Search

Amyotrophic lateral sclerosis (ALS) is a rare neuromuscular disease characterized by severe muscle weakness mainly due to degeneration and death of motor neurons. A peculiarity of the neurodegenerative processes is the variable susceptibility among distinct neuronal populations, exemplified by the contrasting resilience of motor neurons innervating the ocular motor system and the more vulnerable facial and hypoglossal motor neurons. The crucial role of vascular endothelial growth factor (VEGF) as a neuroprotective factor in the nervous system is well-established since a deficit of VEGF has been related to motoneuronal degeneration. In this study, we investigated the survival of ocular, facial, and hypoglossal motor neurons utilizing the murine SOD1 G93A ALS model at various stages of the disease. Our primary objective was to determine whether the survival of the different brainstem motor neurons was linked to disparate VEGF expression levels in resilient and susceptible motor neurons throughout neurodegeneration. Our findings revealed a selective loss of motor neurons exclusively within the vulnerable nuclei. Furthermore, a significantly higher level of VEGF was detected in the more resistant motor neurons, the extraocular ones. We also examined whether TDP-43 dynamics in the brainstem motor neuron of SOD mice was altered. Our data suggests that the increased VEGF levels observed in extraocular motor neurons may potentially underlie their resistance during the neurodegenerative processes in ALS in a TDP-43-independent manner. Our work might help to better understand the underlying mechanisms of selective vulnerability of motor neurons in ALS., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Angel M Pastor reports financial support was provided by Government of Andalusia. Angel M Pastor reports financial support was provided by Ministry of Science Technology and Innovations. David Pozo reports financial support was provided by Ministry of Science Technology and Innovations. David Pozo reports financial support from the following foundations: CONTRA LA ELA (Reto Todos Unidos), Fundació Catalana d'ELA Miquel Valls, and Juntos Contra la ELA. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The single flagellum of African trypanosomes is essential in multiple aspects of the parasites' development. The FLAgellar Member 8 protein (FLAM8), localised to the tip of the flagellum in cultured insect forms of Trypanosoma brucei, was identified as a marker of the locking event that controls flagellum length. Here, we investigated whether FLAM8 could also reflect the flagellum maturation state in other parasite cycle stages. We observed that FLAM8 distribution extended along the entire flagellar cytoskeleton in mammalian-infective forms. Then, a rapid FLAM8 concentration to the distal tip occurs during differentiation into early insect forms, illustrating the remodelling of an existing flagellum. In the tsetse cardia, FLAM8 further localises to the entire length of the new flagellum during an asymmetric division. Strikingly, in parasites dividing in the tsetse midgut and in the salivary glands, the amount and distribution of FLAM8 in the new flagellum were seen to predict the daughter cell fate. We propose and discuss how FLAM8 could be considered a meta-marker of the flagellum stage and maturation state in trypanosomes., (© 2021 Instuit Pasteur. Cellular Microbiology published by John Wiley & Sons Ltd.)

TOF-LisH-PLL motifs define FOP family proteins; some members are involved in flagellum assembly. The critical role of FOP family protein FOR20 is poorly understood. Here, we report relative localisations of the four FOP family proteins in parasitic Trypanosoma brucei: TbRP2, TbOFD1 and TbFOP/FOP1-like are mature basal body proteins whereas TbFOR20 is present on pro- and mature basal bodies - on the latter it localises distal to TbRP2. We discuss how the data, together with published work for another protist Giardia intestinalis, informs on likely FOR20 function. Moreover, our localisation study provides convincing evidence that the antigen recognised by monoclonal antibody YL1/2 at trypanosome mature basal bodies is FOP family protein TbRP2, not tyrosinated α-tubulin as widely stated in the literature. Curiously, FOR20 proteins from T. brucei and closely related African trypanosomes possess short, negatively-charged N-terminal extensions absent from FOR20 in other trypanosomatids and other eukaryotes. The extension is necessary for protein targeting, but insufficient to re-direct TbRP2 to probasal bodies. Yet, FOR20 from the American trypanosome T. cruzi, which lacks any extension, localises to pro- and mature basal bodies when expressed in T. brucei. This identifies unexpected variation in FOR20 architecture that is presently unique to one clade of trypanosomatids., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

