Proteome analysis of protein partners to nucleosomes containing canonical H2A or the variant histones H2A.Z or H2A.X

2012 ◽  
Vol 393 (1-2) ◽  
pp. 47-61 ◽  
Author(s):  
Satoru Fujimoto ◽  
Corrine Seebart ◽  
Tiziana Guastafierro ◽  
Jessica Prenni ◽  
Paola Caiafa ◽  
...  
Keyword(s):  
Regulation Of Gene Expression ◽  
Proteome Analysis ◽  
Strand Break ◽  
Histone Variants ◽  
Histone H2a ◽  
Systematic Analysis ◽  
Cellular Processes ◽  
Nucleosome Structure ◽  
Protein Partners ◽  
Dna Double Strand Break

Abstract Although the existence of histone variants has been known for quite some time, only recently are we grasping the breadth and diversity of the cellular processes in which they are involved. Of particular interest are the two variants of histone H2A, H2A.Z and H2A.X because of their roles in regulation of gene expression and in DNA double-strand break repair, respectively. We hypothesize that nucleosomes containing these variants may perform their distinct functions by interacting with different sets of proteins. Here, we present our proteome analysis aimed at identifying protein partners that interact with nucleosomes containing H2A.Z, H2A.X or their canonical H2A counterpart. Our development of a nucleosome-pull down assay and analysis of the recovered nucleosome-interacting proteins by mass spectrometry allowed us to directly compare nuclear partners of these variant-containing nucleosomes to those containing canonical H2A. To our knowledge, our data represent the first systematic analysis of the H2A.Z and H2A.X interactome in the context of nucleosome structure.

Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

2020 ◽  
Author(s):  
Qinglei Hang ◽  
Liyong Zeng ◽  
Li Wang ◽  
Litong Nie ◽  
Fan Yao ◽  
...  
Keyword(s):  
Radiation Treatment ◽  
Critical Role ◽  
Strand Break ◽  
Histone H2a ◽  
Tumor Resistance ◽  
Dna Double Strand Breaks ◽  
Canonical Function ◽  
Dna Double Strand Break ◽  
Radiation Induced ◽  
Protein Recruitment

Abstract Cells respond to cytotoxic DNA double-strand breaks (DSBs) by recruiting repair proteins to the damaged sites. During the DNA damage response, ubiquitin signaling plays a critical role in coordinating protein recruitment. Here, we find that the microRNA biogenesis factor DGCR8 promotes tumor resistance to X-ray radiation independently of its Drosha-binding ability. In response to radiation, the deubiquitinase USP51 and the kinase ATM mediate the stabilization and activation of DGCR8 through deubiquitination and phosphorylation, respectively. While radiation-induced USP51 binds, deubiquitinates, and stabilizes DGCR8, ATM-dependent phosphorylation of DGCR8 at serine 677 leads to the recruitment of DGCR8 and DGCR8’s binding partner RNF168 to MDC1 and RNF8. This, in turn, promotes ubiquitination of histone H2A, repair of DSBs, and radioresistance. Altogether, these findings reveal the non-canonical function of DGCR8 in DSB repair and suggest that radiation treatment may result in therapy-induced tumor radioresistance through USP51- and ATM-mediated upregulation and activation of DGCR8.

scholarly journals Molecular architecture of the human tRNA ligase complex

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alena Kroupova ◽  
Fabian Ackle ◽  
Igor Asanović ◽  
Stefan Weitzer ◽  
Franziska M Boneberg ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Strand Break ◽  
Molecular Architecture ◽  
Rna Repair ◽  
Cellular Processes ◽  
Unfolded Protein ◽  
Terminal Domain ◽  
Structural Framework ◽  
Dna Double Strand Break ◽  
Protein Response

RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair and mRNA transport. Here we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex, and provide a structural framework for understanding its functions in cellular RNA metabolism.

scholarly journals Yeast ATM and ATR use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

2019 ◽  
Author(s):  
Kevin Li ◽  
Gabriel Bronk ◽  
Jane Kondev ◽  
James E. Haber
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Three Dimensions ◽  
One Dimension ◽  
Histone H2a ◽  
Directed Motion ◽  
Checkpoint Kinases ◽  
Dna Double Strand Break ◽  
Chromosome Iii ◽  
A Site

AbstractOne of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (γ-H2AX) around a DNA double-strand break (DSB). In the budding yeast S. cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATR or Tel1ATM checkpoint kinases. We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of γ-H2AX by ChIP-qPCR in order to uncover the mechanisms by which Mec1ATR and Tel1ATM propagate histone modifications across chromatin. With either kinase, γ-H2AX spreads as far as ∼50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans – at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a 3D diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.

scholarly journals Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

2020 ◽  
Vol 117 (35) ◽  
pp. 21354-21363 ◽  
Author(s):  
Kevin Li ◽  
Gabriel Bronk ◽  
Jane Kondev ◽  
James E. Haber
Keyword(s):  
Three Dimensional ◽  
Strand Break ◽  
Double Strand Break ◽  
Three Dimensions ◽  
Histone H2a ◽  
Directed Motion ◽  
Checkpoint Kinases ◽  
Dna Double Strand Break ◽  
Chromosome Iii ◽  
A Site

