Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination

Abstract We examined the influence of the tetratricopeptide repeat factor XAB2 on chromosomal break repair, and found that XAB2 promotes end resection that generates the 3′ ssDNA intermediate for homologous recombination (HR). Namely, XAB2 is important for chromosomal double-strand break (DSB) repair via two pathways of HR that require end resection as an intermediate step, end resection of camptothecin (Cpt)-induced DNA damage, and RAD51 recruitment to ionizing radiation induced foci (IRIF), which requires end resection. Furthermore, XAB2 mediates specific aspects of the DNA damage response associated with end resection proficiency: CtIP hyperphosphorylation induced by Cpt and BRCA1 IRIF. XAB2 also promotes histone acetylation events linked to HR proficiency. From truncation mutation analysis, the capacity for XAB2 to promote HR correlates with its ability to form a complex with ISY1 and PRP19, which show a similar influence as XAB2 on HR. This XAB2 complex localizes to punctate structures consistent with interchromatin granules that show a striking adjacent-localization to the DSB marker γH2AX. In summary, we suggest that the XAB2 complex mediates DNA damage response events important for the end resection step of HR, and speculate that its adjacent-localization relative to DSBs marked by γH2AX is important for this function.

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Schizosaccharomyces pombe KAT5 contributes to resection and repair of a DNA double strand break

Genetics ◽

10.1093/genetics/iyab042 ◽

2021 ◽

Author(s):

Tingting Li ◽

Ruben C Petreaca ◽

Susan L Forsburg

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Chromatin Remodeling ◽

Dna Damage Response ◽

Histone Acetyltransferase ◽

Strand Break ◽

Double Strand Break ◽

Dna Damage Checkpoint ◽

Dna Double Strand Break ◽

Damage Response

Abstract Chromatin remodeling is essential for effective repair of a DNA double strand break. KAT5 (S. pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that coordinates various DNA damage response activities at a DNA double strand break (DSB), including histone remodeling and activation of the DNA damage checkpoint. In S. pombe, mutations in mst1+ causes sensitivity to DNA damaging drugs. Here we show that Mst1 is recruited to DSBs. Mutation of mst1+ disrupts recruitment of repair proteins and delays resection. These defects are partially rescued by deletion of pku70, which has been previously shown to antagonize repair by homologous recombination. These phenotypes of mst1 are similar to pht1-4KR, a non-acetylatable form of histone variant H2A.Z, which has been proposed to affect resection. Our data suggest that Mst1 functions to direct repair of DSBs towards homologous recombination pathways by modulating resection at the double strand break.

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The DNA damage response regulates the oocyte pool in mammals

10.1101/816488 ◽

2019 ◽

Author(s):

Ana Martínez-Marchal ◽

Maria Teresa Guillot ◽

Mònica Ferrer ◽

Anna Guixé ◽

Montserrat Garcia-Caldés ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Response ◽

Adult Life ◽

Strand Break ◽

Oocyte Number ◽

Genome Wide ◽

Germ Cell Cysts ◽

Damage Response

SummaryMammalian oogonia proliferate without completing cytokinesis producing germ cell cysts. Within these cysts, oocytes differentiate and enter meiosis, promote genome-wide double-strand break (DSBs) formation which repair by homologous recombination leads to synapsis of the homologous chromosomes. Errors in homologous recombination or synapsis trigger the activation of surveillance mechanisms, traditionally called ‘pachytene checkpoint’, to either repair them or send the cells to programmed death. Contrary to what is found in spermatocytes, most oocytes present a remarkable persistence of unrepaired DSBs at pachynema. Simultaneously, there is a massive oocyte death accompanying the oocyte cyst breakdown. This oocyte elimination is thought to be required to properly form the follicles, which constitute the pool of germ cells females will use during their adult life. Based on all the above mentioned, we hypothesized that the apparently inefficient meiotic recombination occurring in mouse oocytes may be required to eliminate most of the oocytes in order to regulate the oocyte number, promote cyst breakdown and follicle formation in mammalian females. To test this idea, we analyzed perinatal ovaries to evaluate the oocyte population, cyst breakdown and follicle formation in control and mutant mice for the effector kinase of the DNA damage response, CHK2. Our results confirm the involvement of CHK2 in the elimination of oocytes that accumulate unrepaired DSBs and show that CHK2 regulates the number of oocytes in fetal ovaries. We also show that CHK2 is required to eliminate oocytes as a result of LINE-1 activation, which was previously shown to be responsible for fetal oocyte loss. Nonetheless, the number of oocytes found in Chk2 mutant ovaries three days after birth was similar to that of control ovaries, suggesting the existence of CHK2-independent mechanisms capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2 mutant ovaries suggesting that CHK1 regulates postnatal oocyte death. Moreover, both CHK1 and CHK2 functions are required to timely breakdown cyst and form follicles. Altogether, we propose the DNA damage response controls the number of oocytes present perinatally and is required to properly break down oocyte cysts and form follicles, highlighting the importance of the DNA damage response in setting the reserve of oocytes each female will use during their entire lifespan.

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The DNA damage response is required for oocyte cyst breakdown and follicle formation in mice

PLoS Genetics ◽

10.1371/journal.pgen.1009067 ◽

2020 ◽

Vol 16 (11) ◽

pp. e1009067

Author(s):

Ana Martínez-Marchal ◽

Yan Huang ◽

Maria Teresa Guillot-Ferriols ◽

Mònica Ferrer-Roda ◽

Anna Guixé ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Response ◽

Strand Break ◽

Double Strand Break ◽

Mutant Mice ◽

Oocyte Number ◽

Prophase I ◽

Damage Response

Mammalian oogonia proliferate without completing cytokinesis, forming cysts. Within these, oocytes differentiate and initiate meiosis, promoting double-strand break (DSBs) formation, which are repaired by homologous recombination (HR) causing the pairing and synapsis of the homologs. Errors in these processes activate checkpoint mechanisms, leading to apoptosis. At the end of prophase I, in contrast with what is observed in spermatocytes, oocytes accumulate unrepaired DSBs. Simultaneously to the cyst breakdown, there is a massive oocyte death, which has been proposed to be necessary to enable the individualization of the oocytes to form follicles. Based upon all the above-mentioned information, we hypothesize that the apparently inefficient HR occurring in the oocytes may be a requirement to first eliminate most of the oocytes and enable cyst breakdown and follicle formation. To test this idea, we compared perinatal ovaries from control and mutant mice for the effector kinase of the DNA Damage Response (DDR), CHK2. We found that CHK2 is required to eliminate ~50% of the fetal oocyte population. Nevertheless, the number of oocytes and follicles found in Chk2-mutant ovaries three days after birth was equivalent to that of the controls. These data revealed the existence of another mechanism capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2-/- mice, implying that CHK1 regulates postnatal oocyte death. Moreover, we found that CHK1 and CHK2 functions are required for the timely breakdown of the cyst and to form follicles. Thus, we uncovered a novel CHK1 function in regulating the oocyte population in mice. Based upon these data, we propose that the CHK1- and CHK2-dependent DDR controls the number of oocytes and is required to properly break down oocyte cysts and form follicles in mammals.

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PP4 phosphatase cooperates in recombinational DNA repair by enhancing double-strand break end resection

10.1101/470427 ◽

2018 ◽

Author(s):

María Teresa Villoria ◽

Pilar Gutiérrez-Escribano ◽

Facundo Ramos ◽

Esmeralda Alonso-Rodríguez ◽

Eva Merino ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Kinase Activity ◽

Strand Break ◽

Double Strand Break ◽

Dna Lesion ◽

Damage Response ◽

End Resection ◽

Recombinational Dna Repair

AbstractThe role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by the Mec1 kinase and its own auto-phosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 phosphorylation and activation has been well documented, less is known about the processes that counteract its kinase activity during the response to DNA damage. Here, we describe that PP4 phosphatase dephosphorylates Rad53 during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects DNA resection and repair by single-strand annealing, defects that are bypassed by reducing the hyper-phosphorylation state of Rad53 observed in the absence of the phosphatase. These results confirm that Rad53 is one of the main targets of PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection.

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Schizosaccharomyces pombe KAT5 contributes to resection and repair of a DNA double strand break

10.1101/2020.06.03.132316 ◽

2020 ◽

Author(s):

Tingting Li ◽

Ruben C. Petreaca ◽

Susan L. Forsburg

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Chromatin Remodeling ◽

Dna Damage Response ◽

Histone Acetyltransferase ◽

Strand Break ◽

Double Strand Break ◽

Dna Damage Checkpoint ◽

Dna Double Strand Break ◽

Damage Response

AbstractChromatin remodeling is essential for effective repair of a DNA double strand break. KAT5 (S. pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that coordinates various DNA damage response activities at a DSB, including histone remodeling and activation of the DNA damage checkpoint. In S. pombe, mutations in mst1+ causes sensitivity to DNA damaging drugs. Here we show that Mst1 is recruited to DSBs. Mutation of mst1+ disrupts recruitment of repair proteins and delays resection. These defects are partially rescued by deletion of pku70, which has been previously shown to antagonize repair by homologous recombination. These phenotypes of mst1 are similar to pht1-4KR, a non-acetylatable form of histone variant H2A.Z, which has been proposed to affect resection. These data suggest that Mst1 functions to direct repair of DSBs towards homologous recombination pathways by modulating resection at the double strand break.

