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J J Champoux

Publications and source records attributed to J J Champoux.

At least 19 recordsLinked to original sources
PubMed2001

The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily.

The phospholipase D (PLD) superfamily is a diverse group of proteins that includes enzymes involved in phospholipid metabolism, a bacterial toxin, poxvirus envelope proteins, and bacterial nucleases. Based on sequence comparisons, we show here that the tyrosyl-DNA phosphodiesterase (Tdp1) that has been implicated in the repair of topoisomerase I covalent complexes with DNA contains two unusual HKD signature motifs that place the enzyme in a distinct class within the PLD superfamily. Mutagenesis studies with the human enzyme in which the invariant histidines and lysines of the HKD motifs are changed confirm that these highly conserved residues are essential for Tdp1 activity. Furthermore, we show that, like other members of the family for which it has been examined, the reaction involves the formation of an intermediate in which the cleaved substrate is covalently linked to the enzyme. These results reveal that the hydrolytic reaction catalyzed by Tdp1 occurs by the phosphoryl transfer chemistry that is common to all members of the PLD superfamily.

Amino Acid Motifs↗
PubMed2001

Structural alterations in the DNA ahead of the primer terminus during displacement synthesis by reverse transcriptases.

Unlike most DNA polymerases, reverse transcriptases can initiate DNA synthesis at a single-strand break and displace the downstream non- template strand simultaneously with extension of the primer. This reaction is important for generation of the long terminal repeat sequences in the duplex DNA product of retroviral reverse transcription. Oligonucleotide-based model displacement constructs were used to study the interaction of human immunodeficiency virus type 1 and Moloney murine leukemia virus reverse transcriptases with the DNA. Under conditions where the DNA is saturated with enzyme, there is no protection against DNase I cleavage of the 5' single-stranded extension that would correspond to the already-displaced strand. However, the DNase I footprint on the non-template strand extends from the +1 to the +9 position for the human immunodeficiency virus type 1 enzyme and from +1 to +7 or +8 for the Moloney enzyme. This extent of protection on the non-template strand is similar to what was observed previously for the template strand downstream from the primer terminus. Use of potassium permanganate as a probe for unpaired bases in the region ahead of the primer terminus reveals that the two base-pairs immediately in front of the enzyme are melted by the bound enzyme. These findings are consistent with a displacement mechanism in which the reverse transcriptase plays an active role in unpairing the DNA ahead of the translocating polymerase. The results are interpreted in light of a recent crystal structure showing the nature of the protein-DNA contacts with the template strand ahead of the primer terminus.

DNA Footprinting↗
PubMed2001

The role of histidine 632 in catalysis by human topoisomerase I.

Based on the crystal structure of human topoisomerase I, we hypothesized that hydrogen bonding between the side chain of the highly conserved His(632) and one of the nonbridging oxygens of the scissile phosphate contributes to catalysis by stabilizing the transition state. This hypothesis has been tested by examining the effects of changing His(632) to glutamine, asparagine, alanine, and tryptophan. The change to glutamine reduced both the relaxation activity and single-turnover cleavage activity by approximately 100-fold, whereas the same change at three other conserved histidines (positions 222, 367, and 406) had no significant effect on the relaxation activity. The properties of the mutant protein containing asparagine instead of histidine at position 632 were similar to those of the glutamine mutant, whereas mutations to alanine or tryptophan reduced the activity by approximately 4 orders of magnitude. The reduction in activity for the mutants was not due to alterations in substrate binding affinities or changes in the cleavage specificities of the proteins. The above results for the glutamine mutation in conjunction with the similar effects of pH on the wild type and the H632Q mutant enzyme rule out the possibility that His(632) acts as a general acid to protonate the leaving 5'-oxygen during the cleavage reaction. Taken together, these data strongly support the hypothesis that the only role for His(632) is to stabilize the pentavalent transition state through hydrogen bonding to one of the nonbridging oxygens.

Amino Acid Substitution↗
PubMed2001

DNA topoisomerases: structure, function, and mechanism.

DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. In addition, these enzymes fine-tune the steady-state level of DNA supercoiling both to facilitate protein interactions with the DNA and to prevent excessive supercoiling that is deleterious. In recent years, the crystal structures of a number of topoisomerase fragments, representing nearly all the known classes of enzymes, have been solved. These structures provide remarkable insights into the mechanisms of these enzymes and complement previous conclusions based on biochemical analyses. Surprisingly, despite little or no sequence homology, both type IA and type IIA topoisomerases from prokaryotes and the type IIA enzymes from eukaryotes share structural folds that appear to reflect functional motifs within critical regions of the enzymes. The type IB enzymes are structurally distinct from all other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. The structural themes common to all topoisomerases include hinged clamps that open and close to bind DNA, the presence of DNA binding cavities for temporary storage of DNA segments, and the coupling of protein conformational changes to DNA rotation or DNA movement. For the type II topoisomerases, the binding and hydrolysis of ATP further modulate conformational changes in the enzymes to effect changes in DNA topology.

Archaea↗
PubMed2000

Analysis of plus-strand primer selection, removal, and reutilization by retroviral reverse transcriptases.

The ability of reverse transcriptase to generate, extend, and remove the primer derived from the polypurine tract (PPT) is vital for reverse transcription, since this process determines one of the ends required for integration of the viral DNA. Based on the ability of the RNase H activity of Moloney murine leukemia virus reverse transcriptase to cleave a long RNA/DNA hybrid containing the PPT, it appears that cleavages that could generate the plus-strand primer can occur by an internal cleavage mechanism without any positioning by an RNA 5'-end, and such cleavages may serve to minimize cleavage events within the PPT itself. If the PPT were to be cleaved inappropriately just upstream of the normal plus-strand origin site, the resulting 3'-ends would not be extended by reverse transcriptase. Extension of the PPT primer by at least 2 nucleotides is sufficient for recognition and correct cleavage by RNase H at the RNA-DNA junction to remove the primer. Specific removal of the PPT primer after polymerase extension deviates from the general observation that primer removal occurs by cleavage one nucleotide away from the RNA-DNA junction and suggests that the same PPT specificity determinants responsible for generation of the PPT primer also direct PPT primer removal. Once the PPT primer has been extended and removed from the nascent plus-strand DNA, reinitiation at the resulting plus-strand primer terminus does not occur, providing a mechanism to prevent the repeated initiation of plus strands.

Base Sequence↗
PubMed2000

Expression of human topoisomerase I with a partial deletion of the linker region yields monomeric and dimeric enzymes that respond differently to camptothecin.

Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH(2)-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine. Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains. Here we demonstrate that deletion of linker residues Asp(660) to Lys(688) yields an active enzyme (topo70DeltaL) that purifies as both a monomer and a dimer. The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits. The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive. However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme. The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker. Yeast cells expressing topo70DeltaL were found to be insensitive to camptothecin.

Baculoviridae↗
PubMed2000

Novel insights into catalytic mechanism from a crystal structure of human topoisomerase I in complex with DNA.

Human topoisomerase I helps to control the level of DNA supercoiling in cells and is vital for numerous DNA metabolic events, including replication, transcription, and recombination. The 2.6 A crystal structure of human topoisomerase I in noncovalent complex with a DNA duplex containing a cytosine at the -1 position of the scissile strand rather than the favored thymine is reported. The hydrogen bond between the O2 position of this -1 base and the epsilon-amino of the conserved Lys-532 residue, the only base-specific contact observed previously in the human topoisomerase I-DNA interaction, is maintained in this complex. Several unique features of this structure, however, have implications for the DNA-binding and active-site mechanisms of the enzyme. First, the ends of the DNA duplex were observed to shift by up to 5.4 A perpendicular to the DNA helical axis relative to structures reported previously, suggesting a novel degree of plasticity in the interaction between human topoisomerase I and its DNA substrate. Second, 12 additional residues at the NH(2) terminus of the protein (Trp-203-Gly-214) could be built in this structure, and they were found to pack against the putative hinge region implicated in the clamping of the enzyme around duplex DNA. Third, a water molecule was observed adjacent to the scissile phosphate and the active-site residues; the potential specific base character of this solvent molecule in the active-site mechanism of the enzyme is discussed. Fourth, the scissile phosphate group was found to be rotated by 75 degrees, bringing Lys-532 into hydrogen-bonding distance of one of the nonbridging phosphate oxygens. This orientation of the scissile phosphate group implicates Lys-532 as a fifth active-site residue, and also mimics the orientation observed for the 3'-phosphotyrosine linkage in the covalent human topoisomerase I-DNA complex structure. The implications of these structural features for the mechanism of the enzyme are discussed, including the potential requirement for a rotation of the scissile phosphate group during DNA strand cleavage and covalent attachment.