One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (γ-H2AX) around a DNA double-strand break (DSB). In the budding yeastSaccharomyces cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATRor Tel1ATMcheckpoint kinases. We induced a site-specific DSB with HO endonuclease at theMATlocus on chromosome III and monitored the formation of γ-H2AX by chromatin immunoprecipitation (ChIP)-qPCR in order to uncover the mechanisms by which Mec1ATRand Tel1ATMpropagate histone modifications across chromatin. With either kinase, γ-H2AX spreads as far as ∼50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin intrans—at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a three-dimensional diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.

scholarly journals Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

2014 ◽  
Vol 206 (7) ◽  
pp. 877-894 ◽  
Author(s):  
Olivia Barton ◽  
Steffen C. Naumann ◽  
Ronja Diemer-Biehs ◽  
Julia Künzel ◽  
Monika Steinlage ◽  
...  
Keyword(s):  
Large Scale ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Interacting Protein ◽  
Genomic Deletions ◽  
Cellular Processes ◽  
Dna Double Strand Break ◽  
Radiation Induced ◽  
Alternative Nhej

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.

The histone codes for meiosis

Reproduction ◽  
2017 ◽  
Vol 154 (3) ◽  
pp. R65-R79 ◽  
Author(s):  
Lina Wang ◽  
Zhiliang Xu ◽  
Muhammad Babar Khawar ◽  
Chao Liu ◽  
Wei Li
Keyword(s):  
Chromatin Remodeling ◽  
Strand Break ◽  
Future Trends ◽  
Functional Roles ◽  
Cellular Processes ◽  
Cell Divisions ◽  
Special Events ◽  
Dna Double Strand Break ◽  
Diploid Cells ◽  
Histone Codes

Meiosis is a specialized process that produces haploid gametes from diploid cells by a single round of DNA replication followed by two successive cell divisions. It contains many special events, such as programmed DNA double-strand break (DSB) formation, homologous recombination, crossover formation and resolution. These events are associated with dynamically regulated chromosomal structures, the dynamic transcriptional regulation and chromatin remodeling are mainly modulated by histone modifications, termed ‘histone codes’. The purpose of this review is to summarize the histone codes that are required for meiosis during spermatogenesis and oogenesis, involving meiosis resumption, meiotic asymmetric division and other cellular processes. We not only systematically review the functional roles of histone codes in meiosis but also discuss future trends and perspectives in this field.

scholarly journals Molecular architecture of the human tRNA ligase complex

2021 ◽  
Author(s):  
Alena Kroupova ◽  
Fabian Ackle ◽  
Franziska M Boneberg ◽  
Alessia Chui ◽  
Stefan Weitzer ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Strand Break ◽  
Molecular Architecture ◽  
Cellular Processes ◽  
Unfolded Protein ◽  
Terminal Domain ◽  
Structural Framework ◽  
Dna Double Strand Break ◽  
Protein Response ◽  
Intersubunit Interactions

RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair and mRNA transport. Here we present a biochemical analysis of the intersubunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex, and provide a structural framework for understanding its functions in cellular RNA metabolism.

scholarly journals RAD50 promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

2020 ◽  
Author(s):  
Ann-Kathrin Mehnert ◽  
Marco Prorocic ◽  
Annick Dujeancourt-Henry ◽  
Sebastian Hutchinson ◽  
Richard McCulloch ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Antigenic Variation ◽  
Strand Break ◽  
Surface Glycoprotein ◽  
Histone H2a ◽  
African Trypanosomes ◽  
Mrn Complex ◽  
Dna Double Strand Break ◽  
Damage Response

ABSTRACTHomologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 is required for RAD51-dependent homologous recombination, phosphorylation of histone H2A and controlled resection following a DNA double strand break (DSB). Perhaps surprisingly, DSB resection in the rad50 nulls was not impaired and appeared to peak earlier than in the parental strains. Finally, we show that RAD50 suppresses DNA repair using donors with short stretches of homology at a subtelomeric locus, with null strains producing a greater diversity of expressed VSG variants following DSB repair. We conclude that RAD50 promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation.

scholarly journals Competing interactions modulate the activity of Sgs1 during DNA end resection

2019 ◽  
Author(s):  
Kristina Kasaciunaite ◽  
Fergus Fettes ◽  
Maryna Levikova ◽  
Peter Daldrop ◽  
Petr Cejka ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Stability ◽  
Strand Break ◽  
Cellular Functions ◽  
Single Stranded Dna ◽  
Single Molecule Level ◽  
Protein Partners ◽  
Dna Double Strand Break ◽  
Dna End Resection ◽  
End Resection

AbstractDNA double-strand break repair by homologous recombination employs long-range resection of the 5’ DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay has been shown, it remains unclear whether and how the proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single molecule level and investigated its regulation by Dna2, the single-stranded DNA binding protein RPA and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection the proteins form a physical complex and couple their activities. Sgs1 unwinds DNA and feeds single-stranded DNA to Dna2 for degradation. RPA is found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We think that the differential regulation of the Sgs1 activity by its protein partners is important to allow diverse cellular functions of Sgs1 during the maintenance of genome stability.

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