In 2011, the Dutch government reimbursed smoking cessation pharmacotherapy with behavioral therapy for quitting smokers. We investigate whether inequalities in the use of pharmacotherapy change and, if not, whether this is due to a relatively positive injunctive norm in lower socioeconomic status (SES) groups. A total of 75,415 participants aged ≥15 years from the Dutch Continuous Survey of Smoking Habits, 2009-2012, were considered with the following measures: SES (education/income), injunctive norm (mostly acceptable/neutral/mostly unacceptable), period (2011/all other years), and pharmacotherapy use (yes/no). The proportion of low SES smokers compared with high SES smokers making quit attempts with pharmacotherapy did not differ significantly. The injunctive norm of low SES smokers differed significantly from high SES smokers and nonsmokers of all SES levels. Low income smokers with mostly acceptable injunctive norms were significantly less likely to make quit attempts using pharmacotherapy than those with a neutral or less accepting injunctive norm. The significantly lower use of pharmacotherapy in quit attempts in low income smokers with a positive injunctive norm toward smoking may partly underlie the lack of uptake of reimbursed pharmacotherapy in low SES smokers.

Background: The optimum channel(s) used to recruit smokers living in disadvantaged neighbourhoods for smoking cessation behavioural therapy (SCBT) is unknown. This paper examines the channels through which smokers participating in a free, multi-session SCBT programme heard about and were referred to this service in a disadvantaged neighbourhood, and compares participants' characteristics and attendance between channels., Methods: 109 participants, recruited from free SCBT courses in disadvantaged areas of two cities in the Netherlands, underwent repeated surveys. Participants were asked how they heard about the SCBT and who referred them. Participant characteristics were compared between five channels, including the General Practitioner (GP), a community organisation, word of mouth, another health professional, and media or self-referred. Whether the channels through which people heard about or were referred to the service predicted attendance of ≥4 sessions was investigated with logistic regression analysis., Results: Over a quarter of the participants had no or primary education only, and more than half belonged to ethnic minority populations. Most participants heard through a single channel. More participants heard about (49%) and were referred to (60%) the SCBT by the (GP) than by any other channel. Factors influencing quit success, including psychosocial factors and nicotine dependence, did not differ significantly between channel through which participants heard about the SCBT. No channel significantly predicted attendance., Conclusion: The GP was the single most important source to both hear about and be referred to smoking cessation behavioural therapy in a disadvantaged neighbourhood. A majority of participants of low socioeconomic or ethnic minority status heard about the programme through this channel. Neither the channel through which participants heard about or were referred to the therapy influenced attendance. As such, concentrating on the channel which makes use of the existing infrastructure and which is highest yielding, the GP, would be an appropriate strategy if recruitment resources were scarce.

Background: The Global Financial Crisis (GFC) increased levels of financial strain, especially in those of low socioeconomic status (SES). Financial strain can affect smoking behaviour. This study examines socioeconomic inequalities in current smoking and smoking cessation in The Netherlands before and during the Global Financial Crisis (GFC)., Methods: Participants were 66,960 Dutch adults (≥ 18 years) who took part in the annual national Health Survey (2004-2011). Period was dichotomised: 'pre-' and 'during-GFC'. SES measures used were income, education and neighbourhood deprivation. Outcomes were current smoking rates (smokers/total population) and smoking cessation ratios (former smokers/ever smokers). Multilevel logistic regression models controlled for individual characteristics and tested for interaction between period and SES., Results: In both periods, high SES respondents (in all indicators) had lower current smoking levels and higher cessation ratios than those of middle or low SES. Inequalities in current smoking increased significantly in poorly educated adults of 45-64 years of age (Odds Ratio (OR) low educational level compared with high: 2.00[1.79-2.23] compared to pre-GFC 1.67[1.50-1.86], p for interaction = 0.02). Smoking cessation inequalities by income in 18-30 year olds increased with borderline significance during the GFC (OR low income compared to high income: 0.73[0.58-0.91]) compared to pre-GFC (OR: 0.98[0.80-1.20]), p for interaction = 0.051)., Conclusions: Overall, socioeconomic inequalities in current smoking and smoking cessation were unchanged during the GFC. However, current smoking inequalities by education, and smoking cessation inequalities by income, increased in specific age groups. Increased financial strain caused by the crisis may disproportionately affect smoking behaviour in some disadvantaged groups.

Background: Attendance of a behavioural support programme facilitates smoking cessation. Disadvantaged smokers have been shown to attend less than their more affluent peers. We need to gain in-depth insight into underlying reasons for differing attendance behaviour in disadvantaged smokers, to better address this issue. This study aims to explore the underlying motivations, barriers and social support of smokers exhibiting different patterns of attendance at a free smoking cessation behavioural support programme in a disadvantaged neighbourhood of The Netherlands., Methods: In 29 smokers undertaking smoking cessation group therapy or telephone counselling in a disadvantaged neighbourhood, qualitative interviews were completed, coded and analysed. Major themes were motivations, barriers to attend and social support. Motivations and social support were analysed with reference to the self-determination theory., Results: Two distinct patterns of attendance emerged: those who missed up to two sessions ("frequent attenders"), and those who missed more than two sessions ("infrequent attenders"). The groups differed in their motivations to attend, barriers to attendance, and in the level of social support they received. In comparison with the infrequent attenders, frequent attenders more often had intrinsic motivation to attend (e.g. enjoyed attending), and named more self-determined extrinsic motivations to attend, such as commitment to attendance and wanting to quit. Most of those mentioning intrinsic motivation did not mention a desire to quit as a motivation for attendance. No organizational barriers to attendance were mentioned by frequent attenders, such as misunderstandings around details of appointments. Frequent attenders experienced more social support within and outside the course., Conclusion: Motivation to attend behavioural support, as distinct from motivation to quit smoking, is an important factor in attendance of smoking cessation courses in disadvantaged areas. Some focus on increasing motivation to attend may help to prevent participants missing sessions.

In the present study, we have correlated plasma TDP-43 levels, as measured by ELISA, with the presence of TDP-43 pathological changes in the brains of 28 patients with frontotemporal lobar degeneration (FTLD) (14 with FTLD-TDP and 14 with FTLD-tau) and 24 patients with pathologically confirmed AD (8 with, and 16 without, TDP-43 pathological changes). Western blotting revealed full-length TDP-43, including a phosphorylated form, and a phosphorylated C-terminal fragment, in all samples examined. Both ELISA and immunohistochemistry were performed using phospho-dependent and phospho-independent TDP-43 antibodies for detection of phosphorylated and total TDP-43, respectively. Over all 52 cases, plasma levels of TDP-43, and scores of brain TDP-43 pathology, determined using TDP-43 phospho-dependent antibody correlated with the equivalent measure determined using the TDP phospho-independent antibody. In FTLD, but not AD, TDP-43 plasma levels correlated significantly with the pathology score when using the TDP-43 phospho-dependent antibody, but a similar correlation was not seen in either FTLD or AD using the TDP-43 phospho-independent antibody. With the TDP-43 phospho-independent antibody, there were no significant differences in median plasma TDP-43 levels between FTLD, or AD, patients with or without TDP-43 pathology. Using TDP-43 phospho-dependent antibody, median plasma TDP-43 levels were greater in patients with, than in those without, TDP-43 pathology for FTLD patients, though not significantly so, but not for AD patients. Present assays for TDP-43 do not differentiate between FTLD, or AD, patients with or without TDP-43 pathological changes in their brains. However, the levels of phosphorylated TDP-43 in plasma do correlate with the extent of TDP-43 brain pathology in FTLD, and therefore might be a useful surrogate marker for tracking changes in TDP-43 brain pathology during the course of this disease.

There is clear evidence implicating oxidative stress in the pathology of many neurodegenerative diseases. Reactive oxygen species (ROS) are the primary mediators of oxidative stress, and hydrogen peroxide, a key ROS, is generated during aggregation of the amyloid proteins associated with some of these diseases. Hydrogen peroxide is catalytically converted to the aggressive hydroxyl radical in the presence of Fe(II) and Cu(I), which renders amyloidogenic proteins such as beta-amyloid and alpha-synuclein (implicated in Alzheimer's disease (AD) and Parkinson's disease (PD), respectively) vulnerable to self-inflicted hydroxyl radical attack. Here, we report some of the peptide-derived radicals, detected by electron spin resonance spectroscopy employing sodium 3,5-dibromo-4-nitrosobenzenesulfonate as a spin-trap, following hydroxyl radical attack on Abeta(1-40), alpha-synuclein and some other related peptides. Significantly, we found that sufficient hydrogen peroxide was self-generated during the early stages of aggregation of Abeta(1-40) to produce detectable peptidyl radicals, on addition of Fe(II). Our results support the hypothesis that oxidative damage to Abeta (and surrounding molecules) in the brain in AD could be due, at least in part, to the self-generation of ROS. A similar mechanism could operate in PD and some other "protein conformational" disorders.

Convergent biochemical and genetic evidence suggests that the formation of alpha-synuclein (alpha-syn) protein deposits is an important and, probably, seminal step in the development of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). It has been reported that transgenic animals overexpressing human alpha-syn develop lesions similar to those found in the brain in PD, together with a progressive loss of dopaminergic cells and associated abnormalities of motor function. Inhibiting and/or reversing alpha-syn self-aggregation could, therefore, provide a novel approach to treating the underlying cause of these diseases. We synthesized a library of overlapping 7-mer peptides spanning the entire alpha-syn sequence, and identified amino acid residues 64-100 of alpha-syn as the binding region responsible for its self-association. Modified short peptides containing alpha-syn amino acid sequences from part of this binding region (residues 69-72), named alpha-syn inhibitors (ASI), were found to interact with full-length alpha-syn and block its assembly into both early oligomers and mature amyloid-like fibrils. We also developed a cell-permeable inhibitor of alpha-syn aggregation (ASID), using the polyarginine peptide delivery system. This ASID peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with alpha-syn(A53T), a familial PD-associated mutation. ASI peptides without this delivery system did not reverse levels of Fe(II)-induced DNA damage. Furthermore, the ASID peptide increased (P<0.0005) the number of cells stained positive for Bcl-2, while significantly (P<0.05) decreasing the percentage of cells stained positive for BAX. These short peptides could serve as lead compounds for the design of peptidomimetic drugs to treat PD and related disorders.

Bloom's syndrome (BS) is a genetic disorder associated with short stature, fertility defects, and a predisposition to the development of cancer. BS cells are characterized by genomic instability; in particular, a high rate of reciprocal exchanges between sister-chromatids and homologous chromosomes. The BS gene product, BLM, is a helicase belonging to the highly conserved RecQ family. BLM is known to form a complex with the RAD51 recombinase, and to act upon DNA intermediates that form during homologous recombination, including D-loops and Holliday junctions. Here, we show that BLM also makes a direct physical association with the RAD51L3 protein (also known as RAD51D), a so-called RAD51 paralog that shows limited sequence similarity to RAD51 itself. This interaction is mediated through the N-terminal domain of BLM. To analyze functional interactions between BLM and RAD51L3, we have purified a heteromeric complex comprising RAD51L3 and a second RAD51 paralog, XRCC2. We show that the RAD51L3-XRCC2 complex stimulates BLM to disrupt synthetic 4-way junctions that model the Holliday junction. We also show that a truncated form of BLM, which retains helicase activity but is unable to bind RAD51L3, is not stimulated by the RAD51L3-XRCC2 complex. Our data indicate that the activity of BLM is modulated through an interaction with the RAD51L3-XRCC2 complex, and that this stimulatory effect on BLM is dependent upon a direct physical association between the BLM and RAD51L3 proteins. We propose that BLM co-operates with RAD51 paralogs during the late stages of homologous recombination processes that serve to restore productive DNA replication at sites of damaged or stalled replication forks.

The mechanisms by which the progression of eukaryotic replication forks is controlled after DNA damage are unclear. We have found that fork progression is slowed by cisplatin or UV treatment in intact vertebrate cells and in replication assays in vitro. Fork slowing is reduced or absent in irs1SF CHO cells and XRCC3(-/-) chicken DT40 cells, indicating that fork slowing is an active process that requires the homologous recombination protein XRCC3. The addition of purified human Rad51C-XRCC3 complex restores fork slowing in permeabilized XRCC3(-/-) cells. Moreover, the requirement for XRCC3 for fork slowing can be circumvented by addition of human Rad51. These data demonstrate that the recombination proteins XRCC3 and Rad51 cooperatively modulate the progression of replication forks on damaged vertebrate chromosomes.

