Identification of bacteriophage T4 gene 60 product and a role for this protein in DNA topoisomerase

Bacteriophage T4 DNA topoisomerase has been isolated and shown to contain the proteins coded by the DNA-delay genes 39 and 52 (Liu, L. F., Liu, C.-C., and Alberts, B. M. (1979) Nature (Lond.) 281, 456-461 and Stetler, G. L., King, G. J., and Huang, W. M. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3737-3741). From complementation measurements in vitro and from earlier genetic evidence, these workers suggested that the product of gene 60 (p60) was also a component of the DNA topoisomerase complex. This paper now establishes the identity of p60 and unequivocally shows that this protein is a component of the enzyme complex. T4 DNA topoisomerase was purified by a simplified two-column procedure and found to be a stable complex of p39, p52, and a protein with a relative molecular weight of 18,000. The 18,000-dalton chain has been unambiguously shown to be the product of gene 60 through the use of an amber mutant of gene 60 with Sup+ and Sup- hosts and analyses by two-dimensional gel electrophoresis. While p39 and p52 were tightly associated in the wild type enzyme complex, they were readily separated on a hydroxylapatite column from extracts of cells infected by an amber mutant of gene 60. These findings suggest that p60 plays a structural/functional role in the enzyme complex by holding the larger p39 and p52 in juxtaposition.     

Identification of the Xenopus DNA2 protein as a major nuclease for the 5'->3' strand-specific processing of DNA ends

The first step of homology-dependent DNA double-strand break (DSB) repair is the 5′ strand-specific processing of DNA ends to generate 3′ single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5′ strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5′→3′ degradation of single-strand tails. The unwinding step is catalyzed by DNA helicases, the major one of which is the Xenopus Werner syndrome protein (xWRN), a member of the RecQ helicase family. In this study, we report the purification and identification of the Xenopus DNA2 (xDNA2) as one of the nucleases responsible for the 5′→3′ degradation of single-strand tails. Immunodepletion of xDNA2 resulted in a significant reduction in end processing and homology-dependent DSB repair. These results provide strong evidence that xDNA2 is a major nuclease for the resection of DNA ends for homology-dependent DSB repair in eukaryotes.     

Identification of the gene encoding DNA topoisomerase I from Candida albicans

Abstract A gene encoding a type I topoisomerase (TOP1) was isolated from Candida albicans , sequenced, and expressed in Saccharomyces cerevisiae . The TOP1 gene was identified from a C. albicans genomic library by hybridization with the product of a polymerase chain reaction with degenerate primer sets encoding regions conserved in other TOP1 genes. A clone containing an open reading frame of 2463 bp and predicted to encode a protein of 778 amino acids with sequence similarity to eukaryotic type I topoisomerases was identified. The C. albicans TOP1 gene restored camptothecin sensitivity and increased the topoisomerase activity in S. cerevisiae , indicating that the DNA fragment encodes a functional C. albicans topoisomerase I.     

Identification of the iroA gene product of Neisseria meningitidis as a lactoferrin receptor.

The iroA gene product is an iron limitation-inducible outer membrane protein of Neisseria meningitidis. A spontaneous mutant lacking the gene was unable to bind lactoferrin. Furthermore, Escherichia coli strains expressing the IroA protein were capable of binding lactoferrin. Apparently, the IroA protein functions as a lactoferrin receptor.     

Thermophilic topoisomerase I on a single DNA molecule

Control of DNA topology is critical in thermophilic organisms in which heightened ambient temperatures threaten the stability of the double helix. An important role in this control is played by topoisomerase I, a member of the type IA family of topoisomerases. We investigated the binding and activity of this topoisomerase from the hyperthermophilic bacterium Thermotoga maritima on duplex DNA using single molecule techniques, presenting it with various substrates such as (+) plectonemes, (-) plectonemes, and denaturation bubbles. We found the topoisomerase inactive on both types of plectonemes, but active on denaturation bubbles produced at increased stretching forces in underwound DNA. The relaxation rate depended sensitively on the applied force and the protein concentration. These observations could be understood in terms of a preference of the topoisomerase for single-stranded DNA over double-stranded DNA and allowed for a better understanding of activity of the topoisomerase in bulk experiments on circular plasmids. Binding experiments on a single duplex molecule using a mutant unable to perform cleavage confirmed this interpretation and suggested that T.maritima topoisomerase I behaves like an SSB by lowering the denaturation threshold of underwound DNA. Finally, experiments with a unique single-stranded DNA showed that both ends of the cleaved DNA are tightly maintained by the enzyme, supporting an enzyme-bridged mechanism for this topoisomerase.     

Identification of the Escherichia coli recN gene product as a major SOS protein.

The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified.     

AFM characterization of single strand-specific endonuclease activity on linear DNA

The specificity of nucleases for nicked and un-nicked double-stranded DNA has been characterized using atomic force microscopy (AFM). We have found that AFM has advantages over the usual macroscopic analyses, such as sucrose gradient centrifugation or electrophoresis, in characterizing nuclease digestion. In particular, short DNA fragments resulting from non-specific digestion were detected and, thus, the true length distribution of digested DNA was revealed. A simple numerical method is proposed to estimate the number of nicked sites per DNA molecule based on AFM images.     

Identification of the ftsA gene product.

A nonsense mutation was identified in the essential cell division gene ftsA of Escherichia coli. A gamma-transducing phage was isolated which complemented this mutation. This phage programmed the synthesis of four bacterial proteins in UV-irradiated cells. By substituting the nonsense mutation for the ftsA+ allele in this transducing phage and comparing the proteins programmed by it in UV-treated Su+ and Su- cells, the product of the ftsA gene was identified as a protein with a molecular weight of 50,000.     

Identification of a single nuclear localization signal in the C-terminal domain of an Aspergillus DNA topoisomerase II

DNA topoisomerase II (topo II) is a major nuclear protein that plays an important role in DNA metabolism. We have isolated the gene for topo II ( TOP2) from the filamentous fungus Aspergillus terreus. The deduced amino acid sequence revealed that topo II consists of 1,587 amino acids and has a calculated molecular weight of 180 kDa; the protein expressed in Escherichia coli has an estimated molecular weight of 185 kDa. Expression of topo II polypeptides tagged with yellow fluorescent protein (YFP) in budding yeast suggests that the C-terminal region of the topo II is essential for transport of the fusion protein into the nucleus. The nuclear localization signal (NLS) sequence of topo II is a non-classical bipartite type containing two interdependent, positively charged clusters separated by 15 amino acids. Alanine scanning mutagenesis and deletion analyses showed further that a stretch of 23 amino acid residues (positions 1,234-1,256) is necessary for nuclear import. In addition, we confirmed, using co-immunoprecipitation and two-hybrid analysis, that this non-classical NLS interacts with importin alpha in budding yeast. These results suggest that the fungal topo II NLS is functional in yeast cells.     

The nucleotide sequence of the fission yeast DNA topoisomerase II gene: structural and functional relationships to other DNA topoisomerases.

We have determined the complete nucleotide sequence of a 5.3-kb long genomic DNA fragment of the fission yeast Schizosaccharomyces pombe that encodes DNA topoisomerase II. It contains a 4293 bp long single open reading frame. The predicted polypeptide has 1431 residues (mol. wt 162,000) and shows three characteristic domains; the large C-terminal region, which consists of alternating acidic-basic stretches and might be a chromatin-binding domain, the NH2 half domain homologous to the ATP-binding gyrB subunit of bacterial gyrase and the central-to-latter part which is homologous to the NH2 domain of the catalytic gyrA subunit, suggesting a possible evolutionary consequence of the gene fusion of the bacterial gyrase subunits into the eucaryotic DNA topoisomerase II gene. We have found that the cloned fission yeast TOP2 gene can complement the budding yeast top2 mutation, although the fission yeast TOP2 protein sequence is only 50% homologous to the recently determined sequence of budding yeast (J.C. Wang, personal communication). Conversely, the budding yeast TOP2 gene can complement the fission yeast top2 mutations, indicating that their DNA topoisomerase II genes are functionally exchangeable.     

Mutant isolation of mouse DNA topoisomerase II alpha in yeast.

For characterizing in vivo functions of a mammalian protein, it is informative to obtain conditional mutations and apply them to the mouse genetic system. However, the isolation of conditional mutations has been quite difficult in cultured cells. We report here that functional expression of a heterologous mammalian gene in the yeast Saccharomyces cerevisiae provides a system for isolating mutated genes. We found that the cloned mouse TOP2 alpha cDNA, which encodes mouse DNA topoisomerase II (topo II) alpha, could rescue the lethal phenotype caused by yeast top2 null mutation. In order to generate and select temperature-sensitive mouse topo II alpha, an expression plasmid was mutagenized in vitro and was transformed, using the plasmid shuffling method, into the yeast strain, in which the endogenous TOP2 gene had been disrupted. We observed that one of such clone of yeast cells harboring a mutagenized mouse TOP2 alpha showed temperature-sensitive growth. Enzymatic assays and sequencing analysis revealed that this phenotype was caused by the thermosensitive nature of the mutant mouse protein, which has isoleucine at amino acid 961 instead of threonine. Therefore we have isolated the first conditional mutation in the mouse TOP2 alpha.     