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PP4 phosphatase cooperates in recombinational DNA repair by enhancing double-strand break end resection

Nucleic Acids Research ◽

10.1093/nar/gkz794 ◽

2019 ◽

Vol 47 (20) ◽

pp. 10706-10727 ◽

Cited By ~ 2

Author(s):

María Teresa Villoria ◽

Pilar Gutiérrez-Escribano ◽

Esmeralda Alonso-Rodríguez ◽

Facundo Ramos ◽

Eva Merino ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Repair Process ◽

Strand Break ◽

Double Strand Break ◽

Dna Lesion ◽

Damage Response ◽

End Resection ◽

Recombinational Dna Repair

Abstract The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by Mec1 and its own autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 activation has been well documented, less is known about the processes that counteract its activity along the repair of a DNA adduct. Here, we describe that PP4 phosphatase is required to avoid Rad53 hyper-phosphorylation during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects resection and repair by single-strand annealing, defects that are bypassed by reducing Rad53 hyperphosphorylation. These results confirm that Rad53 phosphorylation is controlled by PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection for their success.

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Sites of Acetylation on Newly Synthesized Histone H4 Are Required for Chromatin Assembly and DNA Damage Response Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.00460-13 ◽

2013 ◽

Vol 33 (16) ◽

pp. 3286-3298 ◽

Cited By ~ 19

Author(s):

Zhongqi Ge ◽

Devi Nair ◽

Xiaoyan Guan ◽

Neha Rastogi ◽

Michael A. Freitas ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Histone H3 ◽

Model Organism ◽

Strand Break ◽

Chromatin Assembly ◽

Histone H4 ◽

Histone H4 Acetylation ◽

Damage Response ◽

A Site

The best-characterized acetylation of newly synthesized histone H4 is the diacetylation of the NH2-terminal tail on lysines 5 and 12. Despite its evolutionary conservation, this pattern of modification has not been shown to be essential for either viability or chromatin assembly in any model organism. We demonstrate that mutations in histone H4 lysines 5 and 12 in yeast confer hypersensitivity to replication stress and DNA-damaging agents when combined with mutations in histone H4 lysine 91, which has also been found to be a site of acetylation on soluble histone H4. In addition, these mutations confer a dramatic decrease in cell viability when combined with mutations in histone H3 lysine 56. We also show that mutation of the sites of acetylation on newly synthesized histone H4 results in defects in the reassembly of chromatin structure that accompanies the repair of HO-mediated double-strand breaks. This defect is not due to a decrease in the level of histone H3 lysine 56 acetylation. Intriguingly, mutations that alter the sites of newly synthesized histone H4 acetylation display a marked decrease in levels of phosphorylated H2A (γ-H2AX) in chromatin surrounding the double-strand break. These results indicate that the sites of acetylation on newly synthesized histones H3 and H4 can function in nonoverlapping ways that are required for chromatin assembly, viability, and DNA damage response signaling.

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Association of Homologous Recombination-DNA Damage Response Gene Mutations with Immune Biomarkers in Gastroesophageal Cancers

Molecular Cancer Therapeutics ◽

10.1158/1535-7163.mct-20-0879 ◽

2021 ◽

pp. molcanther.0879.2020

Author(s):

Michael Cerniglia ◽

Joanne Xiu ◽

Axel Grothey ◽

Michael J Pishvaian ◽

Yasmine Baca ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Response ◽

Gene Mutations ◽

Response Gene ◽

Immune Biomarkers ◽

Damage Response

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Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816539115 ◽

2018 ◽

Vol 115 (51) ◽

pp. E11961-E11969 ◽

Cited By ~ 14

Author(s):

Tai-Yuan Yu ◽

Michael T. Kimble ◽

Lorraine S. Symington

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Inhibitory Effects ◽

Checkpoint Signaling ◽

Synthetic Lethal ◽

Strand Breaks ◽

Damage Response ◽

End Resection

The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5′–3′ resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5′ strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1–mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.

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