scholarly journals Rec2 Interplay with both Brh2 and Rad51 Balances Recombinational Repair in Ustilago maydis

2006 ◽  
Vol 26 (2) ◽  
pp. 678-688 ◽  
Author(s):  
Milorad Kojic ◽  
Qingwen Zhou ◽  
Michael Lisby ◽  
William K. Holloman
Keyword(s):  
High Frequency ◽  
Ustilago Maydis ◽  
Radiation Sensitivity ◽  
Recombinational Repair ◽  
Focus Formation ◽  
Transgenic Expression ◽  
Rad51 Focus ◽  
Nuclear Focus ◽  
Extreme Degree ◽  
Radiation Induced

ABSTRACT Rec2 is the single Rad51 paralog in Ustilago maydis. Here, we find that Rec2 is required for radiation-induced Rad51 nuclear focus formation but that Rec2 foci form independently of Rad51 and Brh2. Brh2 foci also form in the absence of Rad51 and Rec2. By coprecipitation from cleared extracts prepared from Escherichia coli cells expressing the proteins, we found that Rec2 interacts physically not only with Rad51 and itself but also with Brh2. Transgenic expression of Brh2 in rec2 mutants can effectively restore radiation resistance, but the frequencies of spontaneous Rad51 focus formation and allelic recombination are elevated. The Dss1-independent Brh2-RPA70 fusion protein is also active in restoring radiation sensitivity of rec2 but is hyperactive to an extreme degree in allelic recombination and in suppressing the meiotic block of rec2. However, the high frequency of chromosome missegregation in meiotic products is an indicator of a corrupted process. The results demonstrate that the importance of Rec2 function is not only in stimulating recombination activity but also in ensuring that recombination is properly controlled.

scholarly journals Brh2-Dss1 Interplay Enables Properly Controlled Recombination in Ustilago maydis

2005 ◽  
Vol 25 (7) ◽  
pp. 2547-2557 ◽  
Author(s):  
Milorad Kojic ◽  
Qingwen Zhou ◽  
Michael Lisby ◽  
William K. Holloman
Keyword(s):  
Dna Damage ◽  
Fluorescent Protein ◽  
Ustilago Maydis ◽  
Binding Domain ◽  
Terminal Region ◽  
Recombinational Repair ◽  
Repair System ◽  
Focus Formation ◽  
Terminal Domain ◽  
Rad51 Focus

ABSTRACT Brh2, the BRCA2 homolog in Ustilago maydis, functions in recombinational repair of DNA damage by regulating Rad51 and is, in turn, regulated by Dss1. Dss1 is not required for Brh2 stability in vivo, nor for Brh2 to associate with Rad51, but is required for formation of green fluorescent protein (GFP)-Rad51 foci following DNA damage by gamma radiation. To understand more about the interplay between Brh2 and Dss1, we isolated mutant variants of Brh2 able to bypass the requirement for Dss1. These variants were found to lack the entire C-terminal DNA-Dss1 binding domain but to maintain the N-terminal region harboring the Rad51-interacting BRC element. GFP-Rad51 focus formation was nearly normal in brh2 mutant cells expressing a representative Brh2 variant with the C-terminal domain deleted. These findings suggest that the N-terminal region of Brh2 has an innate ability to organize Rad51. Survival after DNA damage was almost fully restored by a chimeric form of Brh2 having a DNA-binding domain from RPA70 fused to the Brh2 N-terminal domain, but Rad51 focus formation and mitotic recombination were elevated above wild-type levels. The results provide evidence for a mechanism in which Dss1 activates a Brh2-Rad51 complex and balances a finely regulated recombinational repair system.

scholarly journals The BRCA2-Interacting Protein BCCIP Functions in RAD51 and BRCA2 Focus Formation and Homologous Recombinational Repair

2005 ◽  
Vol 25 (5) ◽  
pp. 1949-1957 ◽  
Author(s):  
Huimei Lu ◽  
Xu Guo ◽  
Xiangbing Meng ◽  
Jingmei Liu ◽  
Chris Allen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
Focus Formation ◽  
Interacting Protein ◽  
Strand Breaks ◽  
Homologous Recombinational Repair ◽  
Radiation Induced ◽  
Nuclear Foci

ABSTRACT Homologous recombinational repair (HRR) of DNA damage is critical for maintaining genome stability and tumor suppression. RAD51 and BRCA2 colocalization in nuclear foci is a hallmark of HRR. BRCA2 has important roles in RAD51 focus formation and HRR of DNA double-strand breaks (DSBs). We previously reported that BCCIPα interacts with BRCA2. We show that a second isoform, BCCIPβ, also interacts with BRCA2 and that this interaction occurs in a region shared by BCCIPα and BCCIPβ. We further show that chromatin-bound BRCA2 colocalizes with BCCIP nuclear foci and that most radiation-induced RAD51 foci colocalize with BCCIP. Reducing BCCIPα by 90% or BCCIPβ by 50% by RNA interference markedly reduces RAD51 and BRCA2 foci and reduces HRR of DSBs by 20- to 100-fold. Similarly, reducing BRCA2 by 50% reduces RAD51 and BCCIP foci. These data indicate that BCCIP is critical for BRCA2- and RAD51-dependent responses to DNA damage and HRR.

