22 research outputs found
The ATP-dependent mechanism of cohesin function in chromosome segregation
Equal chromosome distribution during mitotic cell divisions is necessary for maintaining genomic stability in eukaryotes. An essential prerequisite is the alignment of sister chromatid pairs in metaphase. Pairing or cohesion between sister chromatids is established during DNA replication and is promoted by the chromosomal cohesin complex. The budding yeast Saccharomyces cerevisiae cohesin complex consists of four core subunits, Smcl and Smc3, both members of the Structural Maintenance of Chromosome (SMC) protein family, and the Sccl and Scc3 subunits. At the anaphase onset cohesion is suddenly lost by proteolytic cleavage of cohesin's Sccl subunit, leading to dissociation of cohesin from chromosomes and separation of sister chromatids getting pulled towards opposite cell poles by spindle microtubules. The mechanism by which cohesin binds to DNA initially, how cohesion is established during DNA replication and how cohesin dissociates from chromosomes in anaphase, is unknown. In this study, cohesin bound to chromatin which likely represents the functional pool of the complex, was biochemically characterised. Cohesin was found to associate with chromatin in clusters but size, shape and subunit composition does not change during cohesion establishment. This suggests that the molecular function of cohesin is inherent of the complex and may have to be sought in its characteristic architecture and conserved domains. Cohesin's Smc1 and Smc3 subunits are largely composed of long stretches of antiparallel intramolecular coiled coils which are flanked at one end by putative ATP- Binding Cassette (ABC) ATPase head domain. Heterodimerisation of Smc1 and Smc3 results in the formation of a proteinaceous ring, large enough to embrace two strands of DNA which has lead to the hypothesis that cohesion is mediated by entrapment of both sister chromatids within the ring. This study shows that cohesin has indeed ATP binding activity. The two SMC subunits by themselves form a ring, closed at their interacting ATPase head domains in an ATP-dependent and independent fashion. Disruption of this interaction and opening of the ring is triggered by a cleavage fragment of the Scc1 subunit. To assess the role of ATP in cohesion, point mutations were introduced that were designed to prevent ATP binding or hydrolysis by the Smc1 subunit. ATP binding was found to be essential for cohesin complex assembly whereas a motif implicated in ATP hydrolysis is required for loading of cohesin onto DNA. In addition, an intact SMC ring is indispensable for DNA binding, indicating that ATP hydrolysis may be coupled to DNA transport into the ring. These data suggest that ATP hydrolysis is necessary for loading of cohesin onto chromatin, whereas a prerequisite for DNA unloading of cohesin during anaphase is the disruption of the ring promoted by a Scc1 cleavage fragment. The analysis of ATP function in the context of cohesin's ring structure contributes to a biochemical understanding of the establishment and resolution of sister chromatid cohesion. In addition, since ring structure and functional domains appear to be conserved within related Smc protein complexes, similar mechanisms might apply to their multiple roles in chromosome biology like DNA condensation
Molecular architecture of the human tRNA ligase complex
RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B, and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair, and mRNA transport. Here, we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex and provide a structural framework for understanding its functions in cellular RNA metabolism
Polymerase δ deficiency causes syndromic immunodeficiency with replicative stress
Polymerase δ is essential for eukaryotic genome duplication and synthesizes DNA at both the leading and lagging strands. The polymerase δ complex is a heterotetramer comprising the catalytic subunit POLD1 and the accessory subunits POLD2, POLD3, and POLD4. Beyond DNA replication, the polymerase δ complex has emerged as a central element in genome maintenance. The essentiality of polymerase δ has constrained the generation of polymerase δ-knockout cell lines or model organisms and, therefore, the understanding of the complexity of its activity and the function of its accessory subunits. To our knowledge, no germline biallelic mutations affecting this complex have been reported in humans. In patients from 2 independent pedigrees, we have identified what we believe to be a novel syndrome with reduced functionality of the polymerase δ complex caused by germline biallelic mutations in POLD1 or POLD2 as the underlying etiology of a previously unknown autosomal-recessive syndrome that combines replicative stress, neurodevelopmental abnormalities, and immunodeficiency. Patients' cells showed impaired cell-cycle progression and replication-associated DNA lesions that were reversible upon overexpression of polymerase δ. The mutations affected the stability and interactions within the polymerase δ complex or its intrinsic polymerase activity. We believe our discovery of human polymerase δ deficiency identifies the central role of this complex in the prevention of replication-related DNA lesions, with particular relevance to adaptive immunity.</p
The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response
The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the
unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its
ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we
confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a
conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian
tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts
RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an
antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions.
Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus,
we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein
PYROXD1 through an unexpected redox mechanism
The ATP-dependent mechanism of cohesion function in chromosome segregation
EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Time Spent Outdoors and Associations with Sleep, Optimism, Happiness and Health before and during the COVID-19 Pandemic in Austria
Social restriction measures (SRM) implemented during the COVID-19 pandemic led to a reduction in time spent outdoors (TSO). The aim of this study was to describe TSO and evaluate its association with sleep outcomes, optimism, happiness and health-status before and during SRM. Two online surveys were conducted in 2017 (N = 1004) and 2020, during SRM (N = 1010), in samples representative of the age, sex and region of the Austrian population. Information on the duration of TSO, sleep, optimism, happiness and health-status was collected. Multivariable-adjusted logistic regression models were used to study the association of TSO with chronic insomnia, short sleep, late chronotype, optimism, happiness and self-rated health-status. The mean TSO was 3.6 h (SD: 2.18) in 2017 and 2.6 h (SD: 1.87) during times of SRM. Men and participants who were older, married or in a partnership and lived in a rural area reported longer TSO. Participants who spent less time outdoors were more likely to report short sleep or a late chronotype in both surveys and, in 2020, also chronic insomnia. Less TSO was associated with lower happiness and optimism levels and poor health-status. Our findings suggest that TSO may be a protective factor for sleep, mood and health, particularly during stressful and uncertain times
ANGEL2 is a member of the CCR4 family of deadenylases with 2',3'-cyclic phosphatase activity
RNA molecules are frequently modified with a terminal 2',3'-cyclic phosphate group as a result of endonuclease cleavage, exonuclease trimming, or de novo synthesis. During pre-transfer RNA (tRNA) and unconventional messenger RNA (mRNA) splicing, 2',3'-cyclic phosphates are substrates of the tRNA ligase complex, and their removal is critical for recycling of tRNAs upon ribosome stalling. We identified the predicted deadenylase angel homolog 2 (ANGEL2) as a human phosphatase that converts 2',3'-cyclic phosphates into 2',3'-OH nucleotides. We analyzed ANGEL2's substrate preference, structure, and reaction mechanism. Perturbing ANGEL2 expression affected the efficiency of pre-tRNA processing, X-box-binding protein 1 (XBP1) mRNA splicing during the unfolded protein response, and tRNA nucleotidyltransferase 1 (TRNT1)-mediated CCA addition onto tRNAs. Our results indicate that ANGEL2 is involved in RNA pathways that rely on the ligation or hydrolysis of 2',3'-cyclic phosphates