Cholesterol hemisuccinate: a selective inhibitor of family X DNA polymerases

Cholesterol hemisuccinate (compound 5), which consists of succinic acid esterified to the beta-hydroxyl group of cholesterol, selectively and strongly inhibited the activities of mammalian DNA polymerases (pols) such as pol beta, pol lambda, and terminal deoxynucleotidyltransferase (TdT), which are family X pols, in vitro, and the IC50 values were 2.9, 6.3, and 6.5 microM, respectively. The compound moderately suppressed the activities of other mammalian pols such as pol A (i.e., pol gamma), pol B (i.e., pols alpha, delta, and epsilon), and pol Y (i.e., pols iota, eta, and kappa) with 50% inhibition observed at concentrations of 131, 89.2-98.0, and 120-125 microM, respectively. The compound had no influence on the activities of plant pols alpha and beta, prokaryotic pols and other DNA metabolic enzymes tested. Since other cholesterol-related compounds such as cholesterol, cholesteryl chloride, cholesteryl bromide, cholesteryl acetate, and cholesteryl-5alpha, 6alpha-epoxide (compounds 1-4 and 6, respectively) did not influence the activities of any enzymes tested, the hemisuccinate group of compound 5 could be important for inhibition of the pol X family. Surface plasmon resonance analysis demonstrated that compound 5 bound selectively to the C-terminal 31 kDa domain of pol beta and pol lambda containing a pol beta-like region. On the basis of these results, the inhibitory mechanism of compound 5 on the pol X family was discussed.     

De novo DNA synthesis by human DNA polymerase lambda, DNA polymerase mu and terminal deoxyribonucleotidyl transferase

DNA polymerases (pols) catalyse the synthesis of DNA. This reaction requires a primer-template DNA in order to grow from the 3'OH end of the primer along the template. On the other hand terminal deoxyribonucleotidyl transferase (TdT) catalyses the addition of nucleotides at the 3'OH end of a DNA strand, without the need of a template. Pol lambda and pol micro are ubiquitous enzymes, possess both DNA polymerase and terminal deoxyribonucleotidyl transferase activities and belong to pol X family, together with pol beta and TdT. Here we show that pol lambda, pol micro and TdT, all possess the ability to synthesise in vitro short fragments of DNA in the absence of a primer-template or even a primer or a template in the reaction. The DNA synthesised de novo by pol lambda, pol micro and TdT appears to have an unusual structure. Furthermore we found that the amino acid Phe506 of pol lambda is essential for the de novo synthesis. This novel catalytic activity might be related to the proposed functions of these three pol X family members in DNA repair and DNA recombination.     

In situ detection of neuronal DNA strand breaks using the Klenow fragment of DNA polymerase I reveals different mechanisms of neuron death after global cerebral ischemia

Ischemic cell injury in the brain may involve a cascade of programmed cell death. DNA damage may be either a catalyst or a consequence of this cascade. Therefore, the induction of DNA strand breaks in the rat brain following transient global ischemia was examined using (a) the Klenow labeling assay, identifying DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) with protruding 5' termini, and (b) terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), detecting DNA DSBs with protruding 3' termini or blunt ends. Klenow-positive staining occurred within 2 h of reperfusion and increased with increasing durations of reperfusion. DNA damage detected with the Klenow labeling assay preceded that of TUNEL expression in the caudate putamen, reticular thalamus, thalamus, and cortex. However, in CA1, DNA SSBs were not detected until 72 h of reperfusion and occurred simultaneously with DSBs. Thus, the time course and fragmentation characteristics of DNA damage differ between the hippocampal CA1 and other selectively vulnerable brain regions. This distinct pattern suggests that the delayed neuronal death in CA1 following transient global ischemia may occur via an apoptotic mechanism different from that of other brain regions.     

Expansion of CAG triplet repeats by human DNA polymerases λ and β in vitro,is regulated by flap endonuclease 1 and DNA ligase 1

Huntington's disease (HD) is a neurological genetic disorder caused by the expansion of the CAG trinucleotide repeats (TNR) in the N-terminal region of coding sequence of the Huntingtin's (HTT) gene. This results in the addition of a poly-glutamine tract within the Huntingtin protein, resulting in its pathological form. The mechanism by which TRN expansion takes place is not yet fully understood. We have recently shown that DNA polymerase (Pol) β can promote the microhomology-mediated end joining and triplet expansion of a substrate mimicking a double strand break in the TNR region of the HTT gene. Here we show that TNR expansion is dependent on the structure of the DNA substrate, as well as on the two essential Pol β co-factors: flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). We found that Fen1 significantly stimulated TNR expansion by Pol β, but not by the related enzyme Pol λ, and subsequent ligation of the DNA products by Lig1. Interestingly, the deletion of N-terminal domains of Pol λ, resulted in an enzyme which displayed properties more similar to Pol β, suggesting a possible evolutionary mechanism. These results may suggest a novel mechanism for somatic TNR expansion in HD.     

The semi-quantitative comparison of oxidative stress mediated DNA single and double strand breaks using terminal deoxynucleotidyl transferase mediated end labeling combined with a slot blot technique

The accumulation of DNA damages by environmental stresses is represented by the steady state level of single strand breaks (SSBs) and double strand breaks (DSBs). Terminal deoxynucleotidyl transferase (TdT) mediated end labeling is suitable in detecting DSBs, but is unsuitable for SSBs due to its catalyzing characteristics. However, the sensitivity of TdT to detect SSBs may be significantly improved by first denaturing the double strands and expose all the DNA nicks as potential substrates for TdT. By coupling DNA denaturation to slot blot southern hybridization, the authors demonstrate the sensitive detection of SSBs as well as DSBs in 20 ng DNA samples derived from a retinal pigment epithelial cell line treated with tert-butyl hydroperoxide. The signal intensity of denatured and TdT-treated DNA in slot blot hybridization correlated to the amount of SSBs calculated in an S1 nuclease digestion assay. The signal ratio between denatured and non-denatured DNA likely approximates the SSBs/DSBs ratio in genomic DNA. The combination of DNA denaturing, TdT treatment and slot blot hybridization could be a useful method to assess oxidative stress-induced DNA strand damages.     

Design of reactive-end DNA oligomers via incorporation of oxanine into oligonucleotides using terminal deoxynucleotidyl transferase

Oxanine (Oxa, O), a modified nucleobase, has a novel O-acylisourea structure. Oxa-incorporated oligodeoxynucleotides (ODNs) are reactive DNA oligomers that permit conjugation with various nucleophilic molecules in an activation-free manner. In this study, we developed a new procedure for enzymatic preparation of reactive-end DNA oligomers, using terminal deoxynucleotidyl transferase (TdT), in which a reactive Oxa base is incorporated into the 3′-end of ODNs. One limitation of TdT, an enzyme widely used for end labeling of DNA oligomers, is that it is difficult to control the number of incorporated labels, because it shows template-independent extension with random nucleotides. Notably, TdT showed a rate and efficiency of incorporation of the modified nucleobase, Oxa, different from that of natural bases. We investigated the conditions of TdT-mediated DNA incorporation of Oxa and achieved incorporation of Oxa at the 3′-end of ODNs by optimizing reaction parameters such as temperature and enzyme, cofactor, and substrate concentrations. We also confirmed the reactive functionality of Oxa after incorporation into ODNs by amide bonding conjugation with a polyamine (spermine) under physiological conditions, without need for an additional activation step.     

Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

There exist two major base excision DNA repair (BER) pathways, namely single-nucleotide or “short-patch” (SP-BER), and “long-patch” BER (LP-BER). Both pathways appear to be involved in the repair of small base lesions such as uracil, abasic sites and oxidized bases. In addition to DNA polymerase β (Polβ) as the main BER enzyme for repair synthesis, there is evidence for a minor role for DNA polymerase lambda (Polλ) in BER. In this study we explore the potential contribution of Polλ to both SP- and LP-BER in cell-free extracts. We measured BER activity in extracts of mouse embryonic fibroblasts using substrates with either a single uracil or the chemically stable abasic site analog tetrahydrofuran residue. The addition of purified Polλ complemented the pronounced BER deficiency of POLB-null cell extracts as efficiently as did Polβ itself. We have developed a new approach for determining the relative contributions of SP- and LP-BER pathways, exploiting mass-labeled nucleotides to distinguish single- and multinucleotide repair patches. Using this method, we found that uracil repair in wild-type and in Polβ-deficient cell extracts supplemented with Polλ was ∼80% SP-BER. The results show that recombinant Polλ can contribute to both SP- and LP-BER. However, endogenous Polλ, which is present at a level ˜50% that of Polβ in mouse embryonic fibroblasts, appears to make little contribution to BER in extracts. Thus Polλ in cells appears to be under some constraint, perhaps sequestered in a complex with other proteins, or post-translationally modified in a way that limits its ability to participate effectively in BER.     

Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl transferase activity and can elongate RNA primers: implications for novel functions

DNA polymerase lambda is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5'-deoxyribose-5'-phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase beta suggested a novel beta-like enzyme. All these properties support a role of DNA polymerase lambda in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase lambda are still largely unknown. Here we give evidence that human DNA polymerase lambda has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3'OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase lambda efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase lambda might suggest additional roles for this enzyme in DNA replication and repair processes.     

Multiple functions of DNA polymerases

The primary role of DNA polymerases is to accurately and efficiently replicate the genome in order to ensure the maintenance of the genetic information and its faithful transmission through generations. This is not a simple task considering the size of the genome and its constant exposure to endogenous and environmental DNA damaging agents. Thus, a number of DNA repair pathways operate in cells to protect the integrity of the genome. In addition to their role in replication, DNA polymerases play a central role in most of these pathways. Given the multitude and the complexity of DNA transactions that depend on DNA polymerase activity, it is not surprising that cells in all organisms contain multiple highly specialized DNA polymerases, the majority of which have only recently been discovered. Five DNA polymerases are now recognized in Escherichia coli, 8 in Saccharomyces cerevisiae, and at least 15 in humans. While polymerases in bacteria, yeast and mammalian cells have been extensively studied much less is known about their counterparts in plants. For example, the plant model organism Arabidopsis thaliana is thought to contain 12 DNA polymerases, whose functions are mostly unknown. Here we review the properties and functions of DNA polymerases focusing on yeast and mammalian cells but paying special attention to the plant enzymes and the special circumstances of replication and repair in plant cells.     

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.     

DNA末端定量及其应用的研究

为定量DNA断裂末端评价DNA的降解程度,以饱和标记(TdT)法定量DNA末端最大标记量(L_max);并用流式细胞分析(FCA)、原位DNA末端标记(TUNEL)和琼脂糖电泳检测或标记DNA降解片段.结果发现: a.TdT法最低检测限5 ng DNA,线性范围为5～5 000 ng,较检测DNA降解片段的琼脂糖电泳敏感200倍以上;b.地塞米松(DEX)诱导淋巴瘤(Raji)细胞凋亡产生的L_max呈DEX剂量与诱导时间依赖性增加;c.自发性高血压大鼠(SHR)心肌的L_max呈年龄依赖性增加并与正常对照鼠(WKY)相比差异非常显著(P＜0.01);d.TdT法定量L_max与FCA、TUNEL或电泳分析的相关性良好(r＞0.98).TdT法定量L_max敏感、特异、准确,可应用于分子生物学、细胞生物学,尤其是细胞凋亡动力学DNA降解评价的定量研究.     

Roles of the four DNA polymerases of the crenarchaeon Sulfolobus solfataricus and accessory proteins in DNA replication

The hyperthermophilic crenarchaeon Sulfolobus solfataricus P2 encodes three B-family DNA polymerase genes, B1 (Dpo1), B2 (Dpo2), and B3 (Dpo3), and one Y-family DNA polymerase gene, Dpo4, which are related to eukaryotic counterparts. Both mRNAs and proteins of all four DNA polymerases were constitutively expressed in all growth phases. Dpo2 and Dpo3 possessed very low DNA polymerase and 3' to 5' exonuclease activities in vitro. Steady-state kinetic efficiencies (k(cat)/K(m)) for correct nucleotide insertion by Dpo2 and Dpo3 were several orders of magnitude less than Dpo1 and Dpo4. Both the accessory proteins proliferating cell nuclear antigen and the clamp loader replication factor C facilitated DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis by Dpo2 and Dpo3 was remarkably decreased by single-stranded binding protein, in contrast to Dpo1 and Dpo4. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein was most processive with Dpo1, whereas DNA lesion bypass was most effective with Dpo4. Both Dpo2 and Dpo3, but not Dpo1, bypassed hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypassed uracil and cis-syn cyclobutane thymine dimer, respectively. High concentrations of Dpo2 or Dpo3 did not attenuate DNA synthesis by Dpo1 or Dpo4. We conclude that Dpo2 and Dpo3 are much less functional and more thermolabile than Dpo1 and Dpo4 in vitro but have bypass activities across hypoxanthine, 8-oxoguanine, and either uracil or cis-syn cyclobutane thymine dimer, suggesting their catalytically limited roles in translesion DNA synthesis past deaminated, oxidized base lesions and/or UV-induced damage.     

Distribution and roles of X-family DNA polymerases in eukaryotes

Four types of DNA polymerase (Pol beta, Pol lambda, Pol mu and TdT) have been identified in eukaryotes as members of the polymerase X-family. Only vertebrates have all four types of enzyme. Plants and fungi have one or two X-family polymerases, while protostomes, such as fruit flies and nematodes, do not appear to have any. It is possible that the well-known metabolic pathways in which these enzymes are involved are restricted to the vertebrate world. The distribution of the DNA polymerases involved in DNA repair across the various biological kingdoms differs from that of the DNA polymerases involved in chromosomal DNA replication. In this review, we focus on the interesting pattern of distribution of the X-family enzymes across biological kingdoms and speculate on their roles.     

Inhibition of DNA polymerases and DNA topoisomerase II by triterpenes produced by plant callus

We found that some triterpene compounds could not only selectively inhibit the activities of mammalian DNA polymerase alpha (pol alpha) and beta (pol beta), but could also potently inhibit DNA topoisomerase II (topo II) [Biochem. J. 350 (2000) 757]. Here, we report that natural triterpenes produced by callus from an ancient Chinese medicinal plant were also inhibitors of the enzymes, and some were more selective than others. The natural triterpenes with a carboxyl group equally inhibited the activities of pol alpha, pol beta, and topo II, while the olide-type triterpenes with a ketone group suppressed the activities of pol beta and topo II, but not pol alpha. The other triterpenes from the callus hardly influenced these enzyme activities. As also described previously [J. Biochem. 130 (2001) 657], pol beta and topo II have a three-dimensionally similar triterpene-binding region, which is a pocket in which specific compounds can insert. The newly found triterpene inhibitors might structure-dependently insert into the pocket, and the pocket structure of each enzyme might, three-dimensionally but slightly, differ among them. The triterpene frames could be used for screening new inhibitors of the enzymes, and computer-simulated drug design using the frame and pocket structure may in theory be a possible approach to develop new inhibitors.     

DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases

DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'----5' exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively. Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication.     

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and β are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, β, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and β, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol β inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and β are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and β.     

Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors

The inhibitory activities against DNA polymerases (pols) of catechin derivatives (i.e., flavan-3-ols) such as (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, (+)-catechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-epigallocatechin gallate (EGCg) were investigated. Among the eight catechins, some catechins inhibited mammalian pols, with EGCg being the strongest inhibitor of pol alpha and lambda with IC(50) values of 5.1 and 3.8 microM, respectively. EGCg did not influence the activities of plant (cauliflower) pol alpha and beta or prokaryotic pols, and further had no effect on the activities of DNA metabolic enzymes such as calf terminal deoxynucleotidyl transferase, T7 RNA polymerase, and bovine deoxyribonuclease I. EGCg-induced inhibition of pol alpha and lambda was competitive with respect to the DNA template-primer and non-competitive with respect to the dNTP (2'-deoxyribonucleotide 5'-triphosphate) substrate. Tea catechins also suppressed TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation, and the tendency of the pol inhibitory activity was the same as that of anti-inflammation. EGCg at 250 microg was the strongest suppressor of inflammation (65.6% inhibition) among the compounds tested. The relationship between the structure of tea catechins and the inhibition of mammalian pols and inflammation was discussed.