Topoisomerase II from Chlorella virus PBCV-1 has an exceptionally high DNA cleavage activity.

Chlorella virus PBCV-1 topoisomerase II is the only functional type II enzyme known to be encoded by a virus that infects eukaryotic cells. However, it has not been established whether the protein is expressed following viral infection or whether the enzyme has any catalytic features that distinguish it from cellular type II topoisomerases. Therefore, the present study characterized the physiological expression of PBCV-1 topoisomerase II and individual reaction steps catalyzed by the enzyme. Results indicate that the topoisomerase II gene is widely distributed among Chlorella viruses and that the protein is expressed 60-90 min after viral infection of algal cells. Furthermore, the enzyme has an extremely high DNA cleavage activity that sets it apart from all known eukaryotic type II topoisomerases. Levels of DNA scission generated by the viral enzyme are approximately 30 times greater than those observed with human topoisomerase IIalpha. The high levels of cleavage are not due to inordinately tight enzyme-DNA binding or to impaired DNA religation. Thus, they most likely reflect an elevated forward rate of scission. The robust DNA cleavage activity of PBCV-1 topoisomerase II provides a unique tool for studying the catalytic functions of type II topoisomerases.

Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates.

Topoisomerase II from Chlorella virus PBCV-1. Characterization of the smallest known type II topoisomerase.

Type II topoisomerases, a family of enzymes that govern topological DNA interconversions, are essential to many cellular processes in eukaryotic organisms. Because no data are available about the functions of these enzymes in the replication of viruses that infect eukaryotic hosts, this led us to express and characterize the first topoisomerase II encoded by one of such viruses. Paramecium bursaria chlorella virus 1 (PBCV-1) infects certain chlorella-like green algae and encodes a 120-kDa protein with a similarity to type II topoisomerases. This protein was expressed in Saccharomyces cerevisiae and was highly active in relaxation of both negatively and positively supercoiled plasmid DNA, catenation of plasmid DNA, and decatenation of kinetoplast DNA networks. Its optimal activity was determined, and the omission of Mg(2+) or its replacement with other divalent cations abolished DNA relaxation. All activities of the recombinant enzyme were ATP dependent. Increasing salt concentrations shifted DNA relaxation from a normally processive mechanism to a distributive mode. Thus, even though the PBCV-1 enzyme is considerably smaller than other eukaryotic topoisomerase II enzymes (whose molecular masses are typically 160-180 kDa), it displays all the catalytic properties expected for a type II topoisomerase.

Mutagenic replication in a human cell extract of DNAs containing site-specific and stereospecific benzo(a)pyrene-7,8-diol-9,10-epoxide DNA adducts placed on the leading and lagging strands.

The environmental carcinogen benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) forms DNA adducts with unique stereochemistries that may have divergent biological fates, depending on how they are processed within a cell. To investigate the effect of DNA bulky adduct stereoisomerism on the mutagenic outcome of translesion DNA replication in a human cell extract, oligonucleotides were synthesized that contained (+)- and (-)-anti-cis-BPDE enantiomers on N6 adenine at position 2 of the human N-ras 61 codon. Both the nonadducted and BPDE-adducted oligonucleotides were introduced into two double-stranded vectors, replicative forms M13mp2SVoriL and M13mp2SVoriR, which contain SV40 origins of replication in two different orientations relative to the adduct insertion site. Nonadducted and adduct-containing vector DNA constructs were replicated in HeLa cytoplasmic extracts and then screened in bacteria for base substitutions at the adduct site. The mutation frequencies for the adducted DNAs were at least 10 times higher than for the nonadducted DNA and ranged from 5.5 x 10(-4) to 1.5 x 10(-3). The (-)anti-cis enantiomer was more than twice as mutagenic as the (+)-enantiomer. All three possible base mutations were present, with the A-->G being the predominant one. No dramatic differences in replication fidelity were found when the adducts were placed on the leading versus the lagging strand of the vector.