Cleavage of ultraviolet light-irradiated DNA by single strand-specific S1 endonuclease

It was evidenced that the single strand-specific S1 endonuclease could cleave the ultraviolet light-irradiated T7 DNA. The cleavage of ultraviolet light-irradiated T7 DNA by S1 endonuclease was studied by sucrose density gradient centrifugation. The extent of cleavage was proportional to the dose of ultraviolet light given, the concentration of endonuclease and the ionic strength in the reaction. The cleavage consisted of both single-strand and double-strand breaks. The double-strand breaks were observed even at relatively lower dose of ultraviolet light. It seems likely that S1 endonuclease can recognize the alteration in the double-helical structure produced by ultraviolet light-irradiation rather than specifically attack ultraviolet light-induced photoproducts.     

Curcumin as a DNA topoisomerase II poison

Curcumin, the major active component of the spice turmeric, is recognised as a safe compound with great potential for cancer chemoprevention and cancer therapy. It induces apoptosis, but its initiation mechanism remains poorly understood. Curcumin has been assessed on the human cancer cell lines, TK-10, MCF-7 and UACC-62, and their IC50 values were 12.16, 3.63, 4.28 microM respectively. The possibility of this compound being a topoisomerase II poison has also been studied and it was found that 50 microM of curcumin is active in a similar fashion to the antineoplastic agent etoposide. These results point to DNA damage induced by topoisomerase II poisoning as a possible mechanism by which curcumin initiates apoptosis, and increase the evidence suggesting its possible use in cancer therapy.     

Specific hydrolysis of the cohesive ends of bacteriophage lambda DNA by three single strand-specific nucleases.

Procedures have been worked out for Aspergillus nuclease S1 and mung been nuclease to quantitatively cleave off both of the 12-nucleotide long, single-stranded cohesive ends of lambdaDNA. This cleavage is indicated by the almost complete elimination of the repair incorporation of radioactive nucleotides by DNA polymerase into the digested DNA. With S1 nuclease, cleavage was complete at 10 degrees as well as at 30 degrees. Under the conditions for quantitative cleavage of the single-stranded regions there was no digestion of the double-stranded lambdaDNA. The mung bean nuclease cleaved off the cohesive ends completely at 30 degrees but at 5 degrees, the cleavage was not complete even at high enzyme concentration. The nearest neighbor analysis of the repaired DNA indicates that at 5 degrees about four nucleotides remained undigested. The mung bean nuclease also introduced, under the conditions used, some nicks into double-stranded DNA as determined by the repair incorporation. The Escherichia coli exonuclease VII cleaved off part of the cohesive ends of lambdaDNA, leaving two nucleotides on each end as single-stranded tails.     

Isolation of yeast tRNALeu genes. DNA sequence of a cloned tRNALeu3 gene.

A library of cloned yeast DNA fragments generated by digestion of yeast DNA with the restriction endonuclease Bam HI has been screened by colony hybridization to total yeast [32P]tRNA. Four hundred colonies carrying yeast tRNA genes were isolated. By hybridization to 125I-tRNALeu3, we have isolated from this collection 14 colonies carrying fragments containing yeast tRNALeu genes. The size of the yeast Bam HI inserts ranged from 2.45 x 10(6) to 14 x 10(6) daltons. One of these fragments was mapped in detail by restriction endonuclease digestion and hybridization to 125I-tRNALeu3. The presence of a tRNALeu3 gene was confirmed by DNA sequence. The results indicate that the tRNALeu3 coding region is not co-linear with the tRNALeu3. An intervening tract of 33 base pairs interrupts the coding sequences 1 base pair past the anticodon coding region. The putative structure of a tRNALeu3 precursor is deduced in which the anticodon base pairs with residues from the intervening sequence.     

Identification of a cell cycle-dependent gene product as a sialic acid-binding protein

A Ca2+-dependent sialic acid-binding protein was purified on fetuin-Sepharose from various types of human tissue. The molecular mass was determined to be 10,315 Da by laser desorption mass spectrometry. Partial sequence analysis after cyanogen bromide cleavage that yielded one N-terminus accessible for Edman degradation revealed an identity to an internal stretch following the only methionine residue within a putative amino acid sequence (Mr 10,048), deduced from the cDNA of a cell cycle-specific gene. The reported biochemical identification is a prerequisite to infer the biological role of the so far undetected gene product. Initial glycohistochemical studies with sialic acid-(BSA-biotin) raised evidence for nuclear localization of sialic acid-binding sites that might reflect, at least in part, detection of this protein.