Genetic defects in polymerase H (pol H; hRad30a gene) result in xeroderma pigmentosum variant syndrome (XP-V), and XP-V patients are sensitive to sunlight and highly prone to cancer development. Here, we show that pol H plays a significant role in modulating cellular sensitivity to DNA-targeting anticancer agents. When compared with normal human fibroblast cells, pol H -deficient cells derived from XP-V patients were 3-fold more sensitive to B-D-arabinofuranosylcytosine, gemcitabine, or cis-diamminedichloroplatinum (cisplatin) single-agent treatments and at least 10-fold more sensitive to the gemcitabine/cisplatin combination treatment, a commonly used clinical regimen for treating a wide spectrum of cancers. Cellular and biochemical analyses strongly suggested that the higher sensitivity of XP-V cells to these agents was due to the inability of pol H -deficient cells to help resume the DNA replication process paused by the gemcitabine/cisplatin-introduced DNA lesions. These results indicated that pol H can play an important role in determining the cellular sensitivity to therapeutic agents. The findings not only illuminate pol H as a potential pharmacologic target for developing new anticancer agents but also provide new directions for improving future chemotherapy regimen design considering the use of nucleoside analogues and cisplatin derivatives. (Mol Cancer Res 2006;4(4):257 -65)

Human DNA apurinic/apyrimidinic endonuclease (APE1) plays a key role in the DNA base excision repair process. In this study, we further characterized the exonuclease activity of APE1. The magnesium requirement and pH dependence of the exonuclease and endonuclease activities of APE1 are significantly different. APE1 showed a similar K m value for matched, 3 mispaired, or nucleoside analog ␤-L-dioxolane-cytidine terminated nicked DNA as well as for DNA containing a tetrahydrofuran, an abasic site analog. The k cat for exonuclease activity on matched, 3 mispaired, and ␤-L-dioxolane-cytidine nicked DNA are 2.3, 61.2, and 98.8 min ؊1 , respectively, and 787.5 min ؊1 for APE1 endonuclease. Site-directed APE1 mutant proteins (E96A, E96Q, D210E, D210N, and H309N), which target amino acid residues in the endonuclease active site, also showed significant decrease in exonuclease activity. DNA base excision repair is the main pathway to repair DNA base damages caused by oxidation, radiation, and the loss of bases (1, 2). There are several enzymes that participate in this pathway, including DNA polymerase ␤, DNA ligase III-XRCC1 complex (1, 2), and apurinic/apyrimidinic endonuclease (APE1) 1 to protect the genome integrity (1-3). The DNA repair activity of APE1 resides in the C-terminal region (4, 5). It endonucleolytically makes a nick immediately adjacent to 5Ј of an apurinic/apyrimidinic (AP) site and generates a hydroxyl group at the 3Ј terminus upstream of the nick and a 5Ј-deoxyribose phosphate moiety downstream (6). DNA polymerase ␤ further processes the product of APE1 by inserting a nucleotide into the gap and releasing the 5Ј-deoxyribose phosphate by its intrinsic lyase activity (7). The repair process is then completed by either DNA ligase I or DNA ligase III/XRCC1 to seal the nicked DNA (1). During DNA base excision repair, DNA polymerase ␤ is the major polymerase that incorporates a nucleotide into the gapped DNA (1). However, DNA polymerase ␤ is a lower fidelity polymerase (8) in comparison to the replicating DNA polymerases ␦ or ⑀ (9). ⌻he error rate of polymerase ␤ is about 1 in 4000 incorporations (1).APE1 is a versatile multifunctional protein (10). In addition to its endonuclease activity, it also possesses 3Ј-phosphatase, 3Ј-phosphodiesterase, RNase H, and 3Ј-5Ј-exonuclease activities (10). APE1 knockout mice die in the early embryonic stage (11), which indicates this protein is critical for development. Previously we reported (12) that the 3Ј-5Ј-exonuclease activity of APE1 is the major exonuclease activity in the human cell nucleus for the removal of the nucleoside analog ␤-L-dioxolanecytidine (L-OddC, BCH-4556, Troxacitabine, and Troxatyl) from the 3Ј termini of DNA. L-OddC is a novel nucleoside analog with L-configuration that is currently under phase III clinical trial and is showing promising activity for the treatment of leukemia (13,14). The incorporation of L-OddC into DNA terminates DNA chain elongation because of the lack of the 3Ј-hydroxyl group on the sugar moiety of L-OddC. The cytotoxicity ...

