共查询到20条相似文献,搜索用时 15 毫秒
1.
Koshiyama A Hamada FN Namekawa SH Iwabata K Sugawara H Sakamoto A Ishizaki T Sakaguchi K 《The FEBS journal》2006,273(17):4003-4012
Sumoylation is a post-translational modification system that covalently attaches the small ubiquitin-related modifier (SUMO) to target proteins. Ubc9 is required as the E2-type enzyme for SUMO-1 conjugation to targets. Here, we show that Ubc9 interacts with the meiosis-specific RecA homolog, Lim15/Dmc1 in the basidiomycete Coprinus cinereus (CcLim15), and mediates sumoylation of CcLim15 during meiosis. In vitro protein-protein interaction assays revealed that CcUbc9 interacts with CcLim15 and binds to the C-terminus (amino acids 105-347) of CcLim15, which includes the ATPase domain. Immunocytochemistry demonstrates that CcUbc9 and CcLim15 colocalize in the nuclei from the leptotene stage to the early pachytene stage during meiotic prophase I. Coimmunoprecipitation experiments indicate that CcUbc9 interacts with CcLim15 in vivo during meiotic prophase I. Furthermore, we show that CcLim15 is a target protein of sumoylation both in vivo and in vitro, and identify the C-terminus (amino acids 105-347) of CcLim15 as the site of sumoylation in vitro. These results suggest that sumoylation is a candidate modulator of meiotic recombination via interaction between Ubc9 and Lim15/Dmc1. 相似文献
2.
The Escherichia coli RecA protein pairs homologous DNA molecules and promotes DNA strand exchange in vitro. We have examined DNA strand exchange between a 70 nucleotide ssDNA fragment and a 40 bp duplex, in which all G and C residues (at 18 positions distributed throughout the 40 bp exchanged region) were replaced with the nonstandard nucleosides 2'-deoxyisoguanosine (iG) and 2'-deoxy-5-methylisocytidine (MiC), respectively. We demonstrate that the nonstandard oligonucleotides are substrates for the RecA protein, permitting DNA strand exchange in vitro at a rate and efficiency comparable to exchange with normal DNA substrates. This observation provides an expanded experimental basis for discussions of potential roles for iG and MiC in a genetic code. Experiments of this type also provide another avenue for exploring RecA-facilitated DNA pairing mechanisms. 相似文献
3.
Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex 总被引:3,自引:0,他引:3
Saccharomyces cerevisiae Dmc1, a meiosis-specific homologue of RecA, catalyzes homologous pairing and strand exchange during meiotic DNA recombination. The purified budding yeast Dmc1 (ScDmc1) protein exhibits much weaker recombinase activity in vitro as compared to that of the Escherichia coli RecA protein. Using atomic force microscopy (AFM) with carbon nanotube tips, we found ScDmc1 forms rings with an external diameter of 18 nm and a central cavity of 4 nm. In the presence of single-stranded DNA (ssDNA), the majority of the ScDmc1 protein (90%) bound DNA as protein rings; only a small faction (10%) was able to form filamentous structure. In contrast, nearly all RecA proteins form fine helical nucleoprotein filaments with ssDNA under identical conditions. RecA-mediated recombinase activity is initiated through the nucleation of RecA onto ssDNA to form helical nucleoprotein filaments. Our results support the notion that ScDmc1 becomes catalytically active only when it forms a helical nucleoprotein filament with ssDNA. 相似文献
4.