BRCA1andBRCA2heterozygosity in embryonic stem cells reduces radiation-induced Rad51 focus formation but is not associated with radiosensitivity

2010 ◽  
Vol 86 (12) ◽  
pp. 1095-1105 ◽  
Author(s):  
George Sioftanos ◽  
Amani Ismail ◽  
Lisa Föhse ◽  
Susan Shanley ◽  
Mulugeta Worku ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Focus Formation ◽  
Rad51 Focus ◽  
Radiation Induced

scholarly journals Role for the Fission Yeast RecQ Helicase in DNA Repair in G2

2003 ◽  
Vol 23 (10) ◽  
pp. 3692-3705 ◽  
Author(s):  
Louise V. Laursen ◽  
Eleni Ampatzidou ◽  
Anni H. Andersen ◽  
Johanne M. Murray
Keyword(s):  
Dna Repair ◽  
Radiation Sensitivity ◽  
Recq Helicase ◽  
Focus Formation ◽  
High Molecular Weight Complex ◽  
Recq Helicases ◽  
Epistasis Analysis ◽  
Rad51 Focus ◽  
Human Genomic ◽  
Kinetics Of

ABSTRACT Members of the RecQ helicase subfamily are mutated in several human genomic instability syndromes, such as Bloom, Werner, and Rothmund-Thomson syndromes. We show that Rqh1, the single Schizosaccharomyces pombe homologue, is a 3′-to-5′ helicase and exists with Top3 in a high-molecular-weight complex. top3 deletion is inviable, and this is suppressed by concomitant loss of rqh1 helicase activity or loss of recombination functions. This is consistent with RecQ helicases in other systems. By using epistasis analysis of the UV radiation sensitivity and by analyzing the kinetics of Rhp51 (Rad51 homologue), Rqh1, and Top3 focus formation in response to UV in synchronized cells, we identify the first evidence of a function for Rqh1 and Top3 in the repair of UV-induced DNA damage in G2. Our data provide evidence that Rqh1 functions after Rad51 focus formation during DNA repair. We also identify a function for Rqh1 upstream of recombination in an Rhp18-dependent (Rad18 homologue) pathway. The model that these data allow us to propose helps to reconcile different interpretations of RecQ family helicase function that have arisen between work based on the S. pombe system and models based on studies of Saccharomyces cerevisiae SGS1 suggesting that RecQ helicases act before Rad51.

scholarly journals Distinct Functional Domains of Nibrin Mediate Mre11 Binding, Focus Formation, and Nuclear Localization

2001 ◽  
Vol 21 (6) ◽  
pp. 2184-2191 ◽  
Author(s):  
Ami Desai-Mehta ◽  
Karen M. Cerosaletti ◽  
Patrick Concannon
Keyword(s):  
Dna Repair ◽  
Nuclear Localization ◽  
Direct Interaction ◽  
Nijmegen Breakage Syndrome ◽  
Radiation Sensitivity ◽  
Focus Formation ◽  
Cellular Survival ◽  
Acid Fragment ◽  
Radiation Induced ◽  
Carboxy Terminal

ABSTRACT The inherited chromosomal instability disorder Nijmegen breakage syndrome (NBS) results from truncating mutations in theNBS1 gene, which encodes the protein nibrin. Nibrin is part of a nuclear multiprotein complex that also contains the DNA repair proteins Mre11 and Rad50. Upon irradiation, this complex redistributes within the nucleus, forming distinct foci that have been implicated as sites of DNA repair. In NBS cells, nibrin is absent and Mre11 and Rad50 are cytoplasmic. In this study, the interacting domains on nibrin and Mre11 were mapped using the yeast two-hybrid system and expression of epitope-tagged constructs in NBS fibroblasts. Deletion of the carboxy-terminal 101 amino acids of nibrin eliminated its ability to interact with Mre11 and to complement the radiation sensitivity of NBS cells. However, this truncated form of nibrin could localize to the nucleus and form radiation-inducible foci. Expression of a carboxy-terminal 354-amino-acid fragment of nibrin was sufficient to direct the nuclear localization of nibrin, as well as that of Mre11 and Rad50. Despite providing some partial complementation of the radiation-sensitive phenotype, the nibrin-Mre11-Rad50 complexes in these cells were unable to form foci. These results indicate that nibrin directs not only the nuclear localization of the nibrin-Mre11-Rad50 complexes but also radiation-induced focus formation. However, direct interaction between nibrin and Mre11 is required for normal cellular survival postirradiation. Distinct domains of nibrin are required for each of these functions, focus formation, nuclear localization, and Mre11 interaction.

Matrin3 promotes homologous recombinational repair by regulation of RAD51

2019 ◽  
Vol 166 (4) ◽  
pp. 343-351 ◽  
Author(s):  
Lin Shi ◽  
Jiying Sun ◽  
Aiko Kinomura ◽  
Atsuhiko Fukuto ◽  
Yasunori Horikoshi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Matrix Protein ◽  
Radiation Sensitivity ◽  
Recombinational Repair ◽  
Nuclear Matrix Protein ◽  
Focus Formation ◽  
Homologous Recombinational Repair ◽  
Multiple Stages

Abstract Matrin3 is a highly conserved inner nuclear matrix protein involved in multiple stages of RNA metabolism. Although Matrin3 may also play a role in DNA repair, its precise roles have remained unclear. In this study, we showed that the depletion of Matrin3 led to decreased homologous recombination (HR) efficiency and increased radiation sensitivity of cells. Matrin3-depleted cells showed impaired DNA damage-dependent focus formation of RAD51, a key protein in HR. These findings suggest that Matrin3 promotes HR by regulating RAD51.

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