scholarly journals S. cerevisiae Srs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates

2019 ◽  
Author(s):  
Laura J Hunt ◽  
Emad Ahmed ◽  
Hardeep Kaur ◽  
Jasvinder Ahuja ◽  
Lydia Hulme ◽  
...  
Keyword(s):  
Mitotic Cell ◽  
Dna Helicase ◽  
Ssdna Binding ◽  
Meiotic Progression ◽  
Intermediate Metabolism ◽  
Ssdna Binding Protein ◽  
Srs2 Helicase ◽  
Strand Invasion ◽  
Functional Dna

We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilizes Rad51-DNA filaments, and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalized with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity, and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards inter-homologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division.

scholarly journals The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Alex Bronstein ◽  
Lihi Gershon ◽  
Gilad Grinberg ◽  
Elisa Alonso-Perez ◽  
Martin Kupiec
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
Dna Helicase ◽  
Main Role ◽  
Helicase Domain ◽  
Helicase Activity ◽  
C Terminus ◽  
Srs2 Helicase

ABSTRACTHomologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. TheSaccharomyces cerevisiaeyeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2’s helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a newSRS2allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of thesrs2(1-850) allele suggest a role for Srs2’s helicase activity in sister chromatid cohesion. Our results also indicate that Srs2’s function becomes more central in diploid cells.IMPORTANCEHomologous recombination (HR) is a key mechanism that repairs damaged DNA. However, this process has to be tightly regulated; failure to regulate it can lead to genome instability. The Srs2 helicase is considered a regulator of HR; it was shown to be able to evict the recombinase Rad51 from DNA. Cells lacking Srs2 exhibit sensitivity to DNA-damaging agents, and in some cases, they display defects in DNA replication. The relative roles of the helicase and Rad51 removal activities of Srs2 in genome stability remain unclear. To address this question, we created a new Srs2 mutant which has only the DNA helicase domain. Our study shows that only the DNA helicase domain is needed to deal with DNA damage and assist in DNA replication during vegetative growth and in meiosis. Thus, our findings shift the view on the role of Srs2 in the maintenance of genome integrity.

scholarly journals Crystal Structure of INTS3/INTS6 complex reveals Functional Importance of INTS3 dimerization in DSB Repair

2020 ◽  
Author(s):  
Yu Jia ◽  
Zixiu Cheng ◽  
Sakshibeedu R Bharath ◽  
Qiangzu Sun ◽  
Nannan Su ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Critical Role ◽  
Repair Process ◽  
Dsb Repair ◽  
Ssdna Binding ◽  
C Terminus ◽  
Ssdna Binding Protein ◽  
Direct Binding ◽  
Biochemical Analyses

AbstractSOSS1 is a single-stranded DNA (ssDNA)-binding protein complex that plays a critical role in double-strand DNA break (DSB) repair. SOSS1 consists of three subunits: INTS3, SOSSC, and hSSB1 with INTS3 serving as a scaffold to stabilize this complex. Moreover, the integrator complex subunit 6 (INTS6) participates in the DNA damage response through direct binding to INTS3 but how INTS3 interacts with INTS6 thereby, impacting DBS repair is not clear. Here, we determined the crystal structure of the C-terminus of INTS3 (INTS3c) in complex with the C-terminus of INTS6 (INTS6c) at a resolution of 2.4 Å. Structure analysis revealed that two INTS3c subunits dimerize and interact with INTS6c via conserved residues. Subsequent biochemical analyses confirmed that INTS3c forms a stable dimer and INTS3 dimerization is important for recognizing the longer ssDNA. Perturbation of INTS3c dimerization and disruption of the INTS3c/INTS6c interaction, impair the DSB repair process. Altogether, these results unravel the underappreciated role of INTS3 dimerization and the molecular basis of INTS3/INTS6 interaction in DSB repair.

scholarly journals Crystal structure of the INTS3/INTS6 complex reveals the functional importance of INTS3 dimerization in DSB repair

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yu Jia ◽  
Zixiu Cheng ◽  
Sakshibeedu R. Bharath ◽  
Qiangzu Sun ◽  
Nannan Su ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Critical Role ◽  
Repair Process ◽  
Dsb Repair ◽  
Ssdna Binding ◽  
C Terminus ◽  
Ssdna Binding Protein ◽  
Direct Binding ◽  
Biochemical Analyses

