Characterization of the Caenorhabditis elegans HIM-6/BLM Helicase: Unwinding Recombination Intermediates

Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3''-5'' direction and needs at least five 3'' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.     

Combinatorial Regulation of Meiotic Holliday Junction Resolution in C. elegans by HIM-6 (BLM) Helicase,SLX-4, and the SLX-1, MUS-81 and XPF-1 Nucleases

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of “univalents” linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process.     

SACY-1 DEAD-Box Helicase Links the Somatic Control of Oocyte Meiotic Maturation to the Sperm-to-Oocyte Switch and Gamete Maintenance in Caenorhabditis elegans

In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. In Caenorhabditis elegans, major sperm protein triggers meiotic resumption through a mechanism involving somatic Gα_s–adenylate cyclase signaling and soma-to-germline gap-junctional communication. Using genetic mosaic analysis, we show that the major effector of Gα_s–adenylate cyclase signaling, protein kinase A (PKA), is required in gonadal sheath cells for oocyte meiotic maturation and dispensable in the germ line. This result rules out a model in which cyclic nucleotides must transit through sheath-oocyte gap junctions to activate PKA in the germ line, as proposed in vertebrate systems. We conducted a genetic screen to identify regulators of oocyte meiotic maturation functioning downstream of Gα_s–adenylate cyclase–PKA signaling. We molecularly identified 10 regulatory loci, which include essential and nonessential factors. sacy-1, which encodes a highly conserved DEAD-box helicase, is an essential germline factor that negatively regulates meiotic maturation. SACY-1 is a multifunctional protein that establishes a mechanistic link connecting the somatic control of meiotic maturation to germline sex determination and gamete maintenance. Modulatory factors include multiple subunits of a CoREST-like complex and the TWK-1 two-pore potassium channel. These factors are not absolutely required for meiotic maturation or its negative regulation in the absence of sperm, but function cumulatively to enable somatic control of meiotic maturation. This work provides insights into the genetic control of meiotic maturation signaling in C. elegans, and the conserved factors identified here might inform analysis in other systems through either homology or analogy.     

Caenorhabditis elegans HIM-18/SLX-4 Interacts with SLX-1 and XPF-1 and Maintains Genomic Integrity in the Germline by Processing Recombination Intermediates

Homologous recombination (HR) is essential for the repair of blocked or collapsed replication forks and for the production of crossovers between homologs that promote accurate meiotic chromosome segregation. Here, we identify HIM-18, an ortholog of MUS312/Slx4, as a critical player required in vivo for processing late HR intermediates in Caenorhabditis elegans. DNA damage sensitivity and an accumulation of HR intermediates (RAD-51 foci) during premeiotic entry suggest that HIM-18 is required for HR–mediated repair at stalled replication forks. A reduction in crossover recombination frequencies—accompanied by an increase in HR intermediates during meiosis, germ cell apoptosis, unstable bivalent attachments, and subsequent chromosome nondisjunction—support a role for HIM-18 in converting HR intermediates into crossover products. Such a role is suggested by physical interaction of HIM-18 with the nucleases SLX-1 and XPF-1 and by the synthetic lethality of him-18 with him-6, the C. elegans BLM homolog. We propose that HIM-18 facilitates processing of HR intermediates resulting from replication fork collapse and programmed meiotic DSBs in the C. elegans germline.     

The Synaptonemal Complex Shapes the Crossover Landscape Through Cooperative Assembly,Crossover Promotion and Crossover Inhibition During Caenorhabditis elegans Meiosis

