Functional and molecular insights into the role of Sae2 C-terminus in the activation of MRX endonuclease.

The yeast Sae2 protein, known as CtIP in mammals, once phosphorylated at Ser267, stimulates the endonuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex to cleave DNA ends that possess hairpin structures or protein blocks, such as the Spo11 transesterase or trapped topoisomerases. Stimulation of the Mre11 endonuclease by Sae2 depends on a Rad50-Sae2 interaction, but the mechanism by which this is achieved remains to be elucidated. Through genetic studies, we show that the absence of the last 23 amino acids from the Sae2 C-terminus specifically impairs MRX-dependent DNA cleavage events, while preserving the other Sae2 functions.

Exo1 cooperates with Tel1/ATM in promoting recombination events at DNA replication forks.

Tel1/ataxia telangiectasia mutated (ATM) kinase plays multiple functions in response to DNA damage, promoting checkpoint-mediated cell-cycle arrest and repair of broken DNA. In addition, Tel1 stabilizes replication forks that arrest upon the treatment with the topoisomerase poison camptothecin (CPT). We discover that inactivation of the Exo1 nuclease exacerbates the sensitivity of Tel1-deficient cells to CPT and other agents that hamper DNA replication.

The Chromatin Landscape around DNA Double-Strand Breaks in Yeast and Its Influence on DNA Repair Pathway Choice.

DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated.

To Fix or Not to Fix: Maintenance of Chromosome Ends Versus Repair of DNA Double-Strand Breaks.

Early work by Muller and McClintock discovered that the physical ends of linear chromosomes, named telomeres, possess an inherent ability to escape unwarranted fusions. Since then, extensive research has shown that this special feature relies on specialized proteins and structural properties that confer identity to the chromosome ends, thus allowing cells to distinguish them from intrachromosomal DNA double-strand breaks. Due to the inability of conventional DNA replication to fully replicate the chromosome ends and the downregulation of telomerase in most somatic human tissues, telomeres shorten as cells divide and lose this protective capacity.

The 9-1-1 Complex Controls Mre11 Nuclease and Checkpoint Activation during Short-Range Resection of DNA Double-Strand Breaks.

Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process in which an initial short-range step is catalyzed by the Mre11-Rad50-Xrs2 (MRX) complex and limited to the vicinity of the DSB end. Then the two long-range resection Exo1 and Dna2-Sgs1 nucleases extend the resected DNA tracts.

How do cells sense DNA lesions?

DNA is exposed to both endogenous and exogenous DNA damaging agents that chemically modify it. To counteract the deleterious effects exerted by DNA lesions, eukaryotic cells have evolved a network of cellular pathways, termed DNA damage response (DDR). The DDR comprises both mechanisms devoted to repair DNA lesions and signal transduction pathways that sense DNA damage and transduce this information to specific cellular targets.

Tel1/ATM Signaling to the Checkpoint Contributes to Replicative Senescence in the Absence of Telomerase.

Telomeres progressively shorten at every round of DNA replication in the absence of telomerase. When they become critically short, telomeres trigger replicative senescence by activating a DNA damage response that is governed by the Mec1/ATR and Tel1/ATM protein kinases. While Mec1/ATR is known to block cell division when extended single-stranded DNA (ssDNA) accumulates at eroded telomeres, the molecular mechanism by which Tel1/ATM promotes senescence is still unclear.

Processing of DNA Double-Strand Breaks by the MRX Complex in a Chromatin Context.

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that must be repaired to ensure genomic stability and avoid cell death. The cellular response to DSBs is initiated by the evolutionarily conserved Mre11-Rad50-Xrs2/NBS1 (MRX/MRN) complex that has structural and catalytic functions. Furthermore, it is responsible for DSB signaling through the activation of the checkpoint kinase Tel1/ATM.

Uncoupling Sae2 Functions in Downregulation of Tel1 and Rad53 Signaling Activities.

The Mre11-Rad50-Xrs2 (MRX) complex acts together with the Sae2 protein to initiate resection of DNA double-strand breaks (DSBs) and to regulate a checkpoint response that couples cell cycle progression with DSB repair. Sae2 supports resistance to DNA damage and downregulates the signaling activities of MRX, Tel1, and Rad53 checkpoint proteins at the sites of damage. How these functions are connected to each other is not known.

Processing of DNA Ends in the Maintenance of Genome Stability.

DNA double-strand breaks (DSBs) are particularly hazardous lesions as their inappropriate repair can result in chromosome rearrangements, an important driving force of tumorigenesis. DSBs can be repaired by end joining mechanisms or by homologous recombination (HR). HR requires the action of several nucleases that preferentially remove the 5'-terminated strands at both DSB ends in a process called DNA end resection.

Alkaline Denaturing Southern Blot Analysis to Monitor Double-Strand Break Processing.

Generation of 3' single-stranded DNA (ssDNA) tails at the ends of a double-strand break (DSB) is essential to repair the break through accurate homology-mediated repair pathways. Several methods have been developed to measure ssDNA accumulation at a DSB in the budding yeast Saccharomyces cerevisiae. Here, we describe one of these assays, which is based on the inability of restriction enzymes to cleave ssDNA.

The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1.

Eukaryotic cells preserve genome integrity upon DNA damage by activating a signaling network that promotes DNA repair and controls cell cycle progression. One of the most severe DNA damage is the DNA double-strand break (DSB), whose 5΄ ends can be nucleolitically resected by multiple nucleases to create 3΄-ended single-stranded DNA tails that trigger DSB repair by homologous recombination. Here, we identify the Saccharomyces cerevisiae RNA binding protein Npl3 as a new player in DSB resection.

Resection is responsible for loss of transcription around a double-strand break in Saccharomyces cerevisiae.

Emerging evidence indicate that the mammalian checkpoint kinase ATM induces transcriptional silencing in cis to DNA double-strand breaks (DSBs) through a poorly understood mechanism. Here we show that in Saccharomyces cerevisiae a single DSB causes transcriptional inhibition of proximal genes independently of Tel1/ATM and Mec1/ATR. Since the DSB ends undergo nucleolytic degradation (resection) of their 5'-ending strands, we investigated the contribution of resection in this DSB-induced transcriptional inhibition.