Binding Sites↗
PubMed2000

RNA degradation and primer selection by Moloney murine leukemia virus reverse transcriptase contribute to the accuracy of plus strand initiation.

During reverse transcription, plus strand DNA synthesis is initiated at a purine-rich RNA primer generated by the RNase H activity of reverse transcriptase (RT). Specific initiation of plus strand synthesis from this polypurine tract (PPT) RNA is essential for the subsequent integration of the linear viral DNA product. Based on current models, it is predicted that priming from sites upstream of the PPT may be tolerated by the virus, whereas efficient extension from RNA primers located downstream from the PPT is predicted to generate dead-end products. By using hybrid duplex substrates derived from the Moloney murine leukemia virus long terminal repeat, we investigated the extent to which RNase H degrades the viral RNA during time course cleavage assays, and we tested the capacity of the polymerase activity of RT to use the resulting cleavage products as primers. We find that the majority of the RNA fragments generated by RNase H are 2-25 nucleotides in length, and only following extensive degradation are most fragments reduced to 10 nucleotides or smaller. Although extensive RNA degradation by RNase H likely eliminates many potential RNA primers, based on thermostability predictions it appears that some RNA fragments remain stably annealed to the DNA template. RNA primers generated by RNase H within the long terminal repeat sequence are found to have the capacity to initiate DNA synthesis by RT; however, the priming efficiency is significantly less than that observed with the PPT primer. We find that Moloney murine leukemia virus nucleocapsid protein reduces RNase H degradation and slightly alters the cleavage specificity of RT; however, nucleocapsid protein does not appear to enhance PPT primer utilization or suppress extension from non-PPT RNA primers.

Escherichia coli↗
PubMed2000

Structure-based analysis of the effects of camptothecin on the activities of human topoisomerase I.

The sole target for the anticancer drug camptothecin (CPT) is the type I topoisomerase. The drug poisons the topoisomerase by slowing the religation step of the reaction, thereby trapping the enzyme in a covalent complex on the DNA. In addition, CPT has been shown to inhibit plasmid DNA relaxation in vitro. The structural bases for these two activities of CPT are explored in relation to the recently published crystal structure of the enzyme with bound DNA.

Antineoplastic Agents, Phytogenic↗
PubMed1999

Polypurine tract primer generation and utilization by Moloney murine leukemia virus reverse transcriptase.

During reverse transcription, the RNase H activity of reverse transcriptase specifically cleaves the viral genome within the polypurine tract (PPT) to create the primer used for the initiation of plus-strand DNA synthesis and nonspecifically cleaves the viral genome to facilitate synthesis of plus-strand DNA. To understand how primer length and sequence affect generation and utilization of the PPT, we employed short hybrid substrates containing or lacking the PPT to evaluate cleavage, extension, and binding by reverse transcriptase. Substrates containing RNAs with the correct 3' end for initiation of plus-strand synthesis were extended equally well by reverse transcriptase, but primer length affected susceptibility to RNase H cleavage. RNA substrates with 3' ends extending beyond the plus-strand initiation site were extended poorly but were specifically cleaved to generate the correct 3' end for initiation of plus-strand synthesis. Substrates containing RNAs lacking the PPT were cleaved nonspecifically and extended inefficiently. Specific cleavages to generate the plus-strand primer and 5'-end-directed cleavages were kinetically favored over cleavages that destroyed the PPT primer or degraded other short RNA fragments. The PPT was not intrinsically resistant to cleavage by the isolated RNase H domain, and the isolated polymerase domain extended RNA primers containing the PPT sequence irrespective of the primer 3' end. These results provide insights into how reverse transcriptase generates and selectively utilizes the PPT primer for initiation of plus-strand DNA synthesis.