Cells defective in any of the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are sensitive to DNA cross-linking agents and to ionizing radiation. Because the paralogs are required for the assembly of DNA damage-induced RAD51 foci, and mutant cell lines are defective in homologous recombination and show genomic instability, their defect is thought to be caused by an inability to promote efficient recombinational repair. Here, we show that the five paralogs exist in two distinct complexes in human cells: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (defined as BCDX2), whereas the other consists of RAD51C with XRCC3. Both protein complexes have been purified to homogeneity and their biochemical properties investigated. BCDX2 binds single-stranded DNA and single-stranded gaps in duplex DNA, in accord with the proposal that the paralogs play an early (pre-RAD51) role in recombinational repair. Moreover, BCDX2 complex binds specifically to nicks in duplex DNA. We suggest that the extreme sensitivity of paralog-defective cell lines to cross-linking agents is owing to defects in the processing of incised cross links and the consequential failure to initiate recombinational repair at these sites.

In vertebrates, the RAD51 protein is required for genetic recombination, DNA repair, and cellular proliferation. Five paralogs of RAD51, known as RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3, have been identified and also shown to be required for recombination and genome stability. At the present time, however, very little is known about their biochemical properties or precise biological functions. As a first step toward understanding the roles of the RAD51 paralogs in recombination, the human RAD51C and XRCC3 proteins were overexpressed and purified from baculovirus-infected insect cells. The two proteins copurify as a complex, a property that reflects their endogenous association observed in HeLa cells. Purified RAD51C--XRCC3 complex binds single-stranded, but not duplex DNA, to form protein--DNA networks that have been visualized by electron microscopy.

The RAD51 gene is a homologue of Escherichia coli recA which plays a central role in homologous recombination and DNA repair. This paper describes the identification of the RAD51 gene from the trypanosomatid parasite Leishmania major. The LmRAD51 gene codes for a 377 amino acid polypeptide with a predicted molecular mass of 41259 Da that is highly homologous to the Rad51 family of proteins. Recombinant L. major Rad51 protein (LmRad51) was over-expressed in a bacterial expression system, purified to homogeneity and shown to bind DNA and exhibit DNA-stimulated ATPase activity, consistent with previously reported biochemical characteristics of Rad51 protein. Although LmRad51 expression is below the level of detection in exponentially growing cultures of Leishmania, high levels of LmRad51 mRNA and protein expression can be detected following exposure to the DNA-damaging agent phleomycin. LmRAD51 is one of the first examples of a DNA damage-inducible gene to be characterised in Leishmania, and will be invaluable in studying the contribution of homologous recombination to Leishmania virulence.

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4].

In the yeast Saccharomyces cerevisiae, mutations in the genes RAD51 or RAD52 result in severe defects in genetic recombination and the repair of double-strand DNA breaks. These genes, and others of the RAD52 epistasis group (RAD50, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2), were first identified by their sensitivity to X-rays. They were subsequently shown to be required for spontaneous and induced mitotic recombination, meiotic recombination, and mating-type switching. Human homologues of RAD50, RAD51, RAD52, RAD54 and MRE11 have been identified. Targeted disruption of the murine RAD51 gene results in an embryonic lethal phenotype, indicating that Rad51 protein is required during cell proliferation. Biochemical studies have shown that human RAD51 encodes a protein of relative molecular mass 36,966 (hRad51) that promotes ATP-dependent homologous pairing and DNA strand exchange. As a structural and functional homologue of the RecA protein from Escherichia coli, hRad51 is thought to play a central role in recombination. Yeast Rad51 has been shown to interact with Rad52 protein, as does the human homologue. Here we show that hRad52 stimulates homologous pairing by hRad51. The DNA-binding properties of hRad52 indicate that Rad52 is involved in an early stage of Rad51-mediated recombination.