The synaptonemal complex (SC) is a highly ordered proteinaceous structure that assembles at the interface between aligned homologous chromosomes during meiotic prophase. The SC has been demonstrated to function both in stabilization of homolog pairing and in promoting the formation of interhomolog crossovers (COs). How the SC provides these functions and whether it also plays a role in inhibiting CO formation has been a matter of debate. Here we provide new insight into assembly and function of the SC by investigating the consequences of reducing (but not eliminating) SYP-1, a major structural component of the SC central region, during meiosis in Caenorhabditis elegans. First, we find an increased incidence of double CO (DCO) meiotic products following partial depletion of SYP-1 by RNAi, indicating a role for SYP-1 in mechanisms that normally limit crossovers to one per homolog pair per meiosis. Second, syp-1 RNAi worms exhibit both a strong preference for COs to occur on the left half of the X chromosome and a significant bias for SYP-1 protein to be associated with the left half of the chromosome, implying that the SC functions locally in promoting COs. Distribution of SYP-1 on chromosomes in syp-1 RNAi germ cells provides strong corroboration for cooperative assembly of the SC central region and indicates that SYP-1 preferentially associates with X chromosomes when it is present in limiting quantities. Further, the observed biases in the distribution of both COs and SYP-1 protein support models in which synapsis initiates predominantly in the vicinity of pairing centers (PCs). However, discontinuities in SC structure and clear gaps between localized foci of PC-binding protein HIM-8 and X chromosome-associated SYP-1 stretches allow refinement of models for the role of PCs in promoting synapsis. Our data suggest that the CO landscape is shaped by a combination of three attributes of the SC central region: a CO-promoting activity that functions locally at CO sites, a cooperative assembly process that enables CO formation in regions distant from prominent sites of synapsis initiation, and CO-inhibitory role(s) that limit CO number.REDUCTION in ploidy during sexual reproduction depends on the ability to form pairwise associations between homologous chromosomes. The homolog pairing process typically culminates in an arrangement in which the homologs are aligned in parallel along their lengths, with a highly ordered proteinaceous structure known as the synaptonemal complex (SC) located at the interface between them. Further, in most organisms, pairwise associations between homologs are solidified through the formation of crossovers (COs) between their DNA molecules, a process that is completed within the context of the SC.The SC has long been recognized as a hallmark cytological feature of meiosis. It was discovered on the basis of its highly ordered structure and location at the interface between aligned chromosomes in electron microscopy images of nuclei at the pachytene stage of meiotic prophase (Moses 1956, 2006). Each of the homologs is associated with one of the two lateral elements (LEs) of the SC, which are composed of cohesin complexes and other meiosis-specific structural and regulatory proteins (reviewed in Mlynarczyk-Evans and Villeneuve 2010). The LEs are connected by a highly ordered latticework of transverse filaments, and often a pronounced central element, that comprise the central region of the SC. The protein components of the SC central region are very poorly conserved at the primary sequence level, but the major central region proteins identified from diverse species share in common extended regions of predicted coiled coil structure.The SC has been demonstrated to have at least two conserved functions in meiotic prophase. First, the SC serves to stabilize and maintain tight associations along the lengths of aligned homologs (reviewed in Mlynarczyk-Evans and Villeneuve 2010). This is true both in organisms in which SC assembly is coupled to formation of recombination intermediates (e.g., budding yeast, mouse, and Arabidopsis) and in organisms in which formation of SC between homologs can occur independently of recombination (e.g., Caenorhabditis elegans and Drosophila). Second, SC central region proteins play a role in promoting maturation of recombination intermediates into crossover products (reviewed in De Boer and Heyting 2006). How the SC functions to promote CO formation is not well understood. Moreover, whether the SC might also have additional functions that help to ensure a successful outcome of meiosis has been a matter of debate.In addition to its roles in stabilization of pairing and promoting CO formation, the SC has also been proposed to function in inhibiting CO formation (Egel 1978, 1995; Maguire 1988). This idea of the SC playing an inhibitory role in recombination dates almost as far back as the discovery of the SC itself. Finding a highly ordered structure with a zipper-like appearance extending along the length of each homolog pair naturally gave rise to speculation that it might play a role in the phenomenon of crossover interference, defined as the ability of a (nascent) CO to inhibit the formation of other COs nearby on the same chromosome pair (Muller 1916; Hillers 2004). It was variously proposed either that the SC might serve as a conduit of information along a chromosome pair (e.g., undergoing a distance-dependent “change in state” to inhibit COs) or that SC polymerization might itself confer CO inhibition (Egel 1978; Maguire 1988; Sym and Roeder 1994).Early analysis of the budding yeast mutants lacking Zip1, a major structural component of the SC central region, initially seemed to support the idea that the SC central region played a key role in CO interference, as zip1 mutants formed COs at 30–50% of wild-type levels and the residual COs did not display interference (Sym and Roeder 1994). However, these data were subsequently reinterpreted by postulating that the major interference-sensitive meiotic CO pathway is eliminated in the zip1 mutant and that the residual COs form by an alternative pathway that is not subject to interference (Zalevsky et al. 1999; de los Santos et al. 2003). According to this two-pathway view, the lack of interference in the zip1 mutant can be readily explained without invoking a role for Zip1 in the interference mechanism per se. Conversely, Page and Hawley found that Drosophila females expressing a mutant form of the fly SC central region protein C(3)G retained substantial interference between residual COs despite exhibiting incomplete synapsis, implying that complete SC formation was not required for CO interference (Page and Hawley 2001). In light of these and other findings (e.g., Borner et al. 2004; Fung et al. 2004), the idea that the SC might play a role in inhibiting CO formation fell from favor.In this study, we revisit a potential role for the SC central region in inhibiting CO formation, using the C. elegans experimental system. Several features make this an interesting system for investigating factors that promote and/or inhibit COs during meiosis. First, essentially all COs in C. elegans depend on conserved meiotic CO-promoting machinery (i.e., Msh4 and Msh5) and on SC central region proteins (SYP-1, -2, -3, and -4), so analysis is generally not complicated by residual COs forming by alternative pathways (Zalevsky et al. 1999; Kelly et al. 2000; MacQueen et al. 2002; Colaiacovo et al. 2003; Smolikov et al. 2007a, 2009). Second, C. elegans hermaphrodites exhibit robust CO control, with COs usually being limited to one per homolog pair per meiosis (Hillers and Villeneuve 2003; Nabeshima et al. 2004; Hammarlund et al. 2005). Consequently, circumstances that give rise to double crossover (DCO) meiotic products can be inferred to represent impairment of mechanisms that normally inhibit CO formation. Finally, COs are distributed nonuniformly along the lengths of the chromosomes, with each chromosome containing broad domains of relatively high CO frequency flanking a more central domain where CO frequency is low (Brenner 1974; Barnes et al. 1995; Rockman and Kruglyak 2009), providing an opportunity to evaluate how factors that promote and/or inhibit COs contribute to this landscape.Our strategy was to use RNAi to reduce the levels of wild-type SYP-1 protein without eliminating synapsis entirely and then to examine the effects on CO frequency and distribution. This approach indeed revealed a role for SC central region protein SYP-1 in mechanisms that normally limit the number of COs per homolog pair. Further, it also revealed a role for the SC central region in determining CO distribution, presumably by enabling formation of COs in chromosome regions distant from the dominant site of synapsis initiation. Finally, our experimental design also afforded us the opportunity to evaluate spatial distribution of the SC in the context of limiting amounts of a key central region component. This analysis provided additional insight into the process of SC assembly and the role of cis-acting meiotic pairing centers in this process.     

