Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

De novo variants in CDK13 associated with syndromic ID/DD: Molecular and clinical delineation of 15 individuals and a further review.

De novo variants in the gene encoding cyclin-dependent kinase 13 (CDK13) have been associated with congenital heart defects and intellectual disability (ID). Here, we present the clinical assessment of 15 individuals and report novel de novo missense variants within the kinase domain of CDK13. Furthermore, we describe 2 nonsense variants and a recurrent frame-shift variant. We demonstrate the synthesis of 2 aberrant CDK13 transcripts in lymphoblastoid cells from an individual with a splice-site variant. Clinical characteristics of the individuals include mild to severe ID, developmental delay, behavioral problems, (neonatal) hypotonia and a variety of facial dysmorphism. Congenital heart defects were present in 2 individuals of the current cohort, but in at least 42% of all known individuals. An overview of all published cases is provided and does not demonstrate an obvious genotype-phenotype correlation, although 2 individuals harboring a stop codons at the end of the kinase domain might have a milder phenotype. Overall, there seems not to be a clinically recognizable facial appearance. The variability in the phenotypes impedes an à vue diagnosis of this syndrome and therefore genome-wide or gene-panel driven genetic testing is needed. Based on this overview, we provide suggestions for clinical work-up and management of this recently described ID syndrome.

MLL2 mutation detection in 86 patients with Kabuki syndrome: a genotype-phenotype study.

Recently, pathogenic variants in the MLL2 gene were identified as the most common cause of Kabuki (Niikawa-Kuroki) syndrome (MIM#147920). To further elucidate the genotype-phenotype correlation, we studied a large cohort of 86 clinically defined patients with Kabuki syndrome (KS) for mutations in MLL2. All patients were assessed using a standardized phenotype list and all were scored using a newly developed clinical score list for KS (MLL2-Kabuki score 0-10). Sequencing of the full coding region and intron-exon boundaries of MLL2 identified a total of 45 likely pathogenic mutations (52%): 31 nonsense, 10 missense and four splice-site mutations, 34 of which were novel. In five additional patients, novel, i.e. non-dbSNP132 variants of clinically unknown relevance, were identified. Patients with likely pathogenic nonsense or missense MLL2 mutations were usually more severely affected (median 'MLL2-Kabuki score' of 6) as compared to the patients without MLL2 mutations (median 'MLL2-Kabuki score' of 5), a significant difference (p < 0.0014). Several typical facial features such as large dysplastic ears, arched eyebrows with sparse lateral third, blue sclerae, a flat nasal tip with a broad nasal root, and a thin upper and a full lower lip were observed more often in mutation positive patients.

Sublimiting concentration of TFIIH transcription/DNA repair factor causes TTD-A trichothiodystrophy disorder.

The repair-deficient form of trichothiodystrophy (TTD) most often results from mutations in the genes XPB or XPD, encoding helicases of the transcription/repair factor TFIIH. The genetic defect in a third group, TTD-A, is unknown, but is also caused by dysfunctioning TFIIH. None of the TFIIH subunits carry a mutation and TFIIH from TTD-A cells is active in both transcription and repair. Instead, immunoblot and immunofluorescence analyses reveal a strong reduction in the TFIIH concentration. Thus, the phenotype of TTD-A appears to result from sublimiting amounts of TFIIH, probably due to a mutation in a gene determining the complex stability. The reduction of TFIIH mainly affects its repair function and hardly influences transcription.

A temperature-sensitive disorder in basal transcription and DNA repair in humans.

The xeroderma pigmentosum group D (XPD) helicase subunit of TFIIH functions in DNA repair and transcription initiation. Different mutations in XPD give rise to three ultraviolet-sensitive syndromes: the skin cancer-prone disorder xeroderma pigmentosum (XP), in which repair of ultraviolet damage is affected; and the severe neurodevelopmental conditions Cockayne syndrome (CS) and trichothiodystrophy (TTD). In the latter two, the basal transcription function of TFIIH is also presumed to be affected. Here we report four unusual TTD patients with fever-dependent reversible deterioration of TTD features such as brittle hair. Cells from these patients show an in vivo temperature-sensitive defect of transcription and DNA repair due to thermo-instability of TFIIH. Our findings reveal the clinical consequences of impaired basal transcription and mutations in very fundamental processes in humans, which previously were only known in lower organisms.

