Mechanistic Insights into the Multiple Activities of the Rad5 Family of Enzymes.

The budding yeast protein Rad5 is highly conserved among eukaryotes. Rad5 and its orthologs including helicase like transcription factor (HLTF) and SNF2 histone linker PHD RING helicase (SHPRH) in humans constitute a unique family of enzymes that play critical roles in the cellular response to DNA replication stresses. The function of the Rad5 family of enzymes is fulfilled by their multiple activities, including ubiquitin ligase, replication fork regression activities and others. Herein, we review recent studies that provided mechanistic insights into their multiple activities and their coordination.

A skipping rope translocation mechanism in a widespread family of DNA repair helicases.

Mitomycin repair factor A represents a family of DNA helicases that harbor a domain of unknown function (DUF1998) and support repair of mitomycin C-induced DNA damage by presently unknown molecular mechanisms. We determined crystal structures of Bacillus subtilis Mitomycin repair factor A alone and in complex with an ATP analog and/or DNA and conducted structure-informed functional analyses. Our results reveal a unique set of auxiliary domains appended to a dual-RecA domain core. Upon DNA binding, a Zn2+-binding domain, encompassing the domain of unknown function, acts like a drum that rolls out a canopy of helicase-associated domains, entrapping the substrate and tautening an inter-domain linker across the loading strand. Quantification of DNA binding, stimulated ATPase and helicase activities in the wild type and mutant enzyme variants in conjunction with the mode of coordination of the ATP analog suggest that Mitomycin repair factor A employs similar ATPase-driven conformational changes to translocate on DNA, with the linker ratcheting through the nucleotides like a 'skipping rope'. The electrostatic surface topology outlines a likely path for the displaced DNA strand. Our results reveal unique molecular mechanisms in a widespread family of DNA repair helicases linked to bacterial antibiotics resistance.

Highly expressed captured genes and cross-kingdom domains present in Helitrons create novel diversity in Pleurotus ostreatus and other fungi.

BACKGROUND: Helitrons are class-II eukaryotic transposons that transpose via a rolling circle mechanism. Due to their ability to capture and mobilize gene fragments, they play an important role in the evolution of their host genomes. We have used a bioinformatics approach for the identification of helitrons in two Pleurotus ostreatus genomes using de novo detection and homology-based searching. We have analyzed the presence of helitron-captured genes as well as the expansion of helitron-specific helicases in fungi and performed a phylogenetic analysis of their conserved domains with other representative eukaryotic species. RESULTS: Our results show the presence of two helitron families in P. ostreatus that disrupt gene colinearity and cause a lack of synteny between their genomes. Both putative autonomous and non-autonomous helitrons were transcriptionally active, and some of them carried highly expressed captured genes of unknown origin and function. In addition, both families contained eukaryotic, bacterial and viral domains within the helitron's boundaries. A phylogenetic reconstruction of RepHel helicases using the Helitron-like and PIF1-like helicase conserved domains revealed a polyphyletic origin for eukaryotic helitrons. CONCLUSION: P. ostreatus helitrons display features similar to other eukaryotic helitrons and do not tend to capture host genes or gene fragments. The occurrence of genes probably captured from other hosts inside the helitrons boundaries pose the hypothesis that an ancient horizontal transfer mechanism could have taken place. The viral domains found in some of these genes and the polyphyletic origin of RepHel helicases in the eukaryotic kingdom suggests that virus could have played a role in a putative lateral transfer of helitrons within the eukaryotic kingdom. The high similarity of some helitrons, along with the transcriptional activity of its RepHel helicases indicates that these elements are still active in the genome of P. ostreatus.

Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3' to 5' translocase and helicase activities.

Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and a C-terminal DUF1998 domain (containing a putative tetracysteine metal-binding motif). We show that SftH is a monomeric DNA-dependent ATPase/dATPase that translocates 3' to 5' on single-stranded DNA and has 3' to 5' helicase activity. SftH homologs are found in bacteria representing 12 different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis). SftH homologs are evident in more than 30 genera of Archaea. Among eukarya, SftH homologs are present in plants and fungi.

A novel DNA helicase with strand-annealing activity from the crenarchaeon Sulfolobus solfataricus.

To protect their genetic material cells adopt different mechanisms linked to DNA replication, recombination and repair. Several proteins function at the interface of these DNA transactions. In the present study, we report on the identification of a novel archaeal DNA helicase. BlastP searches of the Sulfolobus solfataricus genome database allowed us to identify an open reading frame (SSO0112, 875 amino acid residues) having sequence similarity with the human RecQ5beta. The corresponding protein, termed Hel112 by us, was produced in Escherichia coli in soluble form, purified to homogeneity and characterized. Gel-filtration chromatography and glycerol-gradient sedimentation analyses revealed that Hel112 forms monomers and dimers in solution. Biochemical characterization of the two oligomeric species revealed that only the monomeric form has an ATP-dependent 3'-5' DNA-helicase activity, whereas, unexpectedly, both the monomeric and dimeric forms possess DNA strand-annealing capability. The Hel112 monomeric form is able to unwind forked and 3'-tailed DNA structures with high efficiency, whereas it is almost inactive on blunt-ended duplexes and bubble-containing molecules. This analysis reveals that S. solfataricus Hel112 shares some enzymatic features with the RecQ-like DNA helicases and suggests potential cellular functions of this protein.

Structure and mechanism of helicases and nucleic acid translocases.

Helicases and translocases are a ubiquitous, highly diverse group of proteins that perform an extraordinary variety of functions in cells. Consequently, this review sets out to define a nomenclature for these enzymes based on current knowledge of sequence, structure, and mechanism. Using previous definitions of helicase families as a basis, we delineate six superfamilies of enzymes, with examples of crystal structures where available, and discuss these structures in the context of biochemical data to outline our present understanding of helicase and translocase activity. As a result, each superfamily is subdivided, where appropriate, on the basis of mechanistic understanding, which we hope will provide a framework for classification of new superfamily members as they are discovered and characterized.

Identification of multiple distinct Snf2 subfamilies with conserved structural motifs.

The Snf2 family of helicase-related proteins includes the catalytic subunits of ATP-dependent chromatin remodelling complexes found in all eukaryotes. These act to regulate the structure and dynamic properties of chromatin and so influence a broad range of nuclear processes. We have exploited progress in genome sequencing to assemble a comprehensive catalogue of over 1300 Snf2 family members. Multiple sequence alignment of the helicase-related regions enables 24 distinct subfamilies to be identified, a considerable expansion over earlier surveys. Where information is known, there is a good correlation between biological or biochemical function and these assignments, suggesting Snf2 family motor domains are tuned for specific tasks. Scanning of complete genomes reveals all eukaryotes contain members of multiple subfamilies, whereas they are less common and not ubiquitous in eubacteria or archaea. The large sample of Snf2 proteins enables additional distinguishing conserved sequence blocks within the helicase-like motor to be identified. The establishment of a phylogeny for Snf2 proteins provides an opportunity to make informed assignments of function, and the identification of conserved motifs provides a framework for understanding the mechanisms by which these proteins function.

WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing.

WRN is unique among the five human RecQ DNA helicases in having a functional exonuclease domain (WRN-exo) and being defective in the premature aging and cancer-related disorder Werner syndrome. Here, we characterize WRN-exo crystal structures, biochemical activity and participation in DNA end joining. Metal-ion complex structures, active site mutations and activity assays reveal a nuclease mechanism mediated by two metal ions. The DNA end-binding Ku70/80 complex specifically stimulates WRN-exo activity, and structure-based mutational inactivation of WRN-exo alters DNA end joining in human cells. We furthermore establish structural and biochemical similarities of WRN-exo to DnaQ-family replicative proofreading exonucleases, describing WRN-specific adaptations consistent with double-stranded DNA specificity and functionally important conformational changes. These results indicate WRN-exo is a human DnaQ family member and support DnaQ-like proofreading activities stimulated by Ku70/80, with implications for WRN functions in age-related pathologies and maintenance of genomic integrity.

Expanding the Mot1 subfamily: 89B helicase encodes a new Drosophila melanogaster SNF2-related protein which binds to multiple sites on polytene chromosomes.

Many proteins of the SNF2 family, which share a similar DNA-dependent ATPase/putative helicase domain, are involved in global transcriptional control and processing of DNA damage. We report here the partial cloning and characterization of 89B helicase, a gene encoding a new Drosophila melanogaster member of the SNF2 family. 89B Helicase protein shows a high degree of homology in its ATPase/helicase domain to the global transcriptional activators SNF2 and Brahma and to the DNA repair proteins ERCC6 and RAD54. It is, however, most strikingly similar to the Saccharomyces cerevisiae protein Mot1, a transcriptional repressor with many target genes for which no homologue has yet been described. 89B helicase is expressed throughout fly development and its large transcript encodes a >200 kDa protein. Staining with anti-89B Helicase antibodies reveals that the protein is present uniformly in early embryos and then becomes localized to the ventral nerve cord and brain. On the polytene chromosomes, 89B Helicase is bound to several hundred specific sites that are randomly distributed. The homology of 89B Helicase to Mot1, its widespread developmental expression and its large number of targets on the polytene chromosomes of larval salivary gland cells suggest that 89B Helicase may play a role in chromosomal metabolism, particularly global transcriptional regulation.

RecQ DNA helicase of Escherichia coli. Characterization of the helix-unwinding activity with emphasis on the effect of single-stranded DNA-binding protein.

RecQ protein of Escherichia coli is a DNA helicase implicated in the RecF pathway of genetic recombination. To gain insight into the mode of its action, the effect of single-stranded DNA-binding proteins (SSBs) on the RecQ-mediated unwinding reaction was investigated. When the unwinding of M13-based, circular partially duplex substrates was measured as a function of the enzyme dose, a markedly sigmoidal relation was revealed, with relatively large amounts of the enzyme being necessary for substantial unwinding to occur. For instance, unwinding 50% of a 71 base-pair (bp) partial duplex substrate in ten minutes required an enzyme-to-substrate molar ratio of about 60. However, these features, indicating the enzyme's "inefficiency", were reversed by SSBs: in the presence of a saturating amount of E. coli SSB the sigmoidal relation was converted to a typically hyperbolic one, and the enzyme-to-substrate molar ratio at 50% unwinding of the 71 bp substrate was reduced to as low as 0.5. Phage T4 gene 32 protein also showed similar stimulatory activity. Further, the single-stranded DNA-dependent ATPase activity of RecQ was found to be relatively insensitive to E. coli SSB; its large excess brought about only a 60% inhibition. It is postulated that RecQ helicase is highly adapted to an SSB-rich environment, where the strand exchange reaction mediated by RecA protein, perhaps coupled closely with the RecQ reaction, should also take place.