▪ Abstract 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 a...

DNA topoisomerases: structure, function, and mechanism.

DNA topoisomerases are the magicians of the DNA world — by allowing DNA strands or double helices to pass through each other, they can solve all of the topological problems of DNA in replication, transcription and other cellular transactions. Extensive biochemical and structural studies over the past three decades have provided molecular models of how the various subfamilies of DNA topoisomerase manipulate DNA. In this review, the cellular roles of these enzymes are examined from a molecular point of view.

/pdf/cellular-roles-of-dna-topoisomerases-a-molecular-perspective-472dbio7sm.pdf

Cellular roles of DNA topoisomerases: a molecular perspective.

A small RNA, RyhB, was found as part of a genomewide search for novel small RNAs in Escherichia coli. The RyhB 90-nt RNA down-regulates a set of iron-storage and iron-using proteins when iron is limiting; it is itself negatively regulated by the ferric uptake repressor protein, Fur (Ferric uptake regulator). RyhB RNA levels are inversely correlated with mRNA levels for the sdhCDAB operon, encoding succinate dehydrogenase, as well as five other genes previously shown to be positively regulated by Fur by an unknown mechanism. These include two other genes encoding enzymes in the tricarboxylic acid cycle, acnA and fumA, two ferritin genes, ftnA and bfr, and a gene for superoxide dismutase, sodB. Fur positive regulation of all these genes is fully reversed in an ryhB mutant. Our results explain the previously observed inability of fur mutants to grow on succinate. RyhB requires the RNA-binding protein, Hfq, for activity. Sequences within RyhB are complementary to regions within each of the target genes, suggesting that RyhB acts as an antisense RNA. In sdhCDAB, the complementary region is at the end of the first gene of the sdhCDAB operon; full-length sdhCDAB message disappears and a truncated message, equivalent in size to the region upstream of the complementarity, is detected when RyhB is expressed. RyhB provides a mechanism for the cell to down-regulate iron-storage proteins and nonessential ironcontaining proteins when iron is limiting, thus modulating intracellular iron usage to supplement mechanisms for iron uptake directly regulated by Fur.

https://www.pnas.org/content/pnas/99/7/4620.full.pdf

A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli.

https://www.cell.com/molecular-cell/pdf/S1097-2765(12)00305-X.pdf

R Loops: From Transcription Byproducts to Threats to Genome Stability

Genomic instability in the form of mutations and chromosome rearrangements is usually associated with pathological disorders, and yet it is also crucial for evolution. Two types of elements have a key role in instability leading to rearrangements: those that act in trans to prevent instability--among them are replication, repair and S-phase checkpoint factors--and those that act in cis--chromosomal hotspots of instability such as fragile sites and highly transcribed DNA sequences. Taking these elements as a guide, we review the causes and consequences of instability with the aim of providing a mechanistic perspective on the origin of genomic instability.

Genome instability: a mechanistic view of its causes and consequences

DNA of plasmid pSAS1002TH (F' ilv+ hemD+ hemC+ cya+) was used to clone the hemD gene of Escherichia coli K-12. Due to poor transformability of the heme-deficient mutants, the restriction fragments of the F' plasmid were first cloned into a mobilizable derivative of pBR322, pSAS1211LP, which was then mobilized into a hemD recA mutant (E. coli SASX419AN). One recombinant plasmid, carrying a HindIII fragment of about 5 kilobases (kb), was shown to complement the hemD mutant and also a cya mutant of E. coli K-12, as well as a hemC mutant of Salmonella typhimurium LT2. Further subcloning of the insert enabled us to locate the hemD gene to a BamHI-PstI fragment (approximately 2.3 kb) which also carried the hemC gene. The hemD gene occupies a region close to the PstI end, since the deletion of a 0.6-kb fragment from this end resulted in loss of the ability to complement the hemD mutation. The use of the promoter-probe vector pK01 and the results of complementation showed that the hemD gene was transcribed under physiological conditions from the same promoter as the hemC gene, the direction of transcription being hemC-hemD. This allows us to define a new polycistronic operon of E. coli K-12, for which we propose the designation Uro operon. Sequencing of the hemD gene showed the presence of an open reading frame (ORF) of 738 nucleotides which could code for a protein with a molecular weight of 27,766, which should correspond to the hemD protein; the ORF starts with the last nucleotide of the hemC gene, the two genes having different reading frames. An ORF of at least 480 base pairs follows the hemD gene after a few nucleotides. The corresponding gene X, the function of which is unknown, might represent a third member of the Uro operon.

