Recombination at collapsed replication forks: the payoff for survival

Recombination is believed to assist replication when forks collapse. By using an elegant system, Lambert et al. (2005) address the consequences of recombination at blocked forks. They show that chromosomal rearrangements are the price to pay for cell viability when forks collapse.     

The yeast kinome displays scale free topology with functional hub clusters

Background  

The availability of interaction databases provides an opportunity for researchers to utilize immense amounts of data exclusively in silico. Recently there has been an emphasis on studying the global properties of biological interactions using network analysis. While this type of analysis offers a wide variety of global insights it has surprisingly not been used to examine more localized interactions based on mechanism. In as such we have particular interest in the role of key topological components in signal transduction cascades as they are vital regulators of healthy and diseased cell states.     

To trim or not to trim: Progression and control of DSB end resection

DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage, since they can lead to genome instability and chromosome rearrangements, which are hallmarks of cancer cells. To face this kind of lesion, eukaryotic cells developed two alternative repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Repair pathway choice is influenced by the cell cycle phase and depends upon the 5′-3′ nucleolytic processing of the break ends, since the generation of ssDNA tails strongly stimulates HR and inhibits NHEJ. A large amount of work has elucidated the key components of the DSBs repair machinery and how this crucial process is finely regulated. The emerging view suggests that besides endo/exonucleases and helicases activities required for end resection, molecular barrier factors are specifically loaded in the proximity of the break, where they physically or functionally limit DNA degradation, preventing excessive accumulation of ssDNA, which could be threatening for cell survival.     

A likelihood method for the detection of selection and recombination using nucleotide sequences

Different regions along nucleotide sequences are often subject to different evolutionary forces. Recombination will result in regions having different evolutionary histories, while selection can cause regions to evolve at different rates. This paper presents a statistical method based on likelihood for detecting such processes by identifying the regions which do not fit with a single phylogenetic topology and nucleotide substitution process along the entire sequence. Subsequent reanalysis of these anomalous regions may then be possible. The method is tested using simulations, and its application is demonstrated using the primate psi eta-globin pseudogene, the V3 region of the envelope gene of HIV-1, and argF sequences from Neisseria bacteria. Reanalysis of anomalous regions is shown to reveal possible immune selection in HIV-1 and recombination in Neisseria. A computer program which implements the method is available.      

Variation in the spectrum of Fusarium species on winter wheat

In 1998–2000 a monitoring of the spectrum of Fusarium species on winter wheat was carried out in the Rhineland. The epidemic spread ofFusarium spp. on wheat plants during growing season was investigated as well as the grain contamination after harvest.F avenaceum was the Fusarium species isolated most frequently followed byF culmorum, F poae andF graminearum. Microdochium nivale occurred considerably only in 1998. Both, susceptibility and plant height of the cultivars were correlated to the incidence of Fusarium species /M nivale on harvested kernels; interactions with cropping intensities were detected. The incidence ofF poae seemed to be independent of the cultivar-specific Fusarium susceptibility. Despite the lack of disease symptoms, between growth stages 45–85 mycelium ofFusarium spp. was detectable in the leaves as well as conidia on the leaf surfaces.     

Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest

Saccharomyces cells with one unrepaired double-strand break (DSB) adapt after checkpoint-mediated G2/M arrest. Adaptation is accompanied by loss of Rad53p checkpoint kinase activity and Chk1p phosphorylation. Rad53p kinase remains elevated in yku70delta and cdc5-ad cells that fail to adapt. Permanent G2/M arrest in cells with increased single-stranded DNA is suppressed by the rfa1-t11 mutation, but this RPA mutation does not suppress permanent arrest in cdc5-ad cells. Checkpoint kinase activation and inactivation can be followed in G2-arrested cells, but there is no kinase activation in G1-arrested cells. We conclude that activation of the checkpoint kinases in response to a single DNA break is cell cycle regulated and that adaptation is an active process by which these kinases are inactivated.     

Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage.     

Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.