Complete genome sequence of a novel secovirid infecting cassava in the Americas

We report the complete genome sequence of a field isolate of a novel bipartite secovirid infecting cassava in Colombia, provisionally named "cassava torrado-like virus" (CsTLV). The genome sequence was obtained using Oxford Nanopore Technology, and the 5’ ends were confirmed by RACE. The RNA1 is 7252 nucleotides (nt) long, encoding a polyprotein of 2336 amino acids (aa) containing the typical “replication block”, conserved torradovirus motifs, and a Maf/Ham1 domain, which is not commonly found in viral genomes. The RNA2 is 4469 nt long and contains two overlapping ORFs encoding proteins of 226 and 1179 aa, showing the characteristic genome arrangement of members of the genus Torradovirus.

Multinucleation associated DNA damage blocks proliferation in p53-compromised cells

Nuclear atypia is one of the hallmarks of cancers. Here, we perform single-cell tracking studies to determine the immediate and long-term impact of nuclear atypia. Tracking the fate of newborn cells exhibiting nuclear atypia shows that multinucleation, unlike other forms of nuclear atypia, blocks proliferation in p53-compromised cells. Because ~50% of cancers display compromised p53, we explored how multinucleation blocks proliferation. Multinucleation increases 53BP1-decorated nuclear bodies (DNA damage repair platforms), along with a heterogeneous reduction in transcription and protein accumulation across the multi-nucleated compartments. Multinucleation Associated DNA Damage associated with 53BP1-bodies remains unresolved for days, despite an intact NHEJ machinery that repairs laser-induced DNA damage within minutes. Persistent DNA damage, a DNA replication block, and reduced phospho-Rb, reveal a novel replication stress independent cell cycle arrest caused by mitotic lesions. These findings call for segregating protective and prohibitive nuclear atypia to inform therapeutic approaches aimed at limiting tumour heterogeneity.

Single molecule dynamics at a bacterial replication fork after nutritional downshift

ABSTRACTReplication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and of lagging strand polymerase DnaE in the model bacterium Bacillus subtilis in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability, we show that proteins react differentially to the stress conditions. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after arrest of polymerase PolC, but binds much less often after fork blocks due to DNA damage or to nutritional downshift. These results indicate that binding of the alarmone ppGpp due to the stringent response prevents DnaG from binding to forks rather than blocking bound primase. Dissimilar behaviour of DnaG and of DnaE suggest that both proteins are recruited independently to the forks, rather than jointly. Turnover of all three proteins was increased during replication block after nutritional downshift, different from the situation due to DNA damage or polymerase inhibition, showing high plasticity of forks in response to different stress conditions. Forks persisted during all stress conditions, apparently ensuring rapid return to replication extension.

Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities

Homologous recombination (HR) is essential for faithful repair of DNA lesions yet must be kept in check, as unrestrained HR may compromise genome integrity and lead to premature aging or cancer. To limit unscheduled HR, cells possess DNA helicases capable of preventing excessive recombination. In this study, we show that the human Fbh1 (hFbh1) helicase accumulates at sites of DNA damage or replication stress in a manner dependent fully on its helicase activity and partially on its conserved F box. hFbh1 interacted with single-stranded DNA (ssDNA), the formation of which was required for hFbh1 recruitment to DNA lesions. Conversely, depletion of endogenous Fbh1 or ectopic expression of helicase-deficient hFbh1 attenuated ssDNA production after replication block. Although elevated levels of hFbh1 impaired Rad51 recruitment to ssDNA and suppressed HR, its small interfering RNA–mediated depletion increased the levels of chromatin-associated Rad51 and caused unscheduled sister chromatid exchange. Thus, by possessing both pro- and anti-recombinogenic potential, hFbh1 may cooperate with other DNA helicases in tightly controlling cellular HR activity.

EFFECT OF ALTITUDE AND WOUNDING ON BLOOD DISEASE PROGRESS OF PLANTAIN

Effect of Altitude and Wounding on Blood Disease Progress of Plantain. In the latest decade, the blood disease of banana has spread in almost all provinces in Indonesia and caused wilting of millions banana clusters in several provinces. It is very difficult to control the disease due to the base data about ecology and epidemiology of the pathogen are still poorly understood. This research aimed to evaluate the effect of wounding of inoculation site on blood disease progress of plantain. The experiment was arranged using randomized completely block design It was conducted at three locations with altitude of 100, 1000, and 1600 m above sea levels as replication block. The treatments were wounding, unwounding inoculation site, inoculation, and uninoculation of plantain cv. Kepok Kuning Wounding was applied by stabbing with an injection pin around the corm of 15 stabs/seedling. The seedlings were planted singly in one liter of non sterile soil in plastic bag. Each treatment consisted of 5 seedlings which was replicated 3 times. Inoculation was done by soil drenching of 20 ml bacterial suspension at concentration of 108 cfu/ml two week after planting. The pathogen used for inoculation originated from low land area (about 100 m above sea level). Observation was done weekly for 5 weeks. The variables observed were wilt intensity and area under disease progress (AUDPC). The results showed that blood disease was able to establish at altitude of 1600 m above sea level. The disease progress however was slower that those at 100 and 1000 m above sea level. On wounded seedling, the disease progress was more aggressive than those on unwounded one.

Autorepression of Rfx1 Gene Expression: Functional Conservation from Yeast to Humans in Response to DNA Replication Arrest

ABSTRACT The yeast Saccharomyces cerevisiae Crt1 transcription repressor is an effector of the DNA damage and replication checkpoint pathway. Crt1 binds and represses genes encoding ribonucleotide reductase (RNR) and its own promoter, establishing a negative-feedback pathway. The role of Rfx1, the mammalian Crt1 homologue, remained uncertain. In this study we investigated the possibility that Rfx1 plays a similar function in animal cells. We show here that, like Crt1, Rfx1 binds and represses its own promoter. Furthermore, Rfx1 binding to its promoter is reduced upon induction of a DNA replication block by hydroxyurea, which led to a release of repression. Significantly, like Crt1, Rfx1 binds and represses the RNR-R2 gene. Upon blocking replication and UV treatment, expression of both Rfx1 and RNR-R2 is induced; however, unlike the results seen with the RNR-R2 gene, the derepression of the RFX1 gene is only partially blocked by inhibiting Chk1, the DNA checkpoint kinase. This report provides evidence for a common mechanism for Crt1 and Rfx1 expression and for the conservation of their mode of action in response to a DNA replication block. We suggest that Rfx1 plays a role in the DNA damage response by down-regulating a subset of genes whose expression is increased in response to replication blocking and UV-induced DNA damage.

Requirement of the Mre11 Complex and Exonuclease 1 for Activation of the Mec1 Signaling Pathway

ABSTRACT The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.