In vitro, the human Rad51 protein (hRad51) promotes homologous pairing and strand exchange reactions suggestive of a key role in genetic recombination. To analyse its role in this process, polyclonal antibodies raised against hRad51 were used to study the distribution of Rad51 in human and mouse spermatocytes during meiosis I. In human spermatocytes, hRad51 was found to form discrete nuclear foci from early zygotene to late pachytene. The foci always co-localized with lateral element proteins, components of the synaptonemal complex (SC). During zygotene, the largest foci were present in regions undergoing synapsis, suggesting that Rad51 is a component of early recombination nodules. Pachytene nuclei showed a greatly reduced level of Rad51 labelling, with the exceptions of any asynapsed autosomes and XY segments, which were intensely labelled. The distribution of Rad51 in mouse spermatocytes was similar to that found in human spermatocytes, except that in this case Rad51 was detectable at leptotene. From these results, we conclude that the Rad51 protein has a role in the interhomologue interactions that occur during meiotic recombination. These interactions are spatially and temporally associated with synapsis during meiotic prophase I.

The human Rad51 protein is a structural homolog of Escherichia coli RecA. The exact role of human Rad51 within the cell is poorly understood but, like its bacterial and yeast homologs, hRad51 is believed to play a central role in homologous recombination. However, recent reports that transgenic mice lacking the RAD51 gene die early in development suggest an additional and essential function for mammalian Rad51 in cell proliferation or genome maintenance. In this paper we describe a simple and quick method for the purification of human Rad51 overproduced in E. coli. Dialysis of cell-free extracts against buffer containing low concentrations of spermidine result in the formation of hRad51 microcrystals as observed by light and electron microscopy. The crystals were easily redissolved in phosphate buffer and hRad51 was further purified to homogeneity using hydroxylapatite, affi-gel heparin and Q-sepharose chromatography. When purified by this method hRad51 is free of endo- and exonuclease activities and suitable for enzymological studies. Spermidine precipitation also provides a rapid method for the large scale purification of hRad51 suitable for physical analysis.

The human testis Rad51 protein, a structural homolog of E. coli RecA, binds single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using circular ssDNA and linear dsDNA (3.0 kb in length), we demonstrate that hRad51 promotes homologous pairing and strand exchange reactions in vitro. Joint molecule formation was dependent upon ATP hydrolysis and DNA homology and was stimulated by the single-strand DNA-binding protein RP-A. In these reactions, the 5' terminus of the complementary strand of the linear duplex was efficiently transferred to the ssDNA. However, under standard conditions, extensive strand exchange was not observed. These results establish hRad51 as a functional homolog of RecA, but indicate that the human protein and its bacterial counterpart differ in their ability to promote extensive strand transfer. It is proposed that hRad51 mediates homology recognition and initiates strand exchange, but that extensive heteroduplex formation in higher organisms requires the actions of additional proteins.

In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.

A mammalian endonuclease that resolves Holliday junctions has been partially purified from extracts of calf thymus and Chinese hamster ovary cells. The activity acts upon (i) synthetic Holliday junctions and (ii) recombination intermediates made by the Escherichia coli RecA protein and appears to be functionally analogous to the E. coli RuvC protein. Cleavage occurs by the introduction of symmetrically related nicks in strands of like polarity to produce nicked duplex DNA products. The nicks can be repaired by DNA ligase. The resolvase is specific for Holliday junctions and does not act upon Y junctions, G/A mismatches, or heterologous loops. The substrate specificity is therefore similar to that of E. coli RuvC protein and contrasts with the broad range specificity of other junction resolvases such as T4 endonuclease VII. The mammalian resolvase activity has been observed at normal levels in extracts prepared from a series of DNA repair-defective cells. These include the x-ray or UV-sensitive hamster lines xrs-5, xrs-6, and Chinese hamster ovary 43-3B (defective in ERCC-1), and murine cells that are severely immunodeficient and defective in both V(D)J rejoining and DNA repair.

The specificity of the Escherichia coli RuvC Holliday junction resolvase has been investigated in vitro. RuvC protein cleaves synthetic DNA substrates that model three- or four-stranded recombination intermediates but fails to act upon Y junctions, G/A mismatches, heterologous loop structures, or two-stranded branched junctions. RuvC therefore differs from endonuclease VII of bacteriophage T4 which exhibits broad range specificity. Using related three- and four-stranded synthetic DNA junctions, we show that RuvC cleaves both junctions at the same DNA sequence and requires a region of homology at the junction point. The action of RuvC on three- and four-stranded recombination intermediates made by RecA was also investigated. We found that RuvC fails to resolve three-stranded intermediates in the presence of RecA, although four-stranded intermediates are resolved under the same conditions. However, both three- and four-stranded intermediates are substrates for the nuclease after removal of RecA. We interpret these differences in terms of the contiguity of the RecA nucleoprotein filament which may, under certain conditions, limit access to the Holliday junction resolvase.

