell genome is continuously exposed to endogenous and exogenous insults that modify its DNA. These modifications can be single-base lesions, bulky DNA adducts, base dimers, base alkylation, cytosine deamination, nitrosation, or other types of base alteration which interfere with DNA replication. Mammalian cells have evolved with a robust defense mechanism to repair these base modifications (damages) to preserve genomic stability. Base excision repair (BER) is the major defense mechanism for cells to remove these oxidative or alkylated single-base modifications. The base excision repair process involves replacement of a single-nucleotide residue by two sub-pathways, the single-nucleotide (SN) and the multi-nucleotide or long-patch (LP) base excision repair pathways. These reactions have been reproduced in vitro using cell free extracts or purified recombinant proteins involved in the base excision repair pathway. In the present chapter, we describe the detailed methodology to reconstitute base excision repair assay systems. These reconstitutive BER assay systems use artificially synthesized and modified DNA. These reconstitutive assay system will be a true representation of biologically occurring damages and their repair.

EEPD1 promotes repair of oxidatively-stressed replication forks.

Unrepaired oxidatively-stressed replication forks can lead to chromosomal instability and neoplastic transformation or cell death. To meet these challenges cells have evolved a robust mechanism to repair oxidative genomic DNA damage through the base excision repair (BER) pathway, but less is known about repair of oxidative damage at replication forks. We found that depletion or genetic deletion of EEPD1 decreases clonogenic cell survival after oxidative DNA damage. We demonstrate that EEPD1 is recruited to replication forks stressed by oxidative damage induced by H2O2 and that EEPD1 promotes replication fork repair and restart and decreases chromosomal abnormalities after such damage. EEPD1 binds to abasic DNA structures and promotes resolution of genomic abasic sites after oxidative stress. We further observed that restoration of expression of EEPD1 via expression vector transfection restores cell survival and suppresses chromosomal abnormalities induced by oxidative stress in EEPD1-depleted cells. Consistent with this, we found that EEPD1 preserves replication fork integrity by preventing oxidatively-stressed unrepaired fork fusion, thereby decreasing chromosome instability and mitotic abnormalities. Our results indicate a novel role for EEPD1 in replication fork preservation and maintenance of chromosomal stability during oxidative stress.

Roles of homologous recombination in response to ionizing radiation-induced DNA damage.

PURPOSE: Ionizing radiation induces a vast array of DNA lesions including base damage, and single- and double-strand breaks (SSB, DSB). DSBs are among the most cytotoxic lesions, and mis-repair causes small- and large-scale genome alterations that can contribute to carcinogenesis. Indeed, ionizing radiation is a 'complete' carcinogen. DSBs arise immediately after irradiation, termed 'frank DSBs,' as well as several hours later in a replication-dependent manner, termed 'secondary' or 'replication-dependent DSBs. DSBs resulting from replication fork collapse are single-ended and thus pose a distinct problem from two-ended, frank DSBs. DSBs are repaired by error-prone nonhomologous end-joining (NHEJ), or generally error-free homologous recombination (HR), each with sub-pathways. Clarifying how these pathways operate in normal and tumor cells is critical to increasing tumor control and minimizing side effects during radiotherapy. CONCLUSIONS: The choice between NHEJ and HR is regulated during the cell cycle and by other factors. DSB repair pathways are major contributors to cell survival after ionizing radiation, including tumor-resistance to radiotherapy. Several nucleases are important for HR-mediated repair of replication-dependent DSBs and thus replication fork restart. These include three structure-specific nucleases, the 3' MUS81 nuclease, and two 5' nucleases, EEPD1 and Metnase, as well as three end-resection nucleases, MRE11, EXO1, and DNA2. The three structure-specific nucleases evolved at very different times, suggesting incremental acceleration of replication fork restart to limit toxic HR intermediates and genome instability as genomes increased in size during evolution, including the gain of large numbers of HR-prone repetitive elements. Ionizing radiation also induces delayed effects, observed days to weeks after exposure, including delayed cell death and delayed HR. In this review we highlight the roles of HR in cellular responses to ionizing radiation, and discuss the importance of HR as an exploitable target for cancer radiotherapy.

The splicing component ISY1 regulates APE1 in base excision repair.

The integrity of cellular genome is continuously challenged by endogenous and exogenous DNA damaging agents. If DNA damage is not removed in a timely fashion the replisome may stall at DNA lesions, causing fork collapse and genetic instability. Base excision DNA repair (BER) is the most important pathway for the removal of oxidized or mono-alkylated DNA. While the main components of the BER pathway are well defined, its regulatory mechanism is not yet understood. We report here that the splicing factor ISY1 enhances apurinic/apyrimidinic endonuclease 1 (APE1) activity, the multifunctional enzyme in BER, by promoting its 5'-3' endonuclease activity. ISY1 expression is induced by oxidative damage, which would provide an immediate up-regulation of APE1 activity in vivo and enhance BER of oxidized bases. We further found that APE1 and ISY1 interact, and ISY1 enhances the ability of APE1 to recognize abasic sites in DNA. Using purified recombinant proteins, we reconstituted BER and demonstrated that ISY1 markedly promoted APE1 activity in both the short- and long-patch BER pathways. Our study identified ISY1 as a regulator of the BER pathway, which would be of physiological relevance where suboptimal levels of APE1 are present. The interaction of ISY1 and APE1 also establishes a connection between DNA damage repair and pre-mRNA splicing.

MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers.

Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223-3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers.

ASR352, A potent anticancer agent: Synthesis, preliminary SAR, and biological activities against colorectal cancer bulk, 5-fluorouracil/oxaliplatin resistant and stem cells.

Despite new agent development and short-term benefits in patients with colorectal cancer (CRC), metastatic CRC cure rates have not improved due to high rates of 5-fluorouracil (5-FU)/leucovorin/oxaliplatin (FOLFOX)-resistance and a clinical therapeutic plateau. At the same time, this treatment regime leads to significant toxicity, cost, and patient inconvenience. Drug-resistance is linked to CRC stem cells, which are associated with the epidermal-to-mesenchymal transition (EMT) pathway. Thus, to optimally treat CRC, a therapy that can target the cell survival and EMT pathways in both CRC bulk and stem cell populations is critical. We recently identified a novel small molecule NSC30049 (7a) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC bulk, FOLFOX-resistant, and CRC stem cells both in vitro and in vivo models. In the present study, we report the synthesis and anti-CRC evaluation of several stable and effective 7a analogs. ASR352 (7b) was identified as one of the equipotent 7a analogs that inhibited the growth of CRC bulk cells, sensitized FOLFOX-resistant cells, and reduced the sphere formation capacity of CRC stem cells. It appears that the complex mechanism of cytotoxicity for 7b includes abrogation of 5-FU-induced the S phase, reduction of the phosphorylation of Chk1 at S317P, S345P and S296P, increased γH2AX staining, activation of caspase 3/PARP1 cleavage, and enhancement of Bax/Bcl2 ratio. Further 7b-mediated reduced phosphorylation of Chk1 was an indirect effect, since it did not inhibit Chk1 activity in an in vitro kinase assay. Our findings suggest that 7b as a single agent, or in combination with 5-FU can be developed as a therapeutic agent in CRC bulk, FOLFOX-resistant, and CRC stem cell populations for unmanageable metastatic CRC conditions.

NSC30049 inhibits Chk1 pathway in 5-FU-resistant CRC bulk and stem cell populations.

The 5-fluorouracil (5-FU) treatment induces DNA damage and stalling of DNA replication forks. These stalled replication forks then collapse to form one sided double-strand breaks, leading to apoptosis. However, colorectal cancer (CRC) stem cells rapidly repair the stalled/collapsed replication forks and overcome treatment effects. Recent evidence suggests a critical role of checkpoint kinase 1 (Chk1) in preventing the replicative stress. Therefore, Chk1 kinase has been a target for developing mono or combination therapeutic agents. In the present study, we have identified a novel orphan molecule NSC30049 (NSC49L) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC heterogeneous bulk and FOLFOX-resistant cell lines in culture with minimal effect on normal colonic epithelial cells. It also inhibits the sphere forming activity of CRC stem cells, and decreases the expression levels of mRNAs of CRC stem cell marker genes. Results showed that NSC49L induces 5-FU-mediated S-phase cell cycle arrest due to increased load of DNA damage and increased γ-H2AX staining as a mechanism of cytotoxicity. The pharmacokinetic analysis showed a higher bioavailability of this compound, however, with a short plasma half-life. The drug is highly tolerated by animals with no pathological aberrations. Furthermore, NSC49L showed very potent activity in a HDTX model of CRC stem cell tumors either alone or in combination with 5-FU. Thus, NSC49L as a single agent or combined with 5-FU can be developed as a therapeutic agent by targeting the Chk1 pathway in 5-FU-resistant CRC heterogeneous bulk and CRC stem cell populations.

Synthetic lethality in malignant pleural mesothelioma with PARP1 inhibition.

