Fhit Deficiency-Induced Global Genome Instability Promotes Mutation and Clonal Expansion

Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a genome ‘caretaker’ whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit ^-/- MEFs escape senescence to become immortal more rapidly than Fhit ^+/+ MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenz[a]anthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a ‘mutator’ phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive.     

Loss of Yeast Peroxiredoxin Tsa1p Induces Genome Instability through Activation of the DNA Damage Checkpoint and Elevation of dNTP Levels

Peroxiredoxins are a family of antioxidant enzymes critically involved in cellular defense and signaling. Particularly, yeast peroxiredoxin Tsa1p is thought to play a role in the maintenance of genome integrity, but the underlying mechanism is not understood. In this study, we took a genetic approach to investigate the cause of genome instability in tsa1Δ cells. Strong genetic interactions of TSA1 with DNA damage checkpoint components DUN1, SML1, and CRT1 were found when mutant cells were analyzed for either sensitivity to DNA damage or rate of spontaneous base substitutions. An elevation in intracellular dNTP production was observed in tsa1Δ cells. This was associated with constitutive activation of the DNA damage checkpoint as indicated by phosphorylation of Rad9/Rad53p, reduced steady-state amount of Sml1p, and induction of RNR and HUG1 genes. In addition, defects in the DNA damage checkpoint did not modulate intracellular level of reactive oxygen species, but suppressed the mutator phenotype of tsa1Δ cells. On the contrary, overexpression of RNR1 exacerbated this phenotype by increasing dNTP levels. Taken together, our findings uncover a new role of TSA1 in preventing the overproduction of dNTPs, which is a root cause of genome instability.     

Genome Instability Mediates the Loss of Key Traits by Acinetobacter baylyi ADP1 during Laboratory Evolution

Acinetobacter baylyi ADP1 has the potential to be a versatile bacterial host for synthetic biology because it is naturally transformable. To examine the genetic reliability of this desirable trait and to understand the potential stability of other engineered capabilities, we propagated ADP1 for 1,000 generations of growth in rich nutrient broth and analyzed the genetic changes that evolved by whole-genome sequencing. Substantially reduced transformability and increased cellular aggregation evolved during the experiment. New insertions of IS1236 transposable elements and IS1236-mediated deletions led to these phenotypes in most cases and were common overall among the selected mutations. We also observed a 49-kb deletion of a prophage region that removed an integration site, which has been used for genome engineering, from every evolved genome. The comparatively low rates of these three classes of mutations in lineages that were propagated with reduced selection for 7,500 generations indicate that they increase ADP1 fitness under common laboratory growth conditions. Our results suggest that eliminating transposable elements and other genetic failure modes that affect key organismal traits is essential for improving the reliability of metabolic engineering and genome editing in undomesticated microbial hosts, such as Acinetobacter baylyi ADP1.     

Expression in Escherichia coli and simple purification of human Fhit protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5',5"'-P(1),P(3)-triphosphate (Ap(3)A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His(6)-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as "H6TV," fused to the N-terminus of Fhit. Expression of H6TV-Fhit in BL21(DE3) cells for 3 h at 37 degrees C produced 30 mg of H6TV-Fhit from 1 L of cell culture ( approximately 4 g of cells). The H6TV-Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV-Fhit with rTEV protease at 4 degrees C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4 degrees C without loss of activity. The pure protein was stable at -20 degrees C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K(m) value for Ap(3)A of 0.9 microM and a k(cat)(monomer) value of 7.2 +/- 1.6 s(-1) (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV-Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap(3)A.     

Cyclin-Dependent Kinase Suppression by WEE1 Kinase Protects the Genome through Control of Replication Initiation and Nucleotide Consumption

Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage.     

Instability of Plastid DNA in the Nuclear Genome

Functional gene transfer from the plastid (chloroplast) and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo) readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes.     

BAF180 Promotes Cohesion and Prevents Genome Instability and Aneuploidy

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Causes and Consequences of Genome Instability in Psychiatric and Neurodegenerative Diseases

Molecular Biology - Each neuron has 100–10000 connections (synapses) with other neural cells, therefore genome pathologies affecting a small proportion of brain cells are capable of causing...     

Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant

Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1⁺ dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.     

Examination of Prokaryotic Multipartite Genome Evolution through Experimental Genome Reduction

Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as Vibrio cholerea and Brucella abortis as well as in many N₂-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria Sinorhizobium meliloti. Understanding the evolution and role of this multipartite genome organization will provide significant insight into these important organisms; yet this knowledge remains incomplete, in part, because technical challenges of large-scale genome manipulations have limited experimental analyses. The distinct evolutionary histories and characteristics of the three replicons that constitute the S. meliloti genome (the chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good model to examine this topic. We transferred essential genes from pSymB into the chromosome, and constructed strains that lack pSymB as well as both pSymA and pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a prokaryotic genome to date and the first removal of an essential chromid. Strikingly, strains lacking pSymA and pSymB (ΔpSymAB) lost the ability to utilize 55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur, yet the ΔpSymAB strain grew well in minimal salts media and in sterile soil. This suggests that the core chromosome is sufficient for growth in a bulk soil environment and that the pSymA and pSymB replicons carry genes with more specialized functions such as growth in the rhizosphere and interaction with the plant. These experimental data support a generalized evolutionary model, in which non-chromosomal replicons primarily carry genes with more specialized functions. These large secondary replicons increase the organism''s niche range, which offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution with the chromosome then leads to the formation of a chromid through the acquisition of functions core to all niches (e.g. pSymB).     

Loss of MEC-17 Leads to Microtubule Instability and Axonal Degeneration

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Lack of Major Genome Instability in Tumors of p53 Null Rats

Tumorigenesis is often associated with loss of tumor suppressor genes (such as TP53), genomic instability and telomere lengthening. Previously, we generated and characterized a rat p53 knockout model in which the homozygous rats predominantly develop hemangiosarcomas whereas the heterozygous rats mainly develop osteosarcomas. Using genome-wide analyses, we find that the tumors that arise in the heterozygous and homozygous Tp53^C273X mutant animals are also different in their genomic instability profiles. While p53 was fully inactivated in both heterozygous and homozygous knockout rats, tumors from homozygous animals show very limited aneuploidy and low degrees of somatic copy number variation as compared to the tumors from heterozygous animals. In addition, complex structural rearrangements such as chromothripsis and breakage-fusion-bridge cycles were never found in tumors from homozygous animals, while these were readily detectable in tumors from heterozygous animals. Finally, we measured telomere length and telomere lengthening pathway activity and found that tumors of homozygous animals have longer telomeres but do not show clear telomerase or alternative lengthening of telomeres (ALT) activity differences as compared to the tumors from heterozygous animals. Taken together, our results demonstrate that host p53 status in this rat p53 knockout model has a large effect on both tumor type and genomic instability characteristics, where full loss of functional p53 is not the main driver of large-scale structural variations. Our results also suggest that chromothripsis primarily occurs under p53 heterozygous rather than p53 null conditions.     

Suppression of Genome Instability in pRB-Deficient Cells by Enhancement of Chromosome Cohesion

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A P53-Independent DNA Damage Response Suppresses Oncogenic Proliferation and Genome Instability

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