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CDK targets Sae2 to control DNA-end resection and homologous recombination

DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template. By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs), with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cell-cycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Δ null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.

Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair

In G0 and G1, DNA double strand breaks are repaired by nonhomologous end joining, whereas in S and G2, they are also repaired by homologous recombination. The human CtIP protein controls double strand break (DSB) resection, an event that occurs effectively only in S/G2 and that promotes homologous recombination but not non-homologous end joining. Here, we mutate a highly conserved cyclin-dependent kinase (CDK) target motif in CtIP and reveal that mutating Thr-847 to Ala impairs resection, whereas mutating it to Glu to mimic constitutive phosphorylation does not. Moreover, we show that unlike cells expressing wild-type CtIP, cells expressing the Thr-to-Glu mutant resect DSBs even after CDK inhibition. Finally, we establish that Thr-847 mutations to either Ala or Glu affect DSB repair efficiency, cause hypersensitivity toward DSB-generating agents, and affect the frequency and nature of radiation-induced chromosomal rearrangements. These results suggest that CDK-mediated control of resection in human cells operates by mechanisms similar to those recently established in yeast.

DNA resection in eukaryotes: deciding how to fix the break

DNA double-strand breaks are repaired by different mechanisms, including homologous recombination and nonhomologous end-joining. DNA-end resection, the first step in recombination, is a key step that contributes to the choice of DSB repair. Resection, an evolutionarily conserved process that generates single-stranded DNA, is linked to checkpoint activation and is critical for survival. Failure to regulate and execute this process results in defective recombination and can contribute to human disease. Here I review recent findings on the mechanisms of resection in eukaryotes, from yeast to vertebrates, provide insights into the regulatory strategies that control it, and highlight the consequences of both its impairment and its deregulation.

The THP1-SAC3-SUS1-CDC31 Complex Works in Transcription Elongation-mRNA Export Preventing RNA-mediated Genome Instability

The eukaryotic THO/TREX complex, involved in mRNP biogenesis, plays a key role in the maintenance of genome integrity in yeast. mRNA export factors such as Thp1-Sac3 also affect genome integrity, but their mutations have other phenotypes different from those of THO/TREX. Sus1 is a novel component of SAGA transcription factor that also associates with Thp1-Sac3, but little is known about its effect on genome instability and transcription. Here we show that Thp1, Sac3, and Sus1 form a functional unit with a role in mRNP biogenesis and maintenance of genome integrity that is independent of SAGA. Importantly, the effects of ribozyme-containing transcription units, RNase H, and the action of human activation-induced cytidine deaminase on transcription and genome instability are consistent with the possibility that R-loops are formed in Thp1-Sac3-Sus1-Cdc31 as in THO mutants. Our data reveal that Thp1-Sac3-Sus1-Cdc31, together with THO/TREX, define a specific pathway connecting transcription elongation with export via an RNA-dependent dynamic process that provides a feedback mechanism for the control of transcription and the preservation of genetic integrity of transcribed DNA regions.

Mitotic recombination in Saccharomyces cerevisiae.

Abstract. Mitotic homologous recombination (HR) is an important mechanism for the repair of double-strand breaks and errors occurring during DNA replication. It is likely that the recombinational repair of DNA lesions occurs preferentially by sister chromatid exchanges that have no genetic consequences. However, most genetically detectable HR events occur between homologous DNA sequences located at allelic positions in homologous chromosomes, or between DNA repeats located at ectopic positions in either the same, homologous or heterologous chromosomes. Mitotic recombination may occur by multiple mechanisms, including double-strand break repair, synthesis-dependent strand annealing, break-induced replication and single-strand annealing. The occurrence of one recombination mechanism versus another depends on different elements, including the position of the homologous partner, the initiation event, the length of homology of the recombinant molecules and the genotype. The genetics and molecular biology of the yeast Saccharomyces cerevisiae have proved essential for the understanding of mitotic recombination mechanisms in eukaryotes. Here, we review recent genetic yeast data that contribute to our understanding of the different mechanisms of mitotic recombination and the in vivo role of the recombination proteins.

CtIP Mutations Cause Seckel and Jawad Syndromes.

Seckel syndrome is a recessively inherited dwarfism disorder characterized by microcephaly and a unique head profile. Genetically, it constitutes a heterogeneous condition, with several loci mapped (SCKL1-5) but only three disease genes identified: the ATR, CENPJ, and CEP152 genes that control cellular responses to DNA damage. We previously mapped a Seckel syndrome locus to chromosome 18p11.31-q11.2 (SCKL2). Here, we report two mutations in the CtIP (RBBP8) gene within this locus that result in expression of C-terminally truncated forms of CtIP. We propose that these mutations are the molecular cause of the disease observed in the previously described SCKL2 family and in an additional unrelated family diagnosed with a similar form of congenital microcephaly termed Jawad syndrome. While an exonic frameshift mutation was found in the Jawad family, the SCKL2 family carries a splicing mutation that yields a dominant-negative form of CtIP. Further characterization of cell lines derived from the SCKL2 family revealed defective DNA damage induced formation of single-stranded DNA, a critical co-factor for ATR activation. Accordingly, SCKL2 cells present a lowered apoptopic threshold and hypersensitivity to DNA damage. Notably, over-expression of a comparable truncated CtIP variant in non-Seckel cells recapitulates SCKL2 cellular phenotypes in a dose-dependent manner. This work thus identifies CtIP as a disease gene for Seckel and Jawad syndromes and defines a new type of genetic disease mechanism in which a dominant negative mutation yields a recessively inherited disorder.

An hpr1 Point Mutation That Impairs Transcription and mRNP Biogenesis without Increasing Recombination

ABSTRACT THO/TREX, a conserved eukaryotic protein complex, is a key player at the interface between transcription and mRNP metabolism. The lack of a functional THO complex impairs transcription, leads to transcription-dependent hyperrecombination, causes mRNA export defects and fast mRNA decay, and retards replication fork progression in a transcription-dependent manner. To get more insight into the interconnection between mRNP biogenesis and genomic instability, we searched for HPR1 mutations that differentially affect gene expression and recombination. We isolated mutants that were barely affected in gene expression but exhibited a hyperrecombination phenotype. In addition, we isolated a mutant, hpr1-101, with a strong defect in transcription, as observed for lacZ, and a general defect in mRNA export that did not display a relevant hyperrecombination phenotype. In THO single-null mutants, but not in the hpr1 point mutants studied, THO and its subunits were unstable. Interestingly, in contrast to hyperrecombinant null mutants, hpr1-101 did not cause retardation of replication fork progression. Transcription and mRNP biogenesis can therefore be impaired by THO/TREX dysfunction without increasing recombination, suggesting that it is possible to separate the mechanism(s) responsible for mRNA biogenesis defects from the further step of triggering transcription-dependent recombination.

Different physiological relevance of yeast THO/TREX subunits in gene expression and genome integrity

THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and plays a role in preventing the transcription-associated genetic instability. THO is composed of Tho2, Hpr1, Mft1 and Thp2 subunits, which associate with the Sub2-Yra1 export factors and Tex1 to form the TREX complex. To compare the functional relevance of the different THO/TREX subunits, we determined the effect of their null mutations on mRNA accumulation and recombination. Unexpectedly, we noticed that a full deletion of HPR1, hpr1ΔK, conferred stronger hyper-recombination phenotype and gene expression defects than did hpr1ΔH, the allele encoding a C-terminal truncated protein which was used in most previous studies. We show that tho2Δ and, to a lesser extent, hpr1ΔK are the THO mutations with the highest impact on all phenotypes, and that sub2Δ shows a similar transcription-dependent hyper-recombination phenotype and in vivo transcription impairment as hpr1ΔK and tho2Δ. Recombination and transcription analyses indicate that THO/TREX mutants share a moderate but significant effect on gene conversion and ectopic recombination, as well as transcription impairment of even short and low GC-content genes. Our data provide new information on the relevance of these proteins in mRNP biogenesis and in the maintenance of genomic integrity.

Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice

DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks.

New Tools to Study DNA Double-Strand Break Repair Pathway Choice

A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB) repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences.