Showing papers on "DNA clamp published in 1997"
TL;DR: Crystal structures suggest that pol beta may enhance fidelity by an induced fit mechanism in which correct base pairing between template and incoming dNTP induces alignment of catalytic groups for catalysis (via thumb closure), but incorrect base pairing will not.
Abstract: DNA polymerase β (pol β) fills single nucleotide (nt) gaps in DNA produced by the base excision repair pathway of mammalian cells. Crystal structures have been determined representing intermediates in the 1 nt gap-filling reaction of pol β: the binary complex with a gapped DNA substrate (2.4 A resolution), the ternary complex including ddCTP (2.2 A), and the binary product complex containing only nicked DNA (2.6 A). Upon binding ddCTP to the binary gap complex, the thumb subdomain rotates into the closed conformation to contact the otherwise solvent-exposed ddCTP-template base pair. Thumb movement triggers further conformational changes which poise catalytic residue Asp192, dNTP, and template for nucleotidyl transfer, effectively assembling the active site. In the product nicked DNA complex, the thumb returns to the open conformation as in the gapped binary DNA complex, facilitating dissociation of the product. These findings suggest that pol β may enhance fidelity by an induced fit mechanism in which co...
595 citations
TL;DR: Rad52 functions as a co-factor for the Rad51 recombinase, acting specifically to overcome the apparent competition by RPA for binding to single-stranded DNA.
542 citations
TL;DR: The crystal structures of two enzymes in the F EN‐1 nuclease family have been solved and they provide a structural basis for the interesting steric requirements of FEN‐1 substrates.
Abstract: Unlike the most well-characterized prokaryotic polymerase, E. coli DNA pol l, none of the eukaryotic polymerases have their own 5' to 3' exonuclease domain for nick translation and Okazaki fragment processing. In eukaryotes, FEN-1 is an endo- and exonuclease that carries out this function independently of the polymerase molecules. Only seven nucleases have been cloned from multicellular eukaryotic cells. Among these, FEN-1 is intriguing because it has complex structural preferences; specifically, it cleaves at branched DNA structures. The cloning of FEN-1 permitted establishment of the first eukaryotic nuclease family, predicting that S. cerevisiae RAD2 (S. pombe Rad13) and its mammalian homolog, XPG, would have similar structural specificity. The FEN-1 nuclease family includes several similar enzymes encoded by bacteriophages. The crystal structures of two enzymes in the FEN-1 nuclease family have been solved and they provide a structural basis for the interesting steric requirements of FEN-1 substrates. Because of their unique structural specificities, FEN-1 and its family members have important roles in DNA replication, repair and, potentially, recombination. Recently, FEN-1 was found to specifically associate with PCNA, explaining some aspects of FEN-1 function during DNA replication and potentially in DNA repair.
460 citations
TL;DR: An extended structure-based alignment of eukaryotic DNA polymerase sequences provides structural insights that should be applicable to most eukarian DNA polymerases.
426 citations
TL;DR: It is found that DNA‐PK can itself bind to linear DNA fragments ranging in size from 18 to 841 bp double‐stranded (ds) DNA, as indicated by mobility shifts; crosslinking between the DNA and DNA‐ PK; and atomic‐force microscopy.
Abstract: DNA-dependent protein kinase (DNA-PK or the scid factor) and Ku are critical for DNA end-joining in V(D)J recombination and in general non-homologous double-strand break repair. One model for the function of DNA-PK is that it forms a complex with Ku70/86, and this complex then binds to DNA ends, with Ku serving as the DNA-binding subunit. We find that DNA-PK can itself bind to linear DNA fragments ranging in size from 18 to 841 bp double-stranded (ds) DNA, as indicated by: (i) mobility shifts; (ii) crosslinking between the DNA and DNA-PK; and (iii) atomic-force microscopy. Binding of the 18 bp ds DNA to DNA-PK activates it for phosphorylation of protein targets, and this level of activation is not increased by addition of purified Ku70/86. Ku can stimulate DNA-PK activity beyond this level only when the DNA fragments are long enough for the independent binding to the DNA of both DNA-PK and Ku. Atomic-force microscopy indicates that under such conditions, the DNA-PK binds at the DNA termini, and Ku70/86 assumes a position along the ds DNA that is adjacent to the DNA-PK.
326 citations
TL;DR: The Rad6-Rad18 complex provides the first example wherein a ubiquitin conjugating activity is physically associated with DNA binding and ATPase activities provided by an associated protein factor.
308 citations
TL;DR: The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized, and it exhibited an extension rate 5 times higher and a processivity 10 to 15 times higher than those of PfuDNA polymerase.
Abstract: The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.
296 citations
TL;DR: The technique of splicing by overlap extension by the polymerase chain reaction (SOE by PCR) is presented as an effective mechanism to splice DNA.
283 citations
TL;DR: It is concluded that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands and Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates.
Abstract: The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70/86 heterodimer and an ≈460-kDa catalytic subunit (DNA-PKcs) DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and protein–nucleic acid complexes without staining or metal coating The AFM provides a rapid and direct means of probing the protein–nucleic acid interactions responsible for DNA repair and genetic regulation Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA A significant number of DNA molecules formed loops in the presence of Ku DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates We conclude that, when bound to DNA, Ku is capable of self-association These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands
281 citations
TL;DR: The studied non-homologous end joining of linearized plasmid DNA with different termini configurations following transformation into tobacco cells suggests that double strand break repair in plants involves extensive end degradation, DNA synthesis following invasion of ectopic templates and multiple template switches.
Abstract: Double strand DNA breaks in plants are primarily repaired via non-homologous end joining. However, little is known about the molecular events underlying this process. We have studied non-homologous end joining of linearized plasmid DNA with different termini configurations following transformation into tobacco cells. A variety of sequences were found at novel end junctions. Joining with no sequence alterations was rare. In most cases, deletions were found at both ends, and rejoining usually occurred at short repeats. A distinct feature of plant junctions was the presence of relatively large, up to 1.2 kb long, insertions (filler DNA), in approximately 30% of the analyzed clones. The filler DNA originated either from internal regions of the plasmid or from tobacco genomic DNA. Some insertions had a complex structure consisting of several reshuffled plasmid-related regions. These data suggest that double strand break repair in plants involves extensive end degradation, DNA synthesis following invasion of ectopic templates and multiple template switches. Such a mechanism is reminiscent of the synthesis-dependent recombination in bacteriophage T4. It can also explain the frequent 'DNA scrambling' associated with illegitimate recombination in plants.
270 citations
TL;DR: The identification and characterization of a novel gene, LIG4, which encodes a protein with strong homology to mammalian DNA ligase IV, indicating diversification of function between different eukaryotic DNA ligases and suggesting that the NHEJ pathway is highly conserved throughout the eukARYotic kingdom.
Abstract: DNA ligases catalyse the joining of single and double‐strand DNA breaks, which is an essential final step in DNA replication, recombination and repair. Mammalian cells have four DNA ligases, termed ligases I–IV. In contrast, other than a DNA ligase I homologue (encoded by CDC9 ), no other DNA ligases have hitherto been identified in Saccharomyces cerevisiae . Here, we report the identification and characterization of a novel gene, LIG4 , which encodes a protein with strong homology to mammalian DNA ligase IV. Unlike CDC9 , LIG4 is not essential for DNA replication, RAD52 ‐dependent homologous recombination nor the repair of UV light‐induced DNA damage. Instead, it encodes a crucial component of the non‐homologous end‐joining (NHEJ) apparatus, which repairs DNA double‐strand breaks that are generated by ionizing radiation or restriction enzyme digestion: a function which cannot be complemented by CDC9 . Lig4p acts in the same DNA repair pathway as the DNA end‐binding protein Ku. However, unlike Ku, it does not function in telomere length homeostasis. These findings indicate diversification of function between different eukaryotic DNA ligases. Furthermore, they provide insights into mechanisms of DNA repair and suggest that the NHEJ pathway is highly conserved throughout the eukaryotic kingdom.
TL;DR: The dynamic movement of proliferating cell nuclear antigen on and off the DNA renders this protein an ideal communicator for a variety of proteins that are essential for DNA metabolic events in eukaryotic cells.
Abstract: DNA metabolic events such as replication, repair and recombination require the concerted action of several enzymes and cofactors. Nature has provided a set of proteins that support DNA polymerases in performing processive, accurate and rapid DNA synthesis. Two of them, the proliferating cell nuclear antigen and its adapter protein replication factor C, cooperate to form a moving platform that was initially thought of only as an anchor point for DNA polymerases delta and epsilon. It now appears that proliferating cell nuclear antigen is also a communication point between a variety of important cellular processes including cell cycle control, DNA replication, nucleotide excision repair, post-replication mismatch repair, base excision repair and at least one apoptotic pathway. The dynamic movement of proliferating cell nuclear antigen on and off the DNA renders this protein an ideal communicator for a variety of proteins that are essential for DNA metabolic events in eukaryotic cells.
TL;DR: The crystal structure of the δ′ subunit of the clamp-loader complex of E. coli DNA polymerase III has been determined and a sequence-structure alignment suggests that nucleotides bind to γ at an interdomain interface within the inner surface of the "C."
TL;DR: It is proposed that after Okazaki fragment DNA synthesis is completed by a PCNA-DNA pol delta complex, DNA pol delta is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.
Abstract: Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol δ and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol δ. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol δ does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol δ, disrupts the DNA ligase I–PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA–DNA pol δ complex, DNA pol δ is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.
TL;DR: The formation of DNA/dendrimer complexes is found to be based entirely on charge interaction, which elucidate some aspects of the interaction between PAMAM dendritic polymers and DNA, and could lead to improvements in the design of polymers or formation ofDNA complexes that will increase the efficiency of non-viral gene transfer.
TL;DR: This work has shown that removal of a 3' nonhomologous tail in Saccharomyces cerevisiae depends on the nucleotide excision repair endonuclease Rad1/Rad10, and also on the mismatch repair proteins Msh2 and Msh3, and suggests that Srs2 acts to extend and stabilize the initial nascent joint between the invading single strand and its homolog.
Abstract: During repair of a double-strand break (DSB) by gene conversion, one or both 3' ends of the DSB invade a homologous donor sequence and initiate new DNA synthesis. The use of the invading DNA strand as a primer for new DNA synthesis requires that any nonhomologous bases at the 3' end be removed. We have previously shown that removal of a 3' nonhomologous tail in Saccharomyces cerevisiae depends on the nucleotide excision repair endonuclease Rad1/Rad10, and also on the mismatch repair proteins Msh2 and Msh3. We now report that these four proteins are needed only when the nonhomologous ends of recombining DNA are 30 nucleotides (nt) long or longer. An additional protein, the helicase Srs2, is required for the RAD1-dependent removal of long 3' tails. We suggest that Srs2 acts to extend and stabilize the initial nascent joint between the invading single strand and its homolog. 3' tails shorter than 30 nt are removed by another mechanism that depends at least in part on the 3'-to-5' proofreading activity of DNA polymerase delta.
TL;DR: HeLa nuclear extract was resolved into a depleted fraction incapable of supporting mismatch repair in vitro, and repair activity was restored upon the addition of a purified fraction isolated from HeLa cells by in vitroplementation assay.
TL;DR: Recent research is beginning to indicate that DNA, despite the absence of 2' hydroxyl groups, could rival RNA in its ability to form intricate structures and in its able to function as an enzyme.
TL;DR: It was found that 10,000 times more of either fatty acid was required for it to bind to the 31 kDa catalytic domain or inhibit the DNA polymerase activity, and the possible modes of inhibition by these long-chain fatty acids are discussed.
TL;DR: The 2.2 A resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with cAMP and a 46-bp DNA fragment reveals a second cAMP molecule bound to each protein monomer.
Abstract: The 2.2 A resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with cAMP and a 46-bp DNA fragment reveals a second cAMP molecule bound to each protein monomer. The second cAMP is in the syn conformation and is located on the DNA binding domain interacting with the helix-turn-helix, a β-hairpin from the regulatory domain and the DNA (via water molecules). The presence of this second cAMP site resolves the apparent discrepancy between the NMR and x-ray data on the conformation of cAMP, and explains the cAMP concentration-dependent behaviors of the protein. In addition, this site’s close proximity to mutations affecting transcriptional activation and its water-mediated interactions with a DNA recognition residue (E181) and DNA raise the possibility that this site has biological relevance.
TL;DR: The results suggest that molecular recognition events necessary for kinetochore formation take place at the level of DNA conformation or epigenetic mechanisms rather than DNA sequence per se.
TL;DR: The inhibition by CDVpp of DNA synthesis by HCMV DNA polymerase and the inability of H CMVDNA polymerase to excise incorporated CDV from DNA may account for the potent and long-lasting anti-CMV activity of CDV.