AbstractSOSS1 is a single-stranded DNA (ssDNA)-binding protein complex that plays a critical role in double-strand DNA break (DSB) repair. SOSS1 consists of three subunits: INTS3, SOSSC, and hSSB1, with INTS3 serving as a scaffold to stabilize this complex. Moreover, the integrator complex subunit 6 (INTS6) participates in the DNA damage response through direct binding to INTS3, but how INTS3 interacts with INTS6, thereby impacting DSB repair, is not clear. Here, we determined the crystal structure of the C-terminus of INTS3 (INTS3c) in complex with the C-terminus of INTS6 (INTS6c) at a resolution of 2.4 Å. Structural analysis revealed that two INTS3c subunits dimerize and interact with INTS6c via conserved residues. Subsequent biochemical analyses confirmed that INTS3c forms a stable dimer and INTS3 dimerization is important for recognizing the longer ssDNA. Perturbation of INTS3c dimerization and disruption of the INTS3c/INTS6c interaction impair the DSB repair process. Altogether, these results unravel the underappreciated role of INTS3 dimerization and the molecular basis of INTS3/INTS6 interaction in DSB repair.

Role of Tyr-22 in the Binding of Pf3 ssDNA Binding Protein to Nucleic Acids

Biochemistry ◽  
1995 ◽  
Vol 34 (16) ◽  
pp. 5635-5643 ◽  
Author(s):  
Michael D. Powell ◽  
Donald M. Gray
Keyword(s):  
Nucleic Acids ◽  
Binding Protein ◽  
Ssdna Binding ◽  
Ssdna Binding Protein

Atm deficiency results in severe meiotic disruption as early as leptonema of prophase I

Development ◽  
1998 ◽  
Vol 125 (20) ◽  
pp. 4007-4017 ◽  
Author(s):  
C. Barlow ◽  
M. Liyanage ◽  
P.B. Moens ◽  
M. Tarsounas ◽  
K. Nagashima ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Mitotic Cell ◽  
Cycle Progression ◽  
Meiotic Progression ◽  
Prophase I ◽  
Human Disorder ◽  
Atm Protein ◽  
Mutant Cells ◽  
Deficient Mice

Infertility is a common feature of the human disorder ataxia-telangiectasia and Atm-deficient mice are completely infertile. To gain further insight into the role of ATM in meiosis, we examined meiotic cells in Atm-deficient mice during development. Spermatocyte degeneration begins between postnatal days 8 and 16.5, soon after entry into prophase I of meiosis, while oocytes degenerate late in embryogenesis prior to dictyate arrest. Using electron microscopy and immunolocalization of meiotic proteins in mutant adult spermatocytes, we found that male and female gametogenesis is severely disrupted in Atm-deficient mice as early as leptonema of prophase I, resulting in apoptotic degeneration. A small number of mutant cells progress into later stages of meiosis, but no cells proceed beyond prophase I. ATR, a protein related to ATM, DMC1, a RAD51 family member, and RAD51 are mislocalized to chromatin and have reduced localization to developing synaptonemal complexes in spermatocytes from Atm-deficient mice, suggesting dysregulation of the orderly progression of meiotic events. ATM protein is normally present at high levels primarily in ova cytoplasm of developing ovarian follicles, and in the nucleus of spermatogonia and to a lesser extent in spermatoctyes, but without localization to the synaptonemal complex. We propose a model in which ATM acts to monitor meiosis by participation in the regulation or surveillance of meiotic progression, similar to its role as a monitor of mitotic cell cycle progression.

scholarly journals Compartmentalization of the replication fork by single-stranded DNA binding protein regulates translesion synthesis

2020 ◽  
Author(s):  
Seungwoo Chang ◽  
Elizabeth S. Thrall ◽  
Luisa Laureti ◽  
Vincent Pagès ◽  
Joseph J. Loparo
Keyword(s):  
Dna Replication ◽  
Binding Protein ◽  
Translesion Synthesis ◽  
Genome Maintenance ◽  
Ssdna Binding ◽  
Epsilon Subunit ◽  
Pol Iv ◽  
Ssdna Binding Protein ◽  
Pol Iii

AbstractDNA replication is mediated by the coordinated actions of multiple enzymes within replisomes. Processivity clamps tether many of these enzymes to DNA, allowing access to the primer/template junction. Many clamp-interacting proteins (CLIPs) are involved in genome maintenance pathways including translesion synthesis (TLS). Despite their abundance, DNA replication in bacteria is not perturbed by these CLIPs. Here we show that while the TLS polymerase Pol IV is largely excluded from moving replisomes, the remodeling of ssDNA binding protein (SSB) upon replisome stalling enriches Pol IV at replication forks. This enrichment is indispensable for Pol IV-mediated TLS on both the leading and lagging strands as it enables Pol IV-processivity clamp binding by overcoming the gatekeeping role of the Pol III epsilon subunit. As we have demonstrated for the Pol IV-SSB interaction, we propose that the binding of CLIPs to the processivity clamp must be preceded by interactions with factors that serve as localization markers for their site of action.

scholarly journals Human replication protein A binds single-stranded DNA in two distinct complexes.