Susan R. Ferrari Jennifer Grubb Douglas K. Bishop 《The Journal of biological chemistry》2009,284(18):11766-11770
During homologous recombination, a number of proteins cooperate to catalyze
the loading of recombinases onto single-stranded DNA. Single-stranded
DNA-binding proteins stimulate recombination by coating single-stranded DNA
and keeping it free of secondary structure; however, in order for recombinases
to load on single-stranded-DNA-binding protein-coated DNA, the activity of a
class of proteins known as recombination mediators is required. Mediator
proteins coordinate the handoff of single-stranded DNA from single-stranded
DNA-binding protein to recombinase. Here we show that a complex of Mei5 and
Sae3 from Saccharomyces cerevisiae preferentially binds
single-stranded DNA and relieves the inhibition of the strand assimilation and
DNA binding abilities of the meiotic recombinase Dmc1 imposed by the
single-stranded DNA-binding protein replication protein A. Additionally, we
demonstrate the physical interaction of Mei5-Sae3 with replication protein A.
Our results, together with previous in vivo studies, indicate that
Mei5-Sae3 is a mediator of Dmc1 assembly during meiotic recombination in
S. cerevisiae.During meiosis, recombination between homologous chromosomes ensures proper
segregation into haploid products. Recombination events are initiated by the
formation of double strand breaks
(DSBs)2 in DNA
(1). This is followed by
resection of free DNA ends to yield 3′ single-stranded tails, upon which
recombinase assembles to form nucleoprotein filaments. Following recombinase
assembly, the nucleoprotein filament engages a donor chromatid, searches for
homologous DNA sequences on that chromatid, and promotes strand exchange to
yield a heteroduplex DNA intermediate often referred to as a joint molecule.
Although recombinase alone is capable of promoting homology search and strand
exchange in vitro, genetic and biochemical studies have demonstrated
that normal recombinase function in vivo requires the activity of a
number of accessory factors
(2). These factors enhance the
assembly of nucleoprotein filaments, target capture, homology search, and
dissociation of recombinase from duplex DNA.Most eukaryotes possess two recombinases, both homologues of the
Escherichia coli recombinase RecA: Rad51, which is the major
recombinase in mitotic cells and is also important during meiotic
recombination, and Dmc1, which functions only in meiosis. Dmc1 and Rad51 have
been shown to assemble at DSBs by immunofluorescence and chromatin
immunoprecipitation
(3–6),
and both proteins oligomerize on single-stranded DNA (ssDNA) to form
nucleofilaments that catalyze strand invasion
(7–9).A number of biochemical studies have defined the role of accessory factors
in stimulating the activity of Rad51
(10–12).
Replication protein A (RPA), the yeast ssDNA-binding protein (SSB), removes
secondary structure in ssDNA that otherwise prevents formation of fully
functional nucleoprotein filaments
(13). Both Rad52 protein
(11,
12) and the heterodimeric
protein Rad55/Rad57 (14) can
overcome the inhibitory effect of RPA on Rad51 nucleoprotein filament
formation in purified systems, mediating a handoff between RPA and Rad51. It
is thought that the mechanism for the mediator activity of Rad52 involves
Rad52 recognizing and binding to RPA-coated ssDNA, where it provides
nucleation sites for the recruitment of free molecules of Rad51
(15). The tumor suppressor
protein BRCA2 also serves as an assembly factor for Rad51 during mitosis in a
variety of species that encode orthologues of this protein, including mice
(16), corn smut
(17), and humans
(18).The meiosis-specific recombinase Dmc1 is stimulated by a distinct set of
accessory factors. Immunostaining studies suggest that the Rad51 mediators
Rad52 and Rad55/Rad57 are not required for assembly of Dmc1 foci in
vivo, although Rad51 itself promotes Dmc1 foci
(19–21).
More recently, immunostaining and chromatin immunoprecipitation experiments
demonstrated a role for the Mei5 and Sae3 proteins of Saccharomyces
cerevisiae in assembly of Dmc1 at sites of DSBs in vivo
(22,
23). Consistent with these
observations, mei5 and sae3 mutants display markedly similar
meiotic defects as compared with dmc1 mutants, including defects in
sporulation, spore viability, crossing over, DSB repair, progression through
meiosis, and synaptonemal complex formation
(19,
22–24).
Finally, the three proteins have been shown to physically interact; Mei5 and
Sae3 have been co-purified and co-immunoprecipitated, and an N-terminal
portion of Mei5 has been shown to interact with Dmc1 in a two-hybrid assay
(22).The fission yeast Schizosaccharomyces pombe encodes two proteins,
Swi5 and Sfr1, which share sequence homology with Sae3 and Mei5, respectively
(22). Swi5 and Sfr1 have been
shown to stimulate the strand exchange activity of Rhp51 (the S.
pombe Rad51 homologue) and Dmc1
(25). Although some results
indicate functional similarity of Swi5-Sfr1 and Mei5-Sae3, there are also
clear differences. The Mei5-Sae3 complex of budding yeast is expressed solely
during meiosis, and no mitotic phenotypes have been reported for mei5
or sae3 mutants (22,
24,
26). In contrast, the
Swi5-Sfr1 complex of fission yeast is expressed in mitotic and meiotic cells,
and mutations in SWI5 have been shown to cause defects in mitotic
recombination (27).
Furthermore, although mei5 and sae3 mutants are
phenotypically similar to dmc1 mutants, swi5 and
sfr1 mutants display more severe meiotic defects during fission yeast
meiosis than do dmc1 mutants
(27–29).
These data suggest that although Swi5-Sfr1 clearly contributes to Rad51
activity in fission yeast, it is possible that the activity of Mei5-Sae3 is
restricted to stimulating Dmc1 in budding yeast.In this study, a biochemical approach is used to test the budding yeast
Mei5-Sae3 complex for properties expected of a recombinase assembly mediator.
We show that Mei5-Sae3 binds both ssDNA and double-stranded DNA (dsDNA) but
binds ssDNA preferentially. We also show that Mei5-Sae3 can overcome the
inhibitory effects of RPA on the ssDNA binding and strand assimilation
activities of Dmc1. Finally, we show that Mei5-Sae3 and RPA bind one another
directly. These results indicate that Mei5-Sae3 acts directly as a mediator
protein for assembly of Dmc1. 相似文献
5.
During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called
homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination,
being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair
site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair
site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while
loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears
specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both
RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm
the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of
VDE-initiated homing for the study of meiotic recombination. 相似文献
6.
RecA protein is involved in homology search and strand exchange processes during recombination. Mitotic cells in eukaryotes express one RecA, Rad51, which is essential for the repair of double-strand breaks (DSBs). Additionally, meiotic cells induce the second RecA, Dmc1. Both Rad51 and Dmc1 are necessary to generate a crossover between homologous chromosomes, which ensures the segregation of the chromosomes at meiotic division I. It is largely unknown how the two RecAs cooperate during meiotic recombination. In this review, recent advances on our knowledge about the roles of Rad51 and Dmc1 during meiosis are summarized and discussed. 相似文献
7.
Arun T. John Peter Jens Lachmann Meenakshi Rana Madeleine Bunge Margarita Cabrera Christian Ungermann 《The Journal of cell biology》2013,201(1):97-111
Membrane microcompartments of the early endosomes serve as a sorting and signaling platform, where receptors are either recycled back to the plasma membrane or forwarded to the lysosome for destruction. In metazoan cells, three complexes, termed BLOC-1 to -3, mediate protein sorting from the early endosome to lysosomes and lysosome-related organelles. We now demonstrate that BLOC-1 is an endosomal Rab-GAP (GTPase-activating protein) adapter complex in yeast. The yeast BLOC-1 consisted of six subunits, which localized interdependently to the endosomes in a Rab5/Vps21-dependent manner. In the absence of BLOC-1 subunits, the balance between recycling and degradation of selected cargoes was impaired. Additionally, our data show that BLOC-1 is both a Vps21 effector and an adapter for its GAP Msb3. BLOC-1 and Msb3 interacted in vivo, and both mutants resulted in a redistribution of active Vps21 to the vacuole surface. We thus conclude that BLOC-1 controls the lifetime of active Rab5/Vps21 and thus endosomal maturation along the endocytic pathway. 相似文献
8.