The recombination of DNA molecules has been reconstituted in vitro using two purified enzymes from Escherichia coli. RecA protein catalyses homologous pairing and strand exchange reactions to form intermediate DNA structures that are acted upon by RuvC. The newly identified RuvC protein resolves the intermediates by specific endonucleolytic cleavage to produce recombinant DNA molecules.

In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts. We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose. The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants. Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holliday junctions in vitro. This deficiency was restored by a multicopy plasmid carrying a ruvC+ gene that overexpressed junction-resolving activity. The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.

Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.

Mutation of the ruv gene of E. coli is associated with sensitivity to radiation, and filamentous growth after transient inhibition of DNA synthesis. The filamentation of ruv strains is abolished by mutations in sfiA or sfiB that prevent SOS induced inhibition of cell division, but this does not restore resistance to UV radiation. Double mutants carrying both ruv and uvr mutations are considerably more sensitive to UV radiation than the single mutants, but there is no additive effect of ruv with recA, recF, recB, or recC mutations. ruv mutations have little effect on conjugal recombination in wild-type strains but confer recombination deficiency and extreme sensitivity to ionizing radiation in recBC sbcB strains. These results, together with the fact that ruv strains are excision proficient and mutable by UV light, are interpreted to suggest that the ruv + product is involved in recombinational repair of damaged DNA rather than in cell division as suggested by Otsuji et al. (1974).

The nucleotide sequence of a 2505 bp region of the Escherichia coli chromosome containing the LexA regulated ruv gene has been determined. A sequence of 1631 bp encoding two non-overlapping open reading frames that constitute a single operon and which specify polypeptides with predicted molecular weights of 22172 daltons and 37177 daltons respectively, was identified as the most probable sequence for ruv. Each of the two open reading frames, designated ruvA and ruvB, is preceded by a reasonable Shine-Dalgarno sequence. Two 16 bp sequences (SOS boxes) that match the consensus sequence for binding LexA protein are located 5' to ruvA in a region that provides a possible single promoter for expression of both ruvA and ruvB, with the second SOS box overlapping the putative -35 region. A possible transcriptional terminator is located 137 bp downstream of ruvB. The amino acid sequence predicted for RuvB contains a region that matches a highly conserved sequence found in several DNA repair and recombination proteins that bind ATP.

The genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated. New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen. The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses. The direction of chromosome mobilization from ruv ::Mud( ApRlac ) strains carrying F42lac + established that ruv is transcribed in a counterclockwise direction. Recombinant lambda phages able to restore UV resistance to ruv mutants were identified,and the ruv + region was subcloned into a low copy number plasmid. The ruv + plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains.

The ruv gene of Escherichia coli, which is associated with inducible mechanisms of DNA repair and recombination, has been cloned into the low-copy plasmid vector pHSG415. The recombinant plasmid pPVA101 fully complements the DNA repair-deficient phenotype of ruv mutants. Restriction endonuclease analysis of this plasmid revealed a 10.6-kilobase (kb) HindIII DNA insert which contained a 7.7-kb PstI fragment identified as being from the chromosomal ruv region. Deletion analysis and Tn1000 insertional inactivation of ruv function located the ruv coding region to a 2.2-kb section of the cloned DNA fragment. A comparison of the proteins encoded by ruv wild-type and mutant plasmids identified the gene product as a protein of molecular weight 41,000.

Conjugational recombination in Escherichia coli was investigated by comparing the effects of recN, recO, ruv and lexA mutations on the formation of recombinants in crosses with strains lacking RecBCD enzyme. The results presented reveal that recN and ruv mutations do not abolish residual recombination in a recB mutant, and have only a rather modest effect on recombination in recBC sbcA strains; in these respects they are quite different from recF, recJ and recO mutations. The differences between these two groups of genes are discussed in relation to the molecular exchanges needed to produce viable recombinants.