Malignant pleural mesotheliomas (MPM) are most often surgically unresectable, and they respond poorly to current chemotherapy and radiation therapy. Between 23 and 64% of malignant pleural mesothelioma have somatic inactivating mutations in the BAP1 gene. BAP1 is a homologous recombination (HR) DNA repair component found in the BRCA1/BARD1 complex. Similar to BRCA1/2 deficient cancers, mutation in the BAP1 gene leads to a deficient HR pathway and increases the reliance on other DNA repair pathways. We hypothesized that BAP1-mutant MPM would require PARP1 for survival, similar to the BRCA1/2 mutant breast and ovarian cancers. Therefore, we used the clinical PARP1 inhibitors niraparib and olaparib to assess whether they could induce synthetic lethality in MPM. Surprisingly, we found that all MPM cell lines examined, regardless of BAP1 status, were addicted to PARP1-mediated DNA repair for survival. We found that niraparib and olaparib exposure markedly decreased clonal survival in multiple MPM cell lines, with and without BAP1 mutations. This clonal cell death may be due to the extensive replication fork collapse and genomic instability that PARP1 inhibition induces in MPM cells. The requirement of MPM cells for PARP1 suggests that they may generally arise from defects in HR DNA repair. More importantly, these data demonstrate that the PARP1 inhibitors could be effective in the treatment of MPM, for which little effective therapy exists.

Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks.

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5' end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5'-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5' end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5'-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5' end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks.

Interaction between APC and Fen1 during breast carcinogenesis.

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER - adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) - promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-ß-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase ß and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase ß (Pol-ß) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-ß-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-ß and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-ß-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

Anti-tumor activity of novel biisoquinoline derivatives against breast cancers.

Breast cancer is classified into three groups according to its expression of hormone/growth factor receptors: (i) estrogen receptor (ER) and progesterone receptor (PR)-positive; (ii) human epidermal growth factor receptor 2 (HER2)-positive; and (iii) ER, PR, and HER2-negative (triple-negative). A series of methoxy-substituted biisoquinoline compounds have been synthesized as a potential chemotherapeutic agent for the triple-negative breast cancers which are especially challenging to manage. Structure activity relationship study revealed that rigid 6,6'-dimethoxy biisoquinoline imidazolium compound (1c, DH20931) exhibited the significant growth inhibitory effects on both triple-positive and triple-negative human breast cancer cell lines with IC50 in the range of 0.3-3.9 µM. The 1c (DH20931) is more potent than structurally related noscapine for growth inhibition of MCF7 cell line (IC50=1.3 vs 57 µM) and MDA-MB231 cell line (IC50=3.9 vs 64 µM).

Adenomatous polyposis coli-mediated accumulation of abasic DNA lesions lead to cigarette smoke condensate-induced neoplastic transformation of normal breast epithelial cells.

Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase ß and 5'-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model.

Adenomatous polyposis coli interacts with flap endonuclease 1 to block its nuclear entry and function.

In previous studies, we found that adenomatous polyposis coli (APC) blocks the base excision repair (BER) pathway by interacting with 5'-flap endonuclease 1 (Fen1). In this study, we identify the molecular features that contribute to the formation and/or stabilization of the APC/Fen1 complex that determines the extent of BER inhibition, and the subsequent accumulation of DNA damage creates mutagenic lesions leading to transformation susceptibility. We show here that APC binds to the nuclear localization sequence of Fen1 (Lys(365)Lys(366)Lys(367)), which prevents entry of Fen1 into the nucleus and participation in Pol-ß-directed long-patch BER. We also show that levels of the APC/Fen1 complex are higher in breast tumors than in the surrounding normal tissues. These studies demonstrate a novel role for APC in the suppression of Fen1 activity in the BER pathway and a new biomarker profile to be explored to identify individuals who may be susceptible to the development of mammary and other tumors.

DNA polymerase ß as a novel target for chemotherapeutic intervention of colorectal cancer.

Chemoprevention presents a major strategy for the medical management of colorectal cancer. Most drugs used for colorectal cancer therapy induce DNA-alkylation damage, which is primarily repaired by the base excision repair (BER) pathway. Thus, blockade of BER pathway is an attractive option to inhibit the spread of colorectal cancer. Using an in silico approach, we performed a structure-based screen by docking small-molecules onto DNA polymerase ß (Pol-ß) and identified a potent anti-Pol-ß compound, NSC-124854. Our goal was to examine whether NSC-124854 could enhance the therapeutic efficacy of DNA-alkylating agent, Temozolomide (TMZ), by blocking BER. First, we determined the specificity of NSC-124854 for Pol-ß by examining in vitro activities of APE1, Fen1, DNA ligase I, and Pol-ß-directed single nucleotide (SN)- and long-patch (LP)-BER. Second, we investigated the effect of NSC-124854 on the efficacy of TMZ to inhibit the growth of mismatch repair (MMR)-deficient and MMR-proficient colon cancer cell lines using in vitro clonogenic assays. Third, we explored the effect of NSC-124854 on TMZ-induced in vivo tumor growth inhibition of MMR-deficient and MMR-proficient colonic xenografts implanted in female homozygous SCID mice. Our data showed that NSC-124854 has high specificity to Pol-ß and blocked Pol-ß-directed SN- and LP-BER activities in in vitro reconstituted system. Furthermore, NSC-124854 effectively induced the sensitivity of TMZ to MMR-deficient and MMR-proficient colon cancer cells both in vitro cell culture and in vivo xenograft models. Our findings suggest a potential novel strategy for the development of highly specific structure-based inhibitor for the prevention of colonic tumor progression.