The Relation between the Axial Complex of Meiotic Prophase Chromosomes and Chromosome Pairing in a Salamander (Plethodon cinereus)

An investigation of the structure of meiotic chromosomes from primary spermatocytes of two salamanders, Plethodon cinereus and Desmognathus fusca, has been made using correlated light and electron microscopy. Feulgen squashes were compared with stained sections and these related to adjacent thin sections in the electron microscope. A transition from the familiar cytological preparation to the electron image was thus effected. A linear complex consisting of three parallel strands has been observed with the electron microscope, passing along the central axis of primary spermatocyte chromosomes. The complex is similar to that found in comparable chromosomes from at least a dozen animal species. The structure in Plethodon is described in detail. Synapsis has been positively identified as the stage of meiotic prophase at which the complex occurs. Thus the complex is a part of bivalent chromosomes. It has not been seen in other stages or other divisions and is thus thought to be exclusively of synaptic occurrence. The term synaptinemal complex is suggested for the entire structure. By virtue of the material condensed around it, the complex is also seen in the light microscope where it appears as a fine, densely Feulgen-positive central core along the chromosome. The complex is thus closely associated with DNA, if not at least in part, composed of it. In the stages studied, homologous chromosomes are not always completely paired. The lateral elements of the complex separate and follow the single chromosome axes at these points. The central element disappears and thus may be a phenomenon of pairing. It is concluded that the lateral elements of the synaptinemal complex may more correctly be a "core" of the single meiotic prophase chromosome, possibly being concerned with its linear organization.     