Abstract: Cidofovir (CDV) (HPMPC) has potent in vitro and in vivo activity against human cytomegalovirus (HCMV), CDV diphosphate (CDVpp), the putative antiviral metabolite of CDV, is an inhibitor and an alternate substrate of HCMV DNA polymerase. CDV is incorporated with the correct complementation to dGMP in the template, and the incorporated CDV at the primer end is not excised by the 3'-to-5' exonuclease activity of HCMV DNA polymerase. The incorporation of a CDV molecule causes a decrease in the rate of DNA elongation for the addition of the second natural nucleotide from the singly incorporated CDV molecule. The reduction in the rate of DNA (36-mer) synthesis from an 18-mer by one incorporated CDV is 31% that of the control. However, the fidelity of HCMV DNA polymerase is maintained for the addition of the nucleotides following a single incorporated CDV molecule. The rate of DNA synthesis by HCMV DNA polymerase is drastically decreased after the incorporation of two consecutive CDV molecules; the incorporation of a third consecutive CDV molecule is not detectable. Incorporation of two CDV molecules separated by either one or two deoxynucleoside monophosphates (dAMP, dGMP, or dTMP) also drastically decreases the rate of DNA chain elongation by HCMV DNA polymerase. The rate of DNA synthesis decreases by 90% when a template which contains one internally incorporated CDV molecule is used. The inhibition by CDVpp of DNA synthesis by HCMV DNA polymerase and the inability of HCMV DNA polymerase to excise incorporated CDV from DNA may account for the potent and long-lasting anti-CMV activity of CDV.
TL;DR: There are five well-characterized nuclear DNA polymerases in eukaryotes (DNA polymerases alpha, beta, delta, epsilon and zeta) and this short review summarizes the current knowledge concerning the participation of each in DNA-repair.
Abstract: There are five well-characterized nuclear DNA polymerases in eukaryotes (DNA polymerases alpha, beta, delta, epsilon and zeta) and this short review summarizes our current knowledge concerning the participation of each in DNA-repair. The three major DNA excision-repair pathways involve a DNA synthesis step that replaces altered bases or nucleotides removed during repair. Base excision-repair removes many modified bases and abasic sites, and in mammalian cells this mainly involves DNA polymerase beta. An alternative means for completion of base excision-repair, involving DNA polymerases delta or epsilon, may also operate and be even more important in yeast. Nucleotide excision-repair uses DNA polymerases delta or epsilon to resynthesize the bases removed during repair of pyrimidine dimers and other bulky adducts in DNA. Similarly, mismatch-repair of replication errors appears to involve DNA polymerases delta or epsilon. DNA polymerase alpha is required for semi-conservative replication of DNA but not for repair of DNA. A more recently discovered enzyme, DNA polymerase zeta, appears to be involved in the bypass of damage, without excision, and occurs during DNA replication of a damaged template.
TL;DR: Comparison of the three Pol I structures reveals no compelling evidence for many of the specific interactions that have been proposed to induce thermostability, but suggests that thermostable arises from innumerable small changes distributed throughout the protein structure.
TL;DR: This work has used a combination of limited proteolysis and mutational analysis to define the smallest soluble fragment of human RPA70 that retains complete DNA binding activity, which comprises residues 181-422.
TL;DR: The identification and characterization of all the DNA polymerases of one archaeon would add considerably to the knowledge of the basic mechanisms of DNA replication in these organisms.
Abstract: Background:
In many respects Archaea are much more like eukaryotes than prokaryotes with respect to the conservation of many of the components involved in transcription, translation and DNA replication. So far, only a few DNA polymerases with structures similar to those of eukaryotic DNA polymerase α have been found in Archaea. The identification and characterization of all the DNA polymerases of one archaeon would add considerably to our knowledge of the basic mechanisms of DNA replication in these organisms.
Results:
We have identified a novel DNA polymerase composed of two proteins, DP1 and DP2, with molecular weights of 69 294 Da and 143 161 Da, respectively, in the hyperthermophilic archaeon, Pyrococcus furiosus, and have cloned the corresponding genes which are tandemly arranged on the Pyrococcus genome. No significant sequence homology was found between these two proteins and other known DNA polymerases. The pol genes were transcribed as part of a single operon that additionally contained genes homologous to the cdc18+/CDC6 and Dmc1/Rad51 family of proteins. We purified the Pyrococcus DNA polymerase from Escherichia coli strains expressing the cloned genes and characterized its activity. It possesses strong 3′ 5′ exonucleolytic activity and has a template-primer preference which is characteristic of a replicative DNA polymerase.
Conclusion:
In P. furiosus, we identified a second DNA polymerase encoded by two genes, neither of which display significant homology to any other known DNA polymerase. Both the enzymatic properties of the enzyme and the gene organization raise the possibility that this enzyme might be the replicative DNA polymerase of P. furiosus.