Fasting before living-kidney donation: effect on donor well-being and postoperative recovery: study protocol of a multicenter randomized controlled trial.

BACKGROUND: One of the main effectors on the quality of life of living-kidney donors is postoperative fatigue. Caloric restriction (CR) and short-term fasting (STF) are associated with improved fitness and increased resistance to acute stress. CR/STF increases the expression of cytoprotective genes, increases immunomodulation via increased anti-inflammatory cytokine production, and decreases the expression of pro-inflammatory markers. As such, nutritional preconditioning by CR or STF represents a non-invasive and cost-effective method that could mitigate the effects of acute surgery-induced stress and postoperative fatigue. To investigate whether preoperative STF contributes to a reduction in fatigue after living-kidney donation, a randomized clinical trial is indicated. METHODS: We aim to determine whether 2.5 days of fasting reduces postoperative fatigue score in subjects undergoing living-kidney donation. In this randomized study, the intervention group will follow a preoperative fasting regime for 2.5 days with a low-dose laxative, while the control group will receive standard care. The main study endpoint is postoperative fatigue, 4 weeks after living-kidney donation. Secondary endpoints include the effect of preoperative fasting on postoperative hospital admission time, the feasibility of STF, and the postoperative recovery of donor and recipient kidney function. This study will provide us with knowledge of the feasibility of STF and confirm its effect on postoperative recovery. DISCUSSION: Our study will provide clinically relevant information on the merits of caloric restriction for living-kidney donors and recipients. We expect to reduce the postoperative fatigue in living-kidney donors and improve the postoperative recovery of living-kidney recipients. It will provide evidence on the clinical merits and potential caveats of preoperative dietary interventions. TRIAL REGISTRATION: Netherlands Trial Register NL9262 . EudraCT 2020-005445-16 . MEC Erasmus MC MEC-2020-0778. CCMO NL74623.078.21.

X-chromosome duplications in males with mental retardation: pathogenic or benign variants?

Studies to identify copy number variants (CNVs) on the X-chromosome have revealed novel genes important in the causation of X-linked mental retardation (XLMR). Still, for many CNVs it is unclear whether they are associated with disease or are benign variants. We describe six different CNVs on the X-chromosome in five male patients with mental retardation that were identified by conventional karyotyping and single nucleotide polymorphism array analysis. One deletion and five duplications ranging in size from 325 kb to 12.5 Mb were observed. Five CNVs were maternally inherited and one occurred de novo. We discuss the involvement of potential candidate genes and focus on the complexity of X-chromosomal duplications in males inherited from healthy mothers with different X-inactivation patterns. Based on size and/or the presence of XLMR genes we were able to classify CNVs as pathogenic in two patients. However, it remains difficult to decide if the CNVs in the other three patients are pathogenic or benign.

Molecular mechanisms of DNA double strand break repair.

DNA double-strand breaks (DSBs) are major threats to the genomic integrity of cells. If not taken care of properly, they can cause chromosome fragmentation, loss and translocation, possibly resulting in carcinogenesis. Upon DSB formation, cell-cycle checkpoints are triggered and multiple DSB repair pathways can be activated. Recent research on the Nijmegen breakage syndrome, which predisposes patients to cancer, suggests a direct link between activation of cell-cycle checkpoints and DSB repair. Furthermore, the biochemical activities of proteins involved in the two major DSB repair pathways, homologous recombination and DNA end-joining, are now beginning to emerge. This review discusses these new findings and their implications for the mechanisms of DSB repair.

Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei.

We have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African Trypanosomiasis Research Organization [EATRO] 427). Endonuclease PstI and S1 nuclease cut the maxi-circle at a single site, allowing its isolation in a linear form with a mol wt of 12.2 x 10(6), determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 x 10(6), determined by gel electrophoresis. The maxi-circle in another T. brucei isolate (EATRO 1125) yields similar fragments but appears to contain a deletion of about 0.7 x 10(6) daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 micron) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 microns) and is completely removed by digestion with endonuclease PstI. 5-10% of the networks are doublets with up to 40 loops of DNA clustered between the two halves of the mini-circle network and probably represent a division stage of the kDNA. Digestion with PstI selectively removes these loops without markedly altering the mini-circle network. We conclude that the long DNA in both single and double networks represents maxi-circles and that long tandemly repeated oligomers of mini-circles are (virtually) absent. kDNA from Trypanosoma equiperdum, a trypanosome species incapable of synthesizing a fully functional mitochondrion, contains single and double networks of dimensions similar to those from T. brucei but without any DNA longer than mini-circle contour length. We conclude that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms.

DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor.

The human BTF2 basic transcription factor (also called TFIIH), which is similar to the delta factor in rat and factor b in yeast, is required for class II gene transcription. A strand displacement assay was used to show that highly purified preparation of BTF2 had an adenosine triphosphate-dependent DNA helicase activity, in addition to the previously characterized carboxyl-terminal domain kinase activity. Amino acid sequence analysis of the tryptic digest generated from the 89-kilodalton subunit of BTF2 indicated that this polypeptide corresponded to the ERCC-3 gene product, a presumed helicase implicated in the human DNA excision repair disorders xeroderma pigmentosum and Cockayne's syndrome. These findings suggest that transcription and nucleotide excision repair may share common factors and hence may be considered to be functionally related.

Action of DNA repair endonuclease ERCC1/XPF in living cells.

To study the nuclear organization and dynamics of nucleotide excision repair (NER), the endonuclease ERCC1/XPF (for excision repair cross complementation group 1/xeroderma pigmentosum group F) was tagged with green fluorescent protein and its mobility was monitored in living Chinese hamster ovary cells. In the absence of DNA damage, the complex moved freely through the nucleus, with a diffusion coefficient (15 +/- 5 square micrometers per second) consistent with its molecular size. Ultraviolet light-induced DNA damage caused a transient dose-dependent immobilization of ERCC1/XPF, likely due to engagement of the complex in a single repair event. After 4 minutes, the complex regained mobility. These results suggest (i) that NER operates by assembly of individual NER factors at sites of DNA damage rather than by preassembly of holocomplexes and (ii) that ERCC1/XPF participates in repair of DNA damage in a distributive fashion rather than by processive scanning of large genome segments.

Photic induction of mPer1 and mPer2 in cry-deficient mice lacking a biological clock.

Mice lacking mCry1 and mCry2 are behaviorally arrhythmic. As shown here, cyclic expression of the clock genes mPer1 and mPer2 (mammalian Period genes 1 and 2) in the suprachiasmatic nucleus and peripheral tissues is abolished and mPer1 and mPer2 mRNA levels are constitutively high. These findings indicate that the biological clock is eliminated in the absence of both mCRY1 and mCRY2 (mammalian cryptochromes 1 and 2) and support the idea that mammalian CRY proteins act in the negative limb of the circadian feedback loop. The mCry double-mutant mice retain the ability to have mPer1 and mPer2 expression induced by a brief light stimulus known to phase-shift the biological clock in wild-type animals. Thus, mCRY1 and mCRY2 are dispensable for light-induced phase shifting of the biological clock.

TFIIH: a key component in multiple DNA transactions.

The transcription factor TFIIH is a versatile, multi-functional protein complex with multiple engagements. Apart from its role in basal transcription, TFIIH is intimately implicated in DNA repair and (probably) in cell cycle control (both of which are required to prevent carcinogenesis) as well as having possible roles in other processes. Thus, it is a striking example of the efficient use of one component for many purposes. Ingeniously, the incorporation of this essential factor into important, but non-essential, mechanisms, such as DNA repair, protects against cancer. The critical role of TFIIH in transcription function renders inactivating TFIIH mutations lethal to cells. Without this transcription connection, such mutations would lead to genetic instability and oncogenesis.

DNA repair. Nucleotide excision-repair in the test tube.

The eukaryotic nucleotide excision-repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway.

Disruption of mouse ERCC1 results in a novel repair syndrome with growth failure, nuclear abnormalities and senescence.