Inhibition of the Smc5/6 Complex during Meiosis Perturbs Joint Molecule Formation and Resolution without Significantly Changing Crossover or Non-crossover Levels

Meiosis is a specialized cell division used by diploid organisms to form haploid gametes for sexual reproduction. Central to this reductive division is repair of endogenous DNA double-strand breaks (DSBs) induced by the meiosis-specific enzyme Spo11. These DSBs are repaired in a process called homologous recombination using the sister chromatid or the homologous chromosome as a repair template, with the homolog being the preferred substrate during meiosis. Specific products of inter-homolog recombination, called crossovers, are essential for proper homolog segregation at the first meiotic nuclear division in budding yeast and mice. This study identifies an essential role for the conserved Structural Maintenance of Chromosomes (SMC) 5/6 protein complex during meiotic recombination in budding yeast. Meiosis-specific smc5/6 mutants experience a block in DNA segregation without hindering meiotic progression. Establishment and removal of meiotic sister chromatid cohesin are independent of functional Smc6 protein. smc6 mutants also have normal levels of DSB formation and repair. Eliminating DSBs rescues the segregation block in smc5/6 mutants, suggesting that the complex has a function during meiotic recombination. Accordingly, smc6 mutants accumulate high levels of recombination intermediates in the form of joint molecules. Many of these joint molecules are formed between sister chromatids, which is not normally observed in wild-type cells. The normal formation of crossovers in smc6 mutants supports the notion that mainly inter-sister joint molecule resolution is impaired. In addition, return-to-function studies indicate that the Smc5/6 complex performs its most important functions during joint molecule resolution without influencing crossover formation. These results suggest that the Smc5/6 complex aids primarily in the resolution of joint molecules formed outside of canonical inter-homolog pathways.     

The Smc5-Smc6 Complex Regulates Recombination at Centromeric Regions and Affects Kinetochore Protein Sumoylation during Normal Growth

The Smc5-Smc6 complex in Saccharomyces cerevisiae is both essential for growth and important for coping with genotoxic stress. While it facilitates damage tolerance throughout the genome under genotoxin treatment, its function during unperturbed growth is mainly documented for repetitive DNA sequence maintenance. Here we provide physical and genetic evidence showing that the Smc5–Smc6 complex regulates recombination at non-repetitive loci such as centromeres in the absence of DNA damaging agents. Mutating Smc6 results in the accumulation of recombination intermediates at centromeres and other unique sequences as assayed by 2D gel analysis. In addition, smc6 mutant cells exhibit increased levels of Rad52 foci that co-localize with centromere markers. A rad52 mutation that decreases centromeric, but not overall, levels of Rad52 foci in smc6 mutants suppresses the nocodazole sensitivity of these cells, suggesting that the Smc6-mediated regulation of recombination at centromeric regions impacts centromere-related functions. In addition to influencing recombination, the SUMO ligase subunit of the Smc5–Smc6 complex promotes the sumoylation of two kinetochore proteins and affects mitotic spindles. These results suggest that the Smc5–Smc6 complex regulates both recombination and kinetochore sumoylation to facilitate chromosomal maintenance during growth.     