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␤-L-Dioxolane-cytidine (L-OddC, BCH-4556, Troxacitabine) is a novel unnatural stereochemical nucleoside analog that is under phase II clinical study for cancer treatment. This nucleoside analog could be phosphorylated and subsequently incorporated into the 3 terminus of DNA. The cytotoxicity of L-OddC was correlated with the amount of L-OddCMP in DNA, which depends on the incorporation by DNA polymerases and the removal by exonucleases. Here we reported the purification and identification of the major enzyme that could preferentially remove L-OddCMP compared with dCMP from the 3 termini of DNA in human cells. Surprisingly, this enzyme was found to be apurinic/apyrimidinic endonuclease (APE1) (1), a well characterized DNA base excision repair protein. APE1 preferred to remove L-over D-configuration nucleosides from 3 termini of DNA. The efficiency of removal of these deoxycytidine analogs were as follows:This report is the first demonstration that an exonuclease can preferentially excise L-configuration nucleoside analogs. This discovery suggests that APE1 could be critical for the activity of L-OddC or other L-nucleoside analogs and may play additional important roles in cells that were not previously known.Deoxyribonucleoside analogs are among the most effective agents for the treatment of cancer and viral diseases. Most of these compounds exert their function by inhibiting cellular or viral DNA synthesis. Since the naturally occurring nucleosides are in the ␤-D-configuration, most of the nucleoside analogs were designed in that configuration. The discovery of ␤-L-2Ј,3Ј-dideoxy-3Ј-thiocytidine (L-SddC, 3TC), 1 a stereochemically unnatural L-nucleoside analog (Fig. 1) inhibitor of HIV and hepatitis B virus replication (2-6), defined a new category for the design of nucleoside analogs. Among these, ␤-L-dioxolane-cytidine (L-OddC) (Fig. 1) is the first to show potential anticancer activity (2, 3), and clinical evaluation demonstrates its effectiveness against both leukemia and solid tumors (4 -7). For activation, most nucleoside analogs require conversion to the triphosphate metabolite by several cellular enzymes. Our previous studies have shown that L-OddC can be phosphorylated by deoxycytidine kinase to its monophosphate metabolite, which is further phosphorylated by cellular kinases to its di-and triphosphate metabolites (2, 3). The latter form of L-OddC can be incorporated into DNA by DNA polymerases ␣, ␤, ␦, ␥, and ⑀ in vitro (8). Since L-OddC lacks a hydroxyl group at the 3Ј-position, it causes premature termination of DNA replication once incorporated and eventually leads to cell death. This chain termination is probably the major mechanism of action of L-OddC. We have also shown previously that the cytotoxicity of this drug is directly related to the steady-state level of L-OddC in DNA (2).The steady-state level of L-OddC in DNA is not only dependent on incorporation by DNA polymerases but also on the excision by DNA repair enzymes. We have previously observed that incorporated L-OddCMP could be excised fr...

The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase (Pol ) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol . Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol could modulate the cytotoxic effect of AraC.Nucleoside analogs are an important class of compounds that are clinically used for anticancer and antiviral treatments (1). For example, 3TC, 3 AZT, and D4T have been used for antiviral treatments (1). AraC ( Fig. 1) is used to treat leukemia (2), and dFdC ( Fig. 1) is used to treat various types of cancer, such as non-small cell lung cancer (3), breast cancer (4), and pancreatic cancer (5). The main mechanism of action of nucleoside analogs is to terminate the DNA elongation process after their incorporation into viral or cellular DNA (6 -8). For example, due to the lack of a 3Ј-hydroxyl group on their sugar moieties, the incorporation of the antiviral compounds 3TC, AZT, and D4T terminates human immunodeficiency virus DNA replication immediately (9).Although both AraC and gemcitabine have a 3Ј-hydroxyl group on their sugar moieties, the incorporation of either compound has been shown to terminate the DNA elongation processes and result in DNA fragmentation (7,10). Studies have shown that the arabinose sugar moiety in AraC and the difluoro group on the 2Ј-position of the sugar moiety of dFdC alter the DNA structure, inhibiting DNA polymerases (11,12). The cytotoxicity of AraC or dFdC is proportional to the amount of the analog incorporated into DNA (6 -8). Therefore, the cellular enzymes that are involved in activating these nucleoside analogs and the DNA polymerases that introduce them into DNA play a critical role in determining their cytotoxic activity.Human DNA polymerase (Pol ) is an exonuclease-deficient, 575-amino acid DNA polymerase that belongs to the X-family (13). It contains a N-terminal BRCT domain that is important for protein partnerships, a proline/serine-rich region that has been suggested to be a target for post-translational modification, and a C-terminal 39-kDa polymerase domain (14). The 39-kDa domain, which is structurally well characterized (15, 16) contains the fingers, palm, and thumb subdomains typical of DNA polymerases (17). It also contains an 8-kDa subdomain with dRP lyase activity (14) that contributes to base excision repair (BER) in vitro. Studies in Pol ␤-deficient mouse embryonic fibroblast extracts indicate that Pol also performs BER in vivo (18,19). Moreover, in a confocal microscopy study, Pol reloca...