scholarly journals A novel nuclear speckle factor, USP42, promotes homologous recombination repair by resolving DNA double-strand break induced R-loop

2019 ◽  
Author(s):  
Misaki Matsui ◽  
Ryo Sakasai ◽  
Masako Abe ◽  
Yusuke Kimura ◽  
Shoki Kajita ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
Nuclear Bodies ◽  
Nuclear Speckles ◽  
Dsb Repair ◽  
Nuclear Speckle ◽  
Spatial Regulation ◽  
Intrinsically Disordered ◽  
Dna Double Strand Break

AbstractThe nucleus of mammalian cells is highly compartmentalized by nuclear bodies, including nuclear speckles. While nuclear bodies are known to function in regulating gene expression, their involvement in DNA repair has not been actively investigated. Here, our focused screen for nuclear speckle factors involved in homologous recombination (HR), which is a faithful DNA double-strand break (DSB) repair mechanism, revealed that nuclear speckle factors regulating transcription are potentially involved in the regulation of HR. Among the top hits, we provide evidence showing that USP42, which is a deubiquitylating enzyme and a hitherto unidentified nuclear speckles factor, promotes HR by facilitating BRCA1 recruitment to DSB sites and DNA-end resection. We further showed that USP42 localizes to nuclear speckles via an intrinsically disordered region, which is required for efficient HR. Furthermore, we established that USP42 interacts with DHX9, which possesses DNA-RNA helicase activity, and is required for efficient resolution of DSB-induced R-loop. Mechanistically, USP42 antagonizes mono-ubiquitylation of DHX9 that is evoked after DSB induction. In conclusion, our data propose a model in which a novel nuclear speckle factor, USP42, facilitates DSB-induced R-loop resolution, BRCA1 loading to DSB sites and preferential DSB repair by HR, indicating the importance of spatial regulation of DSB repair choice mediated by nuclear bodies.Significant statementDefects in the repair of DNA double-strand break (DSB), which is one of the most harmful DNA insults, cause human diseases including cancers. It has been suggested that DSBs generated in the coding region tend to be repaired by homologous recombination (HR) that is error-free DSB repair pathway. To reveal the spatial regulation of HR, in this study, we investigated the potential contribution of nuclear bodies, especially nuclear speckles, to HR, identifying a deubiquitylating enzyme USP42 as a HR promoting factor. We found that USP42 deubiquitylates DHX9, facilitates resolution of DNA-RNA hybrid structure and enhances HR through BRCA1 loading to DSB sites.ClassificationBiological Sciences, Cell Biology

scholarly journals The deSUMOylase SENP2 coordinates homologous recombination and non-homologous end joining by independent mechanisms

2018 ◽  
Author(s):  
Alexander J. Garvin ◽  
Alexandra K. Walker ◽  
Ruth M. Densham ◽  
Anoop Singh Chauhan ◽  
Helen R. Stone ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Non Homologous End Joining

AbstractSUMOylation in the DNA double-strand break (DSB) response regulates recruitment, activity and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and non-homologous enjoining (NHEJ) through the investigation of the deSUMOylase SENP2. We find regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast we show HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 foci retention and increases NHEJ and radioresistance. Collectively our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

scholarly journals The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Anita Collavoli ◽  
Laura Comelli ◽  
Tiziana Cervelli ◽  
Alvaro Galli
Keyword(s):  
Homologous Recombination ◽  
Active Role ◽  
Strand Break ◽  
Double Strand Break ◽  
Human Cells ◽  
Proteasome Activity ◽  
Dsb Repair ◽  
Catalytic Subunits ◽  
Over Expression ◽  
Dna Double Strand Break

By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeastSaccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

scholarly journals β1-Integrin Impacts Rad51 Stability and DNA Double-Strand Break Repair by Homologous Recombination

2018 ◽  
Vol 38 (9) ◽  
Author(s):  
Kazi Mokim Ahmed ◽  
Raj K. Pandita ◽  
Dharmendra Kumar Singh ◽  
Clayton R. Hunt ◽  
Tej K. Pandita
Keyword(s):  
Breast Cancer ◽  
Homologous Recombination ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Strand Break ◽  
Double Strand Break ◽  
Β1 Integrin ◽  
Integrin Expression ◽  
Dsb Repair ◽  
Dna Double Strand Break

ABSTRACT The molecular mechanisms underlying resistance to radiotherapy in breast cancer cells remain elusive. Previously, we reported that elevated β1-integrin is associated with enhanced breast cancer cell survival postirradiation, but how β1-integrin conferred radioresistance was unclear. Ionizing radiation (IR) induced cell killing correlates with the efficiency of DNA double-strand break (DSB) repair, and we found that nonmalignant breast epithelial (S1) cells with low β1-integrin expression have a higher frequency of S-phase-specific IR-induced chromosomal aberrations than the derivative malignant breast (T4-2) cells with high β1-integrin expression. In addition, there was an increased frequency of IR-induced homologous recombination (HR) repairosome focus formation in T4-2 cells compared with that of S1 cells. Cellular levels of Rad51 in T4-2 cells, a critical factor in HR-mediated DSB repair, were significantly higher. Blocking or depleting β1-integrin activity in T4-2 cells reduced Rad51 levels, while ectopic expression of β1-integrin in S1 cells correspondingly increased Rad51 levels, suggesting that Rad51 is regulated by β1-integrin. The low level of Rad51 protein in S1 cells was found to be due to rapid degradation by the ubiquitin proteasome pathway (UPP). Furthermore, the E3 ubiquitin ligase RING1 was highly upregulated in S1 cells compared to T4-2 cells. Ectopic β1-integrin expression in S1 cells reduced RING1 levels and increased Rad51 accumulation. In contrast, β1-integrin depletion in T4-2 cells significantly increased RING1 protein levels and potentiated Rad51 ubiquitination. These data suggest for the first time that elevated levels of the extracellular matrix receptor β1-integrin can increase tumor cell radioresistance by decreasing Rad51 degradation through a RING1-mediated proteasomal pathway.

scholarly journals Formation of Large Palindromic DNA by Homologous Recombination of Short Inverted Repeat Sequences in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 161 (3) ◽  
pp. 1065-1075
Author(s):  
David K Butler ◽  
David Gillespie ◽  
Brandi Steele
Keyword(s):  
Homologous Recombination ◽  
Inverted Repeat ◽  
Excision Repair ◽  
Strand Break ◽  
Double Strand Break ◽  
Repeat Sequence ◽  
Inverted Repeat Sequence ◽  
Repeat Sequences ◽  
Dna Double Strand Break ◽  
Intermolecular Reaction

Abstract Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.

scholarly journals RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

2014 ◽  
Vol 35 (2) ◽  
pp. 406-416 ◽  
Author(s):  
Su Chen ◽  
Chen Wang ◽  
Luxi Sun ◽  
Da-Liang Wang ◽  
Lu Chen ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Open Chromatin ◽  
Dsb Repair ◽  
Heterochromatin Protein ◽  
Dna Double Strand Break ◽  
Recombination Repair ◽  
Ubiquitin Conjugating Enzyme

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients.

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

Genetics ◽  
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.

scholarly journals Human SIRT6 Promotes DNA End Resection Through CtIP Deacetylation

Science ◽  
2010 ◽  
Vol 329 (5997) ◽  
pp. 1348-1353 ◽  
Author(s):  
Abderrahmane Kaidi ◽  
Brian T. Weinert ◽  
Chunaram Choudhary ◽  
Stephen P. Jackson
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Protein A ◽  
Strand Break ◽  
Dsb Repair ◽  
Interacting Protein ◽  
Dna Double Strand Break ◽  
Dna End Resection ◽  
Lysine Deacetylases ◽  
End Resection

SIRT6 belongs to the sirtuin family of protein lysine deacetylases, which regulate aging and genome stability. We found that human SIRT6 has a role in promoting DNA end resection, a crucial step in DNA double-strand break (DSB) repair by homologous recombination. SIRT6 depletion impaired the accumulation of replication protein A and single-stranded DNA at DNA damage sites, reduced rates of homologous recombination, and sensitized cells to DSB-inducing agents. We identified the DSB resection protein CtIP [C-terminal binding protein (CtBP) interacting protein] as a SIRT6 interaction partner and showed that SIRT6-dependent CtIP deacetylation promotes resection. A nonacetylatable CtIP mutant alleviated the effect of SIRT6 depletion on resection, thus identifying CtIP as a key substrate by which SIRT6 facilitates DSB processing and homologous recombination. These findings further clarify how SIRT6 promotes genome stability.

scholarly journals The Roles of Bacterial DNA Double-Strand Break Repair Proteins in Chromosomal DNA Replication

2020 ◽  
Vol 44 (3) ◽  
pp. 351-368 ◽  
Author(s):  
Anurag Kumar Sinha ◽  
Christophe Possoz ◽  
David R F Leach
Keyword(s):  
Dna Replication ◽  
Cell Viability ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Initiation ◽  
Genome Replication ◽  
Chromosomal Dna ◽  
Dsb Repair ◽  
Bacterial Dna ◽  
Dna Double Strand Break

ABSTRACT It is well established that DNA double-strand break (DSB) repair is required to underpin chromosomal DNA replication. Because DNA replication forks are prone to breakage, faithful DSB repair and correct replication fork restart are critically important. Cells, where the proteins required for DSB repair are absent or altered, display characteristic disturbances to genome replication. In this review, we analyze how bacterial DNA replication is perturbed in DSB repair mutant strains and explore the consequences of these perturbations for bacterial chromosome segregation and cell viability. Importantly, we look at how DNA replication and DSB repair processes are implicated in the striking recent observations of DNA amplification and DNA loss in the chromosome terminus of various mutant Escherichia coli strains. We also address the mutant conditions required for the remarkable ability to copy the entire E. coli genome, and to maintain cell viability, even in the absence of replication initiation from oriC, the unique origin of DNA replication in wild type cells. Furthermore, we discuss the models that have been proposed to explain these phenomena and assess how these models fit with the observed data, provide new insights and enhance our understanding of chromosomal replication and termination in bacteria.

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