Assembly of the base excision repair complex on abasic DNA and role of adenomatous polyposis coli on its functional activity.

The assembly and stability of base excision repair (BER) proteins in vivo with abasic DNA and the role of adenomatous polyposis coli (APC) protein in this process are currently unclear. We have studied the assembly of a multiprotein BER complex onto abasic DNA (F-DNA) and characterized the physical and functional activity of the associated proteins. We found that the BER complex contained all the essential components of the long-patch BER system, such as APE1, Pol-ß, Fen1, and DNA ligase I. Interestingly, wild-type APC was also present in the BER complex. Kinetics of the assembly of BER proteins onto the F-DNA were rapid and appeared in sequential order depending upon their requirement in the repair process. The presence of wild-type APC in the BER complex caused a decrease in the level of assembly of BER proteins and negatively affected long-patch BER. These results suggest that major BER proteins in the complex are assembled onto F-DNA and are competent in performing DNA repair. Wild-type APC in the BER complex reduces the repair activity, probably because of interaction with multiple components of the system.

A novel inhibitor of DNA polymerase beta enhances the ability of temozolomide to impair the growth of colon cancer cells.

The recent emerging concept to sensitize cancer cells to DNA-alkylating drugs is by inhibiting various proteins in the base excision repair (BER) pathway. In the present study, we used structure-based molecular docking of DNA polymerase beta (Pol-beta) and identified a potent small molecular weight inhibitor, NSC-666715. We determined the specificity of this small molecular weight inhibitor for Pol-beta by using in vitro activities of APE1, Fen1, DNA ligase I, and Pol-beta-directed single-nucleotide and long-patch BER. The binding specificity of NSC-666715 with Pol-beta was also determined by using fluorescence anisotropy. The effect of NSC-666715 on the cytotoxicity of the DNA-alkylating drug temozolomide (TMZ) to colon cancer cells was determined by in vitro clonogenic and in vivo xenograft assays. The reduction in tumor growth was higher in the combination treatment relative to untreated or monotherapy treatment. NSC-666715 showed a high specificity for blocking Pol-beta activity. It blocked Pol-beta-directed single-nucleotide and long-patch BER without affecting the activity of APE1, Fen1, and DNA ligase I. Fluorescence anisotropy data suggested that NSC-666715 directly and specifically interacts with Pol-beta and interferes with binding to damaged DNA. NSC-666715 drastically induces the sensitivity of TMZ to colon cancer cells both in in vitro and in vivo assays. The results further suggest that the disruption of BER by NSC-666715 negates its contribution to drug resistance and bypasses other resistance factors, such as mismatch repair defects. Our findings provide the "proof-of-concept" for the development of highly specific and thus safer structure-based inhibitors for the prevention of tumor progression and/or treatment of colorectal cancer.

Amino acid Asp181 of 5'-flap endonuclease 1 is a useful target for chemotherapeutic development.

DNA alkylation-induced damage is one of the most efficacious anticancer therapeutic strategies. Enhanced DNA alkylation and weakened DNA repair capacity in cancer cells are responsible for the effectiveness of DNA-alkylating therapies. 5'-Flap endonuclease 1 (Fen1) is an important enzyme involved in base excision repair (BER), specifically in long-patch BER (LP-BER). Using the site-directed mutagenesis approach, we have identified an important role for amino acid Asp181 of Fen1 in its endonuclease activity. Asp181 is thought to be involved in Mg(2+) binding in the active site. Using structure-based molecular docking of Fen1 targeted to its metal binding pocket M2 (Mg(2+) site), we have identified a potent low-molecular weight inhibitor (LMI, NSC-281680) that efficiently blocks LP-BER. In this study, we have demonstrated that the interaction of this LMI with Fen1 blocked its endonuclease activity, thereby blocking LP-BER and enhancing the cytotoxic effect of DNA-alkylating agent Temozolomide (TMZ) in mismatch repair (MMR)-deficient and MMR-proficient colon cancer cells. The results further suggest that blockade of LP-BER by NSC-281680 may bypass other drug resistance mechanisms such as mismatch repair (MMR) defects. Therefore, our findings provide groundwork for the development of highly specific and safer structure-based small molecular inhibitors targeting the BER pathway, which can be used along with existing chemotherapeutic agents, like TMZ, as combination therapy for the treatment of colorectal cancer.