TL;DR: It is suggested that quinolones may act to accelerate the rate of DNA cleavage by stimulating acquisition of this structural perturbation in the ternary complex.
TL;DR: It is proposed that POL3 plays an important role in DNA repair after irradiation, particularly in the error-prone and recombinational pathways.
Abstract: The POL3 encoded catalytic subunit of DNA polymerase δ possesses a highly conserved C-terminal cysteine-rich domain in Saccharomyces cerevisiae. Mutations in some of its cysteine codons display a lethal phenotype, which demonstrates an essential function of this domain. The thermosensitive mutant pol3-13, in which a serine replaces a cysteine of this domain, exhibits a range of defects in DNA repair, such as hypersensitivity to different DNA-damaging agents and deficiency for induced mutagenesis and for recombination. These phenotypes are observed at 24°, a temperature at which DNA replication is almost normal; this differentiates the functions of POL3 in DNA repair and DNA replication. Since spontaneous mutagenesis and spontaneous recombination are efficient in pol3-13, we propose that POL3 plays an important role in DNA repair after irradiation, particularly in the error-prone and recombinational pathways. Extragenic suppressors of pol3-13 are allelic to sdp5-1, previously identified as an extragenic suppressor of pol3-11. SDP5, which is identical to HYS2, encodes a protein homologous to the p50 subunit of bovine and human DNA polymerase δ. SDP5 is most probably the p55 subunit of Polδ of S. cerevisiae and seems to be associated with the catalytic subunit for both DNA replication and DNA repair.
TL;DR: It is shown that binding of large but not small DNAs by the C terminus of p53 negatively regulates sequence-specific DNA binding by the central domain, and that cellular mechanisms to block C-terminal DNA binding would be required.
Abstract: The tumor suppressor p53 has two DNA binding domains: a central sequence-specific domain and a C-terminal sequence-independent domain. Here, we show that binding of large but not small DNAs by the C terminus of p53 negatively regulates sequence-specific DNA binding by the central domain. Four previously described mechanisms for activation of specific DNA binding operate by blocking negative regulation. Deletion of the C terminus of p53 activates specific DNA binding only in the presence of large DNA. Three activator molecules (a small nucleic acid, a monoclonal antibody against the p53 C terminus, and a C-terminal peptide of p53) stimulate sequence-specific DNA binding only in the presence of both large DNA and p53 with an intact C terminus. Our findings argue that interactions of the C terminus of p53 with genomic DNA in vivo would prevent p53 binding to specific promoters and that cellular mechanisms to block C-terminal DNA binding would be required.
TL;DR: It is postulate that As may inhibit DNA break rejoining by interacting with the vicinal dithiols to inactivate DNA ligation in MMS-treated cells.
Abstract: Arsenic has been shown to inhibit methyl methane-sulphonate (MMS)-induced DNA repair but the exact mechanism remains controversial. The purpose of this investigation is to examine which step of DNA repair is most sensitive to arsenite (As) and how As inhibits it. The results from single-cell alkaline electrophoresis, showing post-treatment with As increased DNA strand breaks in MMS-treated cells, suggest that that the excision step seems to be less sensitive to As than later steps. To test this hypothesis, hydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC) were used to block DNA polymerization, allowing the DNA strand breaks to accumulate. These experiments indicated that As had weak inhibitory effects on DNA strand break accumulation. However, As inhibited the rejoining of those DNA strand breaks which could be rejoined within 4 h after release from blockage by Hu plus AraC. To further elucidate this mechanism, a cell extract was used to compare the relative sensitivity of the various steps in DNA repair to As. The potency of the As inhibitory effect as deduced from concentration-response curves were: ligation of poly(rA).oligo(dT) > ligation of poly(dA).oligo(dT) approximately DNA polymerization > or = DNA repair synthesis > excision. As is known to inhibit the activity of pyruvate dehydrogenase by interacting with vicinal dithiol groups. Dithiothreitol could effectively remove As inhibition of both the ligation of poly(rA).oligo(dT) and the activity of pyruvate dehydrogenase but had no obvious effect on As inhibition of poly(dA).oligo(dT) ligation. Since DNA ligase III contains vicinal dithiol groups, we postulate that As may inhibit DNA break rejoining by interacting with the vicinal dithiols to inactivate DNA ligation in MMS-treated cells.