The serotonin receptor SER-1 (5HT2ce) contributes to the regulation of locomotion in Caenorhabditis elegans

Serotonin (5-hydroxytryptamine: 5HT) is an important neuroactive substance in the model roundworm, Caenorhabditis elegans. Aside from having effects in feeding and egg-laying, 5HT inhibits motility and also modulates several locomotory behaviors, notably food-induced slowing and foraging. Recent evidence showed that a serotonergic 5HT2-like receptor named SER-1 (also known as 5HT2ce) was responsible for the effect of 5HT on egg-laying. Here we confirm this observation and show that SER-1 also plays an important role in locomotion. A mutant lacking SER-1 was found to be highly resistant to exogenous 5HT in the absence of food and this resistant phenotype was rescued by reintroducing the SER-1 gene in a mutant background. Pharmacological studies showed that the same antagonists that blocked the activity of recombinant SER-1 in vitro also inhibited the effect of 5HT on motility, suggesting the same receptor was responsible for both effects. When tested for locomotory behaviors, the SER-1 mutant was found to be moderately defective in food-induced slowing. In addition, the mutant changed direction more frequently than the wildtype when searching for food, suggesting that SER-1 may play a role in navigational control during foraging. Both these effects required the presence of MOD-1, a 5HT gated chloride channel, and the results indicate that SER-1 and MOD-1 modulate these behaviors through a common pathway. On the basis of expression analysis of a ser-1::GFP translational fusion, SER-1 is prominently located in central, integrating neurons of the head ganglia (RIA and RIC) but not the body wall musculature. The evidence suggests that SER-1 controls locomotion through indirect modulation of neuromuscular circuits and has effects both on speed and direction of movement.     

The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6 Complex Promotes Sister Chromatid Alignment and Homologous Recombination after DNA Damage in Arabidopsis thaliana

Sister chromatids are often arranged as incompletely aligned entities in interphase nuclei of Arabidopsis thaliana. The STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC) 5/6 complex, together with cohesin, is involved in double-strand break (DSB) repair by sister chromatid recombination in yeasts and mammals. Here, we analyzed the function of genes in Arabidopsis. The wild-type allele of SMC5 is essential for seed development. Each of the two SMC6 homologs of Arabidopsis is required for efficient repair of DNA breakage via intermolecular homologous recombination in somatic cells. Alignment of sister chromatids is enhanced transiently after X-irradiation (and mitomycin C treatment) in wild-type nuclei. In the smc5/6 mutants, the x-ray–mediated increase in sister chromatid alignment is much lower and delayed. The reduced S phase–established cohesion caused by a knockout mutation in one of the α-kleisin genes, SYN1, also perturbed enhancement of sister chromatid alignment after irradiation, suggesting that the S phase–established cohesion is a prerequisite for correct DSB-dependent cohesion. The radiation-sensitive51 mutant, deficient in heteroduplex formation during DSB repair, showed wild-type frequencies of sister chromatid alignment after X-irradiation, implying that the irradiation-mediated increase in sister chromatid alignment is a prerequisite for, rather than a consequence of, DNA strand exchange between sister chromatids. Our results suggest that the SMC5/6 complex promotes sister chromatid cohesion after DNA breakage and facilitates homologous recombination between sister chromatids.     

Localization mechanisms of the axon guidance molecule UNC-6/Netrin and its receptors, UNC-5 and UNC-40, in Caenorhabditis elegans

Netrin is an evolutionarily conserved, secretory axon guidance molecule. Netrin's receptors, UNC-5 and UNC-40/DCC, are single trans-membrane proteins with immunoglobulin domains at their extra-cellular regions. Netrin is thought to provide its positional information by establishing a concentration gradient. UNC-5 and UNC-40 act at growth cones, which are specialized axonal tip structures that are generally located at a long distance from the neural cell body. Thus, the proper localization of both Netrin and its receptors is critical for their function. This review addresses the localization mechanisms of UNC-6/Netrin and its receptors in Caenorhabditis elegans, focusing on our recent reports. These findings include novel insights on cytoplasmic proteins that function upstream of the receptors.     

Protection of specific maternal messenger RNAs by the P body protein CGH-1 (Dhh1/RCK) during Caenorhabditis elegans oogenesis

During oogenesis, numerous messenger RNAs (mRNAs) are maintained in a translationally silenced state. In eukaryotic cells, various translation inhibition and mRNA degradation mechanisms congregate in cytoplasmic processing bodies (P bodies). The P body protein Dhh1 inhibits translation and promotes decapping-mediated mRNA decay together with Pat1 in yeast, and has been implicated in mRNA storage in metazoan oocytes. Here, we have investigated in Caenorhabditis elegans whether Dhh1 and Pat1 generally function together, and how they influence mRNA sequestration during oogenesis. We show that in somatic tissues, the Dhh1 orthologue (CGH-1) forms Pat1 (patr-1)-dependent P bodies that are involved in mRNA decapping. In contrast, during oogenesis, CGH-1 forms patr-1-independent mRNA storage bodies. CGH-1 then associates with translational regulators and a specific set of maternal mRNAs, and prevents those mRNAs from being degraded. Our results identify somatic and germ cell CGH-1 functions that are distinguished by the involvement of PATR-1, and reveal that during oogenesis, numerous translationally regulated mRNAs are specifically protected by a CGH-1-dependent mechanism.     

A Familial Cri-du-Chat/5p Deletion Syndrome Resulted from Rare Maternal Complex Chromosomal Rearrangements (CCRs) and/or Possible Chromosome 5p Chromothripsis

Cri-du-Chat syndrome (MIM 123450) is a chromosomal syndrome characterized by the characteristic features, including cat-like cry and chromosome 5p deletions. We report a family with five individuals showing chromosomal rearrangements involving 5p, resulting from rare maternal complex chromosomal rearrangements (CCRs), diagnosed post- and pre-natally by comprehensive molecular and cytogenetic analyses. Two probands, including a 4½-year-old brother and his 2½-year- old sister, showed no diagnostic cat cry during infancy, but presented with developmental delay, dysmorphic and autistic features. Both patients had an interstitial deletion del(5)(p13.3p15.33) spanning ∼26.22 Mb. The phenotypically normal mother had de novo CCRs involving 11 breakpoints and three chromosomes: ins(11;5) (q23;p14.1p15.31),ins(21;5)(q21;p13.3p14.1),ins(21;5)(q21;p15.31p15.33),inv(7)(p22q32)dn. In addition to these two children, she had three first-trimester miscarriages, two terminations due to the identification of the 5p deletion and one delivery of a phenotypically normal daughter. The unaffected daughter had the maternal ins(11;5) identified prenatally and an identical maternal allele haplotype of 5p. Array CGH did not detect any copy number changes in the mother, and revealed three interstitial deletions within 5p15.33-p13.3, in the unaffected daughter, likely products of the maternal insertions ins(21;5). Chromothripsis has been recently reported as a mechanism drives germline CCRs in pediatric patients with congenital defects. We postulate that the unique CCRs in the phenotypically normal mother could resulted from chromosome 5p chromothripsis, that further resulted in the interstitial 5p deletions in the unaffected daughter. Further high resolution sequencing based analysis is needed to determine whether chromothripsis is also present as a germline structural variation in phenotypically normal individuals in this family.     

The effect of Photorhabdus luminescens (Enterobacteriaceae) on the survival, development, reproduction and behaviour of Caenorhabditis elegans (Nematoda: Rhabditidae)

The free-living soil nematode Caenorhabditis elegans interacts with diverse microorganisms in its natural habitat. These microorganisms may serve as a food source or represent a harmful threat. As such, they constitute one of the most important ecological factors of the worm's natural environment. In this study, we examined the interaction between two natural isolates of C. elegans and three natural isolates of the entomopathogenic bacterium Photorhabdus luminescens. Two of the tested P. luminescens strains were clearly pathogenic. They consistently reduced the worm's developmental, survival and reproductive rate, most likely through the production of nematicidal compounds. The third natural isolate tested did not decrease worm fitness. These differences could be associated with a deletion of one gene (tcdA4) ( approximately 7kb) of the toxin complex D pathogenicity island, which was found in only the non-pathogenic strain. Our study additionally highlights the importance of behaviour in defence. Caenorhabditis elegans showed two distinct avoidance behaviours towards the pathogenic but not the non-pathogenic P. luminescens strains. In particular, nematodes physically escaped and also reduced the ingestion of pathogenic bacteria. Our results strongly suggest that C. elegans possesses the capacity to distinguish between and subsequently respond to harmful and non-harmful strains of the same bacterial species. The underlying mechanisms of such interactions are currently unknown. Their dissection will represent a major challenge for future research and should enhance our knowledge of the ecology of this important model organism in biological research.     

The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the Double-Hairpin Formation Step

A central unanswered question concerning the initial phases of V(D)J recombination has been at which step the 12/23 rule applies. This rule, which governs which variable (V), diversity (D), and joining (J) segments are able to pair during recombination, could operate at the level of signal sequence synapsis after RAG-HMG1 complex binding, signal nicking, or signal hairpin formation. It has also been unclear whether additional proteins are required to achieve adherence to the 12/23 rule. We developed a novel system for the detailed biochemical analysis of the 12/23 rule by using an oligonucleotide-based substrate that can include two signals. Under physiologic conditions, we found that the complex of RAG1, RAG2, and HMG1 can successfully recapitulate the 12/23 rule with the same specificity as that seen intracellularly and in crude extracts. The cleavage complex can bind and nick 12×12 and 23×23 substrates as well as 12×23 substrates. However, hairpin formation occurs at both of the signals only on 12×23 substrates. Moreover, under physiologic conditions, the presence of a partner 23-bp spacer suppresses single-site hairpin formation at a 12-bp spacer and vice versa. Hence, this study illustrates that synapsis suppresses single-site reactions, thereby explaining the high physiologic ratio of paired versus unpaired V(D)J recombination events in lymphoid cells.     

Proteinase Inhibitor 6 (PI-6) Expression in Human Skin: Induction of PI-6 and a PI-6/Proteinase Complex during Keratinocyte Differentiation

Proteinase inhibitor 6 (PI-6) is a 42-kDa intracellular protein present in epithelial cells and endothelial cells. It is capable of inhibiting a number of serine proteinases, including trypsin and chymotrypsin. In this study we examined PI-6 expression in human skin and its primary cell type, the keratinocyte. By immunohistochemical analysis, PI-6 staining is absent from the basal cells, weak in the spinous layer, and strongest in the granulosa layer of human epidermis. Immunoblotting of cultured primary keratinocytes revealed that PI-6 production increases 24-fold on differentiation. Analysis of an immortalized keratinocyte cell line, HaCat, showed a 5-fold increase in PI-6 mRNA and a 7-fold increase in PI-6 protein upon differentiation, and indirect immunofluorescence revealed that this is due to an increase in the number of differentiated cells expressing high levels of PI-6. Of particular interest is the appearance of a preformed complex between PI-6 and an endogenous serine proteinase in differentiating HaCat cells, which was detected by a monoclonal antibody demonstrated to preferentially recognize PI-6 in complex with a proteinase. This identification of a PI-6/proteinase complex is the first example of a serpin bound to a proteinase in keratinocytes. We postulate that a physiological role of PI-6 is to regulate a serine proteinase associated with keratinocyte differentiation.     

The Role of the Human Psoralen 4 (hPso4) Protein Complex in Replication Stress and Homologous Recombination

Psoralen 4 (Pso4) is an evolutionarily conserved protein that has been implicated in a variety of cellular processes including RNA splicing and resistance to agents that cause DNA interstrand cross-links. Here we show that the hPso4 complex is required for timely progression through S phase and transition through the G₂/M checkpoint, and it functions in the repair of DNA lesions that arise during replication. Notably, hPso4 depletion results in delayed resumption of DNA replication after hydroxyurea-induced stalling of replication forks, reduced repair of spontaneous and hydroxyurea-induced DNA double strand breaks (DSBs), and increased sensitivity to a poly(ADP-ribose) polymerase inhibitor. Furthermore, we show that hPso4 is involved in the repair of DSBs by homologous recombination, probably by regulating the BRCA1 protein levels and the generation of single strand DNA at DSBs. Together, our results demonstrate that hPso4 participates in cell proliferation and the maintenance of genome stability by regulating homologous recombination. The involvement of hPso4 in the recombinational repair of DSBs provides an explanation for the sensitivity of Pso4-deficient cells to DNA interstrand cross-links.