1994 ◽  
Vol 14 (6) ◽  
pp. 3993-4001 ◽  
Author(s):  
L J Blackwell ◽  
J A Borowiec
Keyword(s):  
Protein A ◽  
Dna Recombination ◽  
Replication Protein A ◽  
Replication Protein ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Eukaryotic Dna ◽  
Gel Shift

Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.

scholarly journals A New Epistasis Group for the Repair of DNA Damage in Bacteriophage T4: Replication Repair

Genetics ◽  
1987 ◽  
Vol 115 (3) ◽  
pp. 405-417
Author(s):  
Joseph T Wachsman ◽  
John W Drake
Keyword(s):  
Dna Replication ◽  
Excision Repair ◽  
Bacteriophage T4 ◽  
Repair Process ◽  
Dna Helicase ◽  
Host Cells ◽  
Repair System ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Gene 32

ABSTRACT The gene 32 mutation amA453 sensitizes bacteriophage T4 to the lethal effects of ultraviolet (UV) irradiation, methyl methanesulfonate and angelicin-mediated photodynamic irradiation when treated particles are plated on amber-suppressing host cells. The increased UV sensitivity caused by amA453 is additive to that caused by mutations in both the T4 excision repair (denV) and recombination repair (uvsWXY) systems, suggesting the operation of a third kind of repair system. The mutation uvs79, with many similarities to amA453 but mapping in gene 41, is largely epistatic to amA453. The mutation mms1, also with many similarities to amA453, maps close to amA453 within gene 32 and is largely epistatic to uvs79. Neither amA453 nor uvs79 affect the ratio of UV-induced mutational to lethal hits, nor does amA453 affect spontaneous or UV-enhanced recombination frequencies. Gene 32 encodes the major T4 ssDNA-binding protein (the scaffolding of DNA replication) and gene 41 encodes a DNA helicase, both being required for T4 DNA replication. We conclude that a third repair process operates in phage T4 and suggest that it acts during rather than before or after DNA replication.

scholarly journals Inactivation of EWS reduces PGC-1α protein stability and mitochondrial homeostasis

2015 ◽  
Vol 112 (19) ◽  
pp. 6074-6079 ◽  
Author(s):  
Jun Hong Park ◽  
Hong-Jun Kang ◽  
Yun Kyung Lee ◽  
Hyeog Kang ◽  
Jihyun Kim ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Protein Stability ◽  
Energy Homeostasis ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Ssdna Binding ◽  
Cellular Energy ◽  
Mitochondrial Homeostasis ◽  
Ssdna Binding Protein

EWS (Ewing sarcoma) encodes an RNA/ssDNA binding protein that is frequently rearranged in a number of different cancers by chromosomal translocations. Physiologically, EWS has diverse and essential roles in various organ development and cellular processes. In this study, we uncovered a new role of EWS in mitochondrial homeostasis and energy metabolism. Loss of EWS leads to a significant decrease in mitochondria abundance and activity, which is caused by a rapid degradation of Peroxisome proliferator-activated receptor γ Coactivator (PGC-1α), a central regulator of mitochondria biogenesis, function, and cellular energy metabolism. EWS inactivation leads to increased ubiquitination and proteolysis of PGC-1α via proteasome pathway. Complementation of EWS in Ews-deficient cells restores PGC-1α and mitochondrial abundance. We found that expression of E3 ubiquitin ligase, FBXW7 (F-box/WD40 domain protein 7), is increased in the absence of Ews and depletion of Fbxw7 in Ews-null cells restores PGC-1α expression and mitochondrial density. Consistent with these findings, mitochondrial abundance and activity are significantly reduced in brown fat and skeletal muscles of Ews-deficient mice. Furthermore, expression of mitochondrial biogenesis, respiration and fatty acid β-oxidation genes is significantly reduced in the liver of Ews-null mice. These results demonstrate a novel role of EWS in mitochondrial and cellular energy homeostasis by controlling PGC-1α protein stability, and further implicate altered mitochondrial and energy metabolism in cancers harboring the EWS translocation.

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