BACKGROUND: The structure-specific ERCC1/XPF endonuclease complex that contains the ERCC1 and XPF subunits is implicated in the repair of two distinct types of lesions in DNA: nucleotide excision repair (NER) for ultraviolet-induced lesions and bulky chemical adducts; and recombination repair of the very genotoxic interstrand cross-links. RESULTS: Here, we present a detailed analysis of two types of mice with mutations in ERCC1, one in which the gene is 'knocked out', and one in which the encoded protein contains a seven amino-acid carboxy-terminal truncation. In addition to the previously reported symptoms of severe runting, abnormalities of liver nuclei and greatly reduced lifespan (which appeared less severe in the truncation mutant), both types of ERCC1-mutant mouse exhibited an absence of subcutaneous fat, early onset of ferritin deposition in the spleen, kidney malfunction, gross abnormalities of ploidy and cytoplasmic invaginations in nuclei of liver and kidney, and compromised NER and cross-link repair. We also found that heterozygosity for ERCC1 mutations did not appear to provide a selective advantage for chemically induced tumorigenesis. An important clue to the cause of the very severe ERCC1-mutant phenotypes is our finding that ERCC1-mutant cells undergo premature replicative senescence, unlike cells from mice with a defect only in NER. CONCLUSIONS: Our results strongly suggest that the accumulation in ERCC1-mutant mice of endogenously generated DNA interstrand cross-links, which are normally repaired by ERCC1-dependent recombination repair, underlies both the early onset of cell cycle arrest and polyploidy in the liver and kidney. Thus, our work provides an insight into the molecular basis of ageing and highlights the role of ERCC1 and interstrand DNA cross-links.

Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice.

Checkpoints of DNA integrity are conserved throughout evolution, as are the kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia- and Rad-related), which are related to phosphatidylinositol (PI) 3-kinase [1] [2] [3]. The ATM gene is not essential, but mutations lead to ataxia telangiectasia (AT), a pleiotropic disorder characterised by radiation sensitivity and cellular checkpoint defects in response to ionising radiation [4] [5] [6]. The ATR gene has not been associated with human syndromes and, structurally, is more closely related to the canonical yeast checkpoint genes rad3(Sp) and MEC1(Sc) [7] [8]. ATR has been implicated in the response to ultraviolet (UV) radiation and blocks to DNA synthesis [8] [9] [10] [11], and may phosphorylate p53 [12] [13], suggesting that ATM and ATR may have similar and, perhaps, complementary roles in cell-cycle control after DNA damage. Here, we report that targeted inactivation of ATR in mice by disruption of the kinase domain leads to early embryonic lethality before embryonic day 8.5 (E8.5). Heterozygous mice were fertile and had no aberrant phenotype, despite a lower ATR mRNA level. No increase was observed in the sensitivity of ATR(+/-) embryonic stem (ES) cells to a variety of DNA-damaging agents. Attempts to target the remaining wild-type ATR allele in heterozygous ATR(+/-) ES cells failed, supporting the idea that loss of both alleles of the ATR gene, even at the ES-cell level, is lethal. Thus, in contrast to the closely related checkpoint gene ATM, ATR has an essential function in early mammalian development.

Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci formation.

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].

Human and mouse homologs of the Saccharomyces cerevisiae RAD54 DNA repair gene: evidence for functional conservation.

BACKGROUND: Homologous recombination is of eminent importance both in germ cells, to generate genetic diversity during meiosis, and in somatic cells, to safeguard DNA from genotoxic damage. The genetically well-defined RAD52 pathway is required for these processes in the yeast Saccharomyces cerevisiae. Genes similar to those in the RAD52 group have been identified in mammals. It is not known whether this conservation of primary sequence extends to conservation of function. RESULTS: Here we report the isolation of cDNAs encoding a human and a mouse homolog of RAD54. The human (hHR54) and mouse (mHR54) proteins were 48% identical to Rad54 and belonged to the SNF2/SW12 family, which is characterized by amino-acid motifs found in DNA-dependent ATPases. The hHR54 gene was mapped to chromosome 1p32, and the hHR54 protein was located in the nucleus. We found that the levels of hHR54 mRNA increased in late G1 phase, as has been found for RAD54 mRNA. The level of mHR54 mRNA was elevated in organs of germ cell and lymphoid development and increased mHR54 expression correlated with the meiotic phase of spermatogenesis. The hHR54 cDNA could partially complement the methyl methanesulfonate-sensitive phenotype of S. cerevisiae rad54 delta cells. CONCLUSIONS: The tissue-specific expression of mHR54 is consistent with a role for the gene in recombination. The complementation experiments show that the DNA repair function of Rad54 is conserved from yeast to humans. Our findings underscore the fundamental importance of DNA repair pathways: even though they are complex and involve multiple proteins, they seem to be functionally conserved throughout the eukaryotic kingdom.