/pdf/molecular-cloning-and-sequencing-of-the-hemd-gene-of-i1orekv9sb.pdf

Molecular cloning and sequencing of the hemD gene of Escherichia coli K-12 and preliminary data on the Uro operon.

R-loop-dependent hypernegative supercoiling in Escherichia coli topA mutants preferentially occurs at low temperatures and correlates with growth inhibition.

The hemB gene of Escherichia coli K12, coding for porphobilinogen synthase (PBG-S; syn, 5-aminolevulinic acid dehydratase, ALA-D), was cloned following insertion of an EcoRI fragment of plasmid F′13 into the mobilizable vector pCR1 The hybrid plasmid carrying the hemB gene was able to complement a hemB mutant of E coli K12: not only was the PBG-S activity of the mutant restored after the acquisition of the hemB gene, but it was about ten times higher than that of the wild type Subcloning of the original EcoRI fragment (146 kb) enabled us to locate the hemB gene on an Nrul-HpaI fragment of about 11 kb The hemB promoter was located toward the NruI end of the fragment, as shown by the use of the pKO promoter-probe series of vectors Sequencing of the hemB gene indicated the presence of an open reading frame (ORF) of 1051 nucleotides, which should correspond to the HemB protein Primer extension experiments enabled us to identify the 5′ end of the hemB mRNA, and to deduce the-10 and-35 regions of the hemB promoter Protein synthesis performed by an in vitro coupled transcription-translation system, showed the presence of a protein of about 35 kDa This is in agreement with the molecular weight of the HemB protein (356 kDa), as deduced from the nucleotide sequence of the gene Comparison of the amino acid sequences of E coli and human PBG-S allowed the detection of several regions of strong homology between the two proteins Two of these regions correspond, as expected, to the putative zinc-binding and catalytic sites of the human PBG-S

Nucleotide sequence of the hemB gene of Escherichia coli K12.

The hemA gene of Escherichia coli K12 was cloned by complementation of a hemA mutant of this organism. Subcloning of the initial 6.0 kb HindIII fragment allowed the isolation of a 1.5 kb NheI-AvaI fragment which retained the ability to complement the hemA mutant. DNA sequencing by the dideoxy chain terminator method of Sanger showed the presence of an open reading frame (ORF) of 1254 nucleotides, which ends 6 nucleotides beyond the AvaI site. Primer extension experiments showed the existence of a putative transcription initiation site for the hemA gene, located at position 130. A possible promoter sequence was identified upstream from this transcription initiation site, and its functional activity was confirmed by the use of the pK01 promoter-probe vector. Protein synthesis in an in vitro coupled transcription-translation system showed a 46 kDa protein, which corresponds to the mol. wt. of the hemA protein, as deduced from the nucleotide sequence of the gene. No homology was found between the amino acid sequence of the hemA protein of E. coli K12 and known sequences of other Δ-aminolevulinic acid synthases (Δ-ALAS), suggesting that this protein is different from other Δ-ALAS enzymes.

Isolation and nucleotide sequence of the hemA gene of Escherichia coli K12

It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R-loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full-length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R-loops, which, in turn, inhibit RNA synthesis.

Marc Drolet

Papers

Molecular cloning and sequencing of the hemD gene of Escherichia coli K-12 and preliminary data on the Uro operon.

R-loop-dependent hypernegative supercoiling in Escherichia coli topA mutants preferentially occurs at low temperatures and correlates with growth inhibition.

Nucleotide sequence of the hemB gene of Escherichia coli K12.

Isolation and nucleotide sequence of the hemA gene of Escherichia coli K12

RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli.