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Quantitative sensing and signalling of single-stranded DNA during the DNA damage response.

ABSTRACT: The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal - and therefore the cell's DNA damage load - is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection to induce quantitatively different ssDNA signals at a site-specific double strand break in budding yeast and identify two distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is unresponsive to increased amounts of ssDNA, while the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. The global checkpoint signal critically depends on the 9-1-1 and its downstream acting signalling axis, suggesting that ssDNA quantification depends on at least two sensor complexes.

SUBMITTER: Bantele SCS

PROVIDER: S-EPMC6391461 | biostudies-other | 2019 Feb

REPOSITORIES: biostudies-other

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Quantitative sensing and signalling of single-stranded DNA during the DNA damage response.

Bantele Susanne C S SCS Lisby Michael M Pfander Boris B

Nature communications 20190226 1

The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal - and therefore the cell's DNA damage load - is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection to induce quantitatively different ssDNA signals at a site-specific dou ...[more]

PMID: 30808869

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Project description:The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal – and therefore the cells’ DNA damage load – is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection at a site-specific DNA double-strand break (DSB) in budding yeast to generate quantitatively different DNA damage (ssDNA) signals. Interestingly, two major targets of the Mec1-Ddc2 kinase – Rad53 and γH2A – differ in their response to the ssDNA signal, indicating distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is non-quantitative and unresponsive to increased amounts of damage-associated Mec1-Ddc2 kinase. In contrast, the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. We find that not only Mec1-Ddc2 association, but also loading of the 9-1-1 co-sensor complex is enhanced during ongoing resection. Moreover, we can uncouple global checkpoint activation from the amount of Mec1-Ddc2 kinase at the lesion by using mutant conditions that hyper-activate the 9-1-1 signalling axis and at the same time show reduced amounts of damage-associated Mec1-Ddc2 kinase. We therefore propose that a key function of the 9-1-1 complex and the downstream checkpoint mediators is to generate a checkpoint response, which is quantitative and proportional to the cellular DNA damage load. The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal – and therefore the cells’ DNA damage load – is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection at a site-specific DNA double-strand break (DSB) in budding yeast to generate quantitatively different DNA damage (ssDNA) signals. Interestingly, two major targets of the Mec1-Ddc2 kinase – Rad53 and γH2A – differ in their response to the ssDNA signal, indicating distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is non-quantitative and unresponsive to increased amounts of damage-associated Mec1-Ddc2 kinase. In contrast, the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. We find that not only Mec1-Ddc2 association, but also loading of the 9-1-1 co-sensor complex is enhanced during ongoing resection. Moreover, we can uncouple global checkpoint activation from the amount of Mec1-Ddc2 kinase at the lesion by using mutant conditions that hyper-activate the 9-1-1 signalling axis and at the same time show reduced amounts of damage-associated Mec1-Ddc2 kinase. We therefore propose that a key function of the 9-1-1 complex and the downstream checkpoint mediators is to generate a checkpoint response, which is quantitative and proportional to the cellular DNA damage load.

Project description:UnlabelledNeisseria gonorrhoeae is naturally competent for transformation. The first step of the transformation process is the uptake of DNA from the environment into the cell. This transport step is driven by a powerful molecular machine. Here, we addressed the question whether this machine imports single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) at similar rates. The fluorescence signal associated with the uptake of short DNA fragments labeled with a single fluorescent marker molecule was quantified. We found that ssDNA with a double-stranded DNA uptake sequence (DUS) was taken up with a similar efficiency as dsDNA. Imported ssDNA was degraded rapidly, and the thermonuclease Nuc was required for degradation. In a nuc deletion background, dsDNA and ssDNA with a double-stranded DUS were imported and used as the substrates for transformation, whereas the import and transformation efficiencies of ssDNA with single-stranded DUS were below the detection limits. We conclude that the DNA uptake machine requires a double-stranded DUS for efficient DNA recognition and transports ssDNA and dsDNA with comparable efficiencies.ImportanceBacterial transformation enables bacteria to exchange genetic information. It can speed up adaptive evolution and enhances the potential of DNA repair. The transport of DNA through the outer membrane is the first step of transformation in Gram-negative species. It is driven by a powerful molecular machine whose mechanism remains elusive. Here, we show for Neisseria gonorrhoeae that the machine transports single- and double-stranded DNA at comparable rates, provided that the species-specific DNA uptake sequence is double stranded. Moreover, we found that single-stranded DNA taken up into the periplasm is rapidly degraded by the thermonuclease Nuc. We conclude that the secondary structure of transforming DNA is important for the recognition of self DNA but not for the process of transport through the outer membrane.

Dataset Information

Quantitative sensing and signalling of single-stranded DNA during the DNA damage response.

Publications

Quantitative sensing and signalling of single-stranded DNA during the DNA damage response.

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