DNA molecules containing stretches of contiguous guanine residues can assume a stable configuration in which planar quartets of guanine residues joined by Hoogsteen pairing appear in a stacked array. This conformation, called G4 DNA, has been implicated in several aspects of chromosome behavior including immunoglobulin gene rearrangements, promoter activation, and telomere maintenance. Moreover, the ability of the yeast SEP1 gene product to cleave DNA in a G4-DNA-dependent fashion, as well as that of the SGS1 gene product to unwind G4 DNA, has suggested a crucial role for this structure in meiotic synapsis and recombination. Here, we demonstrate that the HOP1 gene product, which plays a crucial role in the formation of synaptonemal complex in Saccharomyces cerevisiae, binds robustly to G4 DNA. The apparent dissociation constant for interaction with G4 DNA is 2 x 10(-10), indicative of binding that is about 1,000-fold stronger than to normal duplex DNA. Oligonucleotides of appropriate sequence bound Hop1 protein maximally if the DNA was first subjected to conditions favoring the formation of G4 DNA. Furthermore, incubation of unfolded oligonucleotides with Hop1 led to their transformation into G4 DNA. Methylation interference experiments confirmed that modifications blocking G4 DNA formation inhibit Hop1 binding. In contrast, neither bacterial RecA proteins that preferentially interact with GT-rich DNA nor histone H1 bound strongly to G4 DNA or induced its formation. These findings implicate specific interactions of Hop1 protein with G4 DNA in the pathway to chromosomal synapsis and recombination in meiosis. 相似文献
9.
Penkner A Portik-Dobos Z Tang L Schnabel R Novatchkova M Jantsch V Loidl J 《The EMBO journal》2007,26(24):5071-5082
Genome stability relies on faithful DNA repair both in mitosis and in meiosis. Here, we report on a Caenorhabditis elegans protein that we found to be homologous to the mammalian repair-related protein CtIP and to the budding yeast Com1/Sae2 recombination protein. A com-1 mutant displays normal meiotic chromosome pairing but forms irregular chromatin aggregates instead of diakinesis bivalents. While meiotic DNA double-strand breaks (DSBs) are formed, they appear to persist or undergo improper repair. Despite the presence of DSBs, the recombination protein RAD-51, which is known to associate with single-stranded DNA (ssDNA) flanking DSBs, does not localize to meiotic chromosomes in the com-1 mutant. Exposure of the mutant to gamma-radiation, however, induces RAD-51 foci, which suggests that the failure of RAD-51 to load is specific to meiotic (SPO-11-generated) DSBs. These results suggest that C. elegans COM-1 plays a role in the generation of ssDNA tails that can load RAD-51, invade homologous DNA tracts and thereby initiate recombination. Extrapolating from the worm homolog, we expect similar phenotypes for mutations in the mammalian tumor suppressor CtIP. 相似文献
10.
Huang G Kaufman AJ Ramanathan Y Singh B 《The Journal of biological chemistry》2011,286(12):10297-10304
SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO(-/-) mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO(-/-) mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12. 相似文献
11.
Sai P Visweshwaran Peter A Thomason Raphael Guerois Sophie Vacher Evgeny V Denisov Lubov A Tashireva Maria E Lomakina Christine Lazennec‐Schurdevin Goran Lakisic Sergio Lilla Nicolas Molinie Veronique Henriot Yves Mechulam Antonina Y Alexandrova Nadezhda V Cherdyntseva Ivan Bièche Emmanuelle Schmitt Robert H Insall Alexis Gautreau 《The EMBO journal》2018,37(13)
The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5β1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled‐coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients. 相似文献
12.
13.
recA1730 is a dominant point mutation preventing SOS mutagenesis. We demonstrate here that: i) RecA1730 fails to produce mutagenesis even though UmuD' is formed, ii) recA1730, when complemented by recA+, can cleave LexA protein and it displays a UmuDC- phenotype in spite of adequate concentrations of matured UmuD' and UmuC proteins, iii) the Mut- phenotype caused by RecA1730 is partially alleviated by MucAB proteins, functional analogs of UmuDC. To explain the mutant phenotype, we postulate that recA1730 impairs a RecA function required for the positioning of the UmuD'C complex within the replisome at the site of lesions. 相似文献
14.
Meiosis-specific mRNAs are transcribed in vegetative fission yeast, and these meiotic mRNAs are selectively removed from mitotic cells to suppress meiosis. This RNA elimination system requires degradation signal sequences called determinant of selective removal (DSR), an RNA-binding protein Mmi1, polyadenylation factors, and the nuclear exosome. However, the detailed mechanism by which meiotic mRNAs are selectively degraded in mitosis but not meiosis is not understood fully. Here we report that Red1, a novel protein, is essential for elimination of meiotic mRNAs from mitotic cells. A red1 deletion results in the accumulation of a large number of meiotic mRNAs in mitotic cells. Red1 interacts with Mmi1, Pla1, the canonical poly(A) polymerase, and Rrp6, a subunit of the nuclear exosome, and promotes the destabilization of DSR-containing mRNAs. Moreover, Red1 forms nuclear bodies in mitotic cells, and these foci are disassembled during meiosis. These results demonstrate that Red1 is involved in DSR-directed RNA decay to prevent ectopic expression of meiotic mRNAs in vegetative cells. 相似文献
15.
Clerici M Mantiero D Lucchini G Longhese MP 《The Journal of biological chemistry》2005,280(46):38631-38638
When eukaryotic chromosomes undergo double strand breaks (DSBs), several evolutionarily conserved proteins, among which the MRX complex, are recruited to the break site, leading to checkpoint activation and DNA repair. The function of the Saccharomyces cerevisiae Sae2 protein, which is known to work together with the MRX complex in meiotic DSB processing and in specific mitotic DSB repair events, is only beginning to be elucidated. Here we provide new insights into the role of Sae2 in mitotic DSB repair. We show that repair by single strand annealing of a single DSB, which is generated by the HO endonuclease between direct repeats, is defective both in the absence of Sae2 and in the presence of the hypomorphic rad50s allele altering the Rad50 subunit of MRX. Moreover, SAE2 overexpression partially suppresses the rad50s single strand annealing repair defects, suggesting that the latter might arise from defective MRX-Sae2 interactions. Finally, SAE2 deletion slows down resection of an HO-induced DSB and impairs DSB end bridging. Thus, Sae2 participates in DSB single strand annealing repair by ensuring both resection and intrachromosomal association of the broken ends. 相似文献
16.
A pathway containing the Ipl1/aurora protein kinase and the spindle midzone protein Ase1 regulates yeast spindle assembly 总被引:2,自引:0,他引:2
It is critical to elucidate the pathways that mediate spindle assembly and therefore ensure accurate chromosome segregation during cell division. Our studies of a unique allele of the budding yeast Ipl1/Aurora protein kinase revealed that it is required for centrosome-mediated spindle assembly in the absence of the BimC motor protein Cin8. In addition, we found that the Ase1 spindle midzone-associated protein is required for bipolar spindle assembly. The cin8 ipl1 and cin8 ase1 double mutant cells exhibit similar defects, and Ase1 overexpression completely restores spindle assembly in cin8 ipl1 strains. Consistent with the possibility that Ipl1 regulates Ase1, an ase1 mutant lacking the Ipl1 consensus phosphorylation sites cannot assemble spindles in the absence of Cin8. In addition, Ase1 phosphorylation and localization were altered in an ipl1 mutant. We therefore propose that Ipl1/Aurora and Ase1 constitute a previously unidentified spindle assembly pathway that becomes essential in the absence of Cin8. 相似文献
17.
Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein 总被引:13,自引:0,他引:13 下载免费PDF全文
Markovtsov V Nikolic JM Goldman JA Turck CW Chou MY Black DL 《Molecular and cellular biology》2000,20(20):7463-7479
Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins. 相似文献
18.
Sellers JA Hou L Athar H Hussain MM Shelness GS 《The Journal of biological chemistry》2003,278(22):20367-20373
The assembly and secretion of triglyceride-rich lipoproteins in vertebrates requires apolipoprotein B (apoB) and the endoplasmic reticulum-localized cofactor, microsomal triglyceride transfer protein (MTP). Invertebrates, particularly insects, transport the majority of their neutral and polar lipids in lipophorins; however, the assembly of lipophorin precursor particles was presumed to be MTP-independent. A Drosophila melanogaster expressed gene sequence (CG9342), displaying 23% identity with human MTP, was recently identified. When coexpressed in COS cells, CG9342 promoted the assembly and secretion of apoB34 and apoB41 (N-terminal 34 and 41% of human apoB). The apoB34-containing particles assembled by human MTP and CG9342 displayed similar peak densities of approximately 1.169 g/ml and similar lipid compositions. However, CG9342 displayed differential sensitivities to two inhibitors of human MTP and low vesicle-based lipid transfer activity, in vitro. In addition, important predicted structural distinctions exist between the human and Drosophila proteins suggesting overlapping but not identical functional roles. We conclude that CG9342 and human MTP are orthologs that share only a subset of functions, consistent with known differences in intracellular and extracellular aspects of vertebrate and invertebrate lipid transport and metabolism. 相似文献
19.
Uracil is removed from DNA by the conserved enzyme uracil DNA N-glycosylase (UNG). Previously, we observed that inhibiting UNG in Xenopus egg extracts blocked assembly of CENP-A, a histone H3 variant. CENP-A is an essential protein in all species, since it is required for chromosome segregation during mitosis. Thus, the implication of UNG in CENP-A assembly implies that UNG would also be essential, but UNG mutants lacking catalytic activity are viable in all species. In this paper, we present evidence that UNG2 colocalizes with CENP-A and H2AX phosphorylation at centromeres in normally cycling cells. Reduction of UNG2 in human cells blocks CENP-A assembly, and results in reduced cell proliferation, associated with increased frequencies of mitotic abnormalities and rapid cell death. Overexpression of UNG2 induces high levels of CENP-A assembly in human cells. Using a multiphoton laser approach, we demonstrate that UNG2 is rapidly recruited to sites of DNA damage. Taken together, our data are consistent with a model in which the N-terminus of UNG2 interacts with the active site of the enzyme and with chromatin. 相似文献
20.
S Schaper M Fromont-Racine P Linder J de la Cruz A Namane M Yaniv 《Current biology : CB》2001,11(23):1885-1890
Cells have a recurrent need for the correct assembly of protein-nucleic acid complexes. We have studied a yeast homolog of the smallest subunit of chromatin assembly factor 1 (CAF1), encoded by YMR131c and termed "RRB1". Unlike other yeast homologs, Msi1p, and Hat2p, Rrb1p is essential for cell viability. Impairment of Rrb1p function results in decreased levels of free 60S ribosomal subunits and the appearance of half-mer polysomes, suggesting its involvement in ribosome biogenesis. Using tandem affinity purification (TAP ) combined with mass spectrometry, we show that Rrb1p is associated with ribosomal protein L3. A fraction of Rrb1p is also found in a protein-precursor rRNA complex containing at least ten other early-assembling ribosomal proteins. We propose that Rrb1p is required for proper assembly of preribosomal particles during early ribosome biogenesis, presumably by targeting L3 onto the 35S precursor rRNA. This action may resemble the mechanism by which CAF1 assembles histones H3/H4 onto newly replicated DNA. 相似文献