Base Sequence↗
PubMed1999

A functional linker in human topoisomerase I is required for maximum sensitivity to camptothecin in a DNA relaxation assay.

Human topoisomerase I is composed of four major domains: the highly charged NH(2)-terminal region, the conserved core domain, the positively charged linker domain, and the highly conserved COOH-terminal domain. Near complete enzyme activity can be reconstituted by combining recombinant polypeptides that approximate the core and COOH-terminal domains, although DNA binding is reduced somewhat for the reconstituted enzyme (Stewart, L., Ireton, G. C., and Champoux, J. J. (1997) J. Mol. Biol. 269, 355-372). A reconstituted enzyme comprising the core domain plus a COOH-terminal fragment containing the complete linker region exhibits the same biochemical properties as a reconstituted enzyme lacking the linker altogether, and thus detachment of the linker from the core domain renders the linker non-functional. The rate of religation by the reconstituted enzyme is increased relative to the forms of the enzyme containing the linker indicating that in the intact enzyme the linker slows religation. Relaxation of plasmid DNA by full-length human topoisomerase I or a 70-kDa form of the enzyme that is missing only the non-essential NH(2)-terminal domain (topo70) is inhibited approximately 16-fold by the anticancer compound, camptothecin, whereas the reconstituted enzyme is nearly resistant to the inhibitory effects of the drug despite similar affinities for the drug by the two forms of the enzyme. Based on these results and in light of the crystal structure of human topoisomerase I, we propose that the linker plays a role in hindering supercoil relaxation during the normal relaxation reaction and that camptothecin inhibition of DNA relaxation depends on a direct effect of the drug on DNA rotation that is also dependent on the linker.

Antineoplastic Agents↗
PubMed1999

Structural flexibility in human topoisomerase I revealed in multiple non-isomorphous crystal structures.

Human topoisomerase I plays a critical role in chromosomal stability by relaxing the DNA superhelical tension that arises from a variety of nuclear processes, including replication, transcription, and chromatin remodeling. Human topoisomerase I is a approximately 91 kDa enzyme composed of four major domains: a 24 kDa N-terminal domain, a56 kDa core domain, a7 kDa linker domain, and a6 kDa C-terminal domain containing the active-site Tyr723 residue. A monoclinic crystal structure of a 70 kDa N-terminally truncated form of human topoisomerase I in non-covalent complex with a 22 bp DNA duplex exhibited remarkable crystal-to-crystal non-isomorphism; shifts in cell constants of up to 9 A in the b -axis length and up to 8.5 degrees in the beta-angle were observed. Eight crystal structures of human topoisomerase I - DNA complexes from this crystal form were determined to between 2.8 and 3.25 A resolution. These structures revealed both dramatic shifts in crystal packing and functionally suggestive regions of conformational flexibility in the structure of the enzyme. Crystal packing shifts of up to 20.5 A combined with rotations of up to 11.5 degrees were observed, helping to explain the variability in cell constants. When the core subdomain III regions of the eight structures are superimposed, the "cap" (core subdomains I and II) of the molecule is observed to rotate by up to 4.6 degrees and to shift by up to 3.6 A. The linker domain shows the greatest degree of conformational flexibility, rotating and shifting by up to 2.5 degrees and 4.6 A, respectively, in five of eight structures, and becoming disordered altogether in the remaining three. These observed regions of conformational flexibility in the cap and the linker domain are consistent with the structural flexibility invoked in the "controled rotation" mechanism proposed for the relaxation of DNA superhelical tension by human topoisomerase I.

Crystallization↗
PubMed1999

Caspase-mediated cleavage of DNA topoisomerase I at unconventional sites during apoptosis.

Previous studies have demonstrated that topoisomerase I is cleaved late during apoptosis, but have not identified the proteases responsible or examined the functional consequences of this cleavage. Here, we have shown that treatment of purified topoisomerase I with caspase-3 resulted in cleavage at DDVD146 downward arrowY and EEED170 downward arrowG, whereas treatment with caspase-6 resulted in cleavage at PEDD123 downward arrowG and EEED170 downward arrowG. After treatment of Jurkat T lymphocytic leukemia cells with anti-Fas antibody or A549 lung cancer cells with topotecan, etoposide, or paclitaxel, the topoisomerase I fragment comigrated with the product that resulted from caspase-3 cleavage at DDVD146 downward arrowY. In contrast, two discrete topoisomerase I fragments that appeared to result from cleavage at DDVD146 downward arrowY and EEED170 downward arrowG were observed after treatment of MDA-MB-468 breast cancer cells with paclitaxel. Topoisomerase I cleavage did not occur in apoptotic MCF-7 cells, which lack caspase-3. Cell fractionation and band depletion studies with the topoisomerase I poison topotecan revealed that the topoisomerase I fragment remains in proximity to the chromatin and retains the ability to bind to and cleave DNA. These observations indicate that topoisomerase I is a substrate of caspase-3 and possibly caspase-6, but is cleaved at sequences that differ from those ordinarily preferred by these enzymes, thereby providing a potential explanation why topoisomerase I cleavage lags behind that of classical caspase substrates such as poly(ADP-ribose) polymerase and lamin B1.

Animals↗
PubMed1999

Structural insights into the function of type IB topoisomerases.

Topoisomerases relax the DNA superhelical tension that arises in cells as a result of several nuclear processes, including transcription, replication and recombination. Recently determined crystal structures of human topoisomerase I in complex with DNA and of the 27 kDa catalytic domain of the vaccinia virus topoisomerase have advanced our understanding of the eukaryotic type IB topoisomerases. These recent structural results provide insights into functional aspects of these topoisomerases, including their DNA binding, strand cleavage and religation activities, as well as the mechanism that these enzymes use to relax DNA superhelical tension. In addition, two proposed models of the anticancer drug camptothecin bound to a covalent complex of human topoisomerase I and DNA suggest a structural basis for the mode of action of the drug.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Properties of strand displacement synthesis by Moloney murine leukemia virus reverse transcriptase: mechanistic implications.

Previous results indicated that Moloney murine leukemia virus reverse transcriptase is capable of extensive synthesis under conditions where it must simultaneously displace a downstream non-template DNA strand. To investigate more fully the mechanistic basis for displacement synthesis and to characterize the activity with natural viral templates, displacement and non-displacement synthesis were compared under a variety of conditions using the viral long terminal repeat plus strand as the template. Although the rates of both displacement and non-displacement synthesis varied regionally over the template, on the average, displacement synthesis was slower by a factor of approximately 3 to 4. Surprisingly, with one particular primer situated downstream of the tRNA primer binding site, displacement synthesis was found to be at least tenfold more processive than non-displacement synthesis, approaching a value of 500 nucleotides. The sequence features associated with pausing during the two modes of synthesis are different in both nucleotide preference and position relative to the enzyme, suggesting that the enzyme contacts the DNA differently under the two modes of synthesis. It was found that pausing during displacement synthesis did not reflect those local regions of DNA with a predicted high degree of thermal stability. Moreover, the very similar effects of temperature on the rates of displacement and non-displacement synthesis make unlikely a strictly passive mechanism of displacement synthesis whereby breathing of the downstream duplex is sufficient for advancement of the polymerase. Together, these results suggest a mechanism of displacement synthesis in which reverse transcriptase actively participates in the process of strand separation in front of the translocating polymerase.

Base Composition↗