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1.
Curr Genet ; 67(2): 225-230, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33237336

RESUMO

The coordinated action of DNA polymerases and DNA helicases is essential at genomic sites that are hard to replicate. Among these are sites that harbour G-quadruplex DNA structures (G4). G4s are stable alternative DNA structures, which have been implicated to be involved in important cellular processes like the regulation of gene expression or telomere maintenance. G4 structures were shown to hinder replication fork progression and cause genomic deletions, mutations and recombination events. Many helicases unwind G4 structures and preserve genome stability, but a detailed understanding of G4 replication and the re-start of stalled replication forks around formed G4 structures is not clear, yet. In our recent study, we identified that Mgs1 preferentially binds to G4 DNA structures in vitro and is associated with putative G4-forming chromosomal regions in vivo. Mgs1 binding to G4 motifs in vivo is partially dependent on the helicase Pif1. Pif1 is the major G4-unwinding helicase in S. cerevisiae. In the absence of Mgs1, we determined elevated gross chromosomal rearrangement (GCR) rates in yeast, similar to Pif1 deletion. Here, we highlight the recent findings and set these into context with a new mechanistic model. We propose that Mgs1's functions support DNA replication at G4-forming regions.


Assuntos
DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Quadruplex G , Conformação de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/genética , DNA Helicases/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura
2.
FASEB J ; 34(9): 12646-12662, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32748509

RESUMO

The integrity of the genetic material is crucial for every organism. One intrinsic attack to genome stability is stalling of the replication fork which can result in DNA breakage. Several factors, such as DNA lesions or the formation of stable secondary structures (eg, G-quadruplexes) can lead to replication fork stalling. G-quadruplexes (G4s) are well-characterized stable secondary DNA structures that can form within specific single-stranded DNA sequence motifs and have been shown to block/pause the replication machinery. In most genomes several helicases have been described to regulate G4 unfolding to preserve genome integrity, however, different experiments raise the hypothesis that processing of G4s during DNA replication is more complex and requires additional, so far unknown, proteins. Here, we show that the Saccharomyces cerevisiae Mgs1 protein robustly binds to G4 structures in vitro and preferentially acts at regions with a strong potential to form G4 structures in vivo. Our results suggest that Mgs1 binds to G4-forming sites and has a role in the maintenance of genome integrity.


Assuntos
DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Quadruplex G , Instabilidade Genômica , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , DNA Fúngico/química , Ligação Proteica
3.
Nucleic Acids Res ; 47(21): 11250-11267, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31566235

RESUMO

The addition of poly(ADP-ribose) (PAR) chains along the chromatin fiber due to PARP1 activity regulates the recruitment of multiple factors to sites of DNA damage. In this manuscript, we investigated how, besides direct binding to PAR, early chromatin unfolding events controlled by PAR signaling contribute to recruitment to DNA lesions. We observed that different DNA-binding, but not histone-binding, domains accumulate at damaged chromatin in a PAR-dependent manner, and that this recruitment correlates with their affinity for DNA. Our findings indicate that this recruitment is promoted by early PAR-dependent chromatin remodeling rather than direct interaction with PAR. Moreover, recruitment is not the consequence of reduced molecular crowding at unfolded damaged chromatin but instead originates from facilitated binding to more exposed DNA. These findings are further substantiated by the observation that PAR-dependent chromatin remodeling at DNA lesions underlies increased DNAse hypersensitivity. Finally, the relevance of this new mode of PAR-dependent recruitment to DNA lesions is demonstrated by the observation that reducing the affinity for DNA of both CHD4 and HP1α, two proteins shown to be involved in the DNA-damage response, strongly impairs their recruitment to DNA lesions.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/metabolismo , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Poli Adenosina Difosfato Ribose/fisiologia , Sítios de Ligação/genética , Células Cultivadas , Cromatina/química , Homólogo 5 da Proteína Cromobox , Humanos , Conformação de Ácido Nucleico , Poli Adenosina Difosfato Ribose/metabolismo , Ligação Proteica
4.
DNA Repair (Amst) ; 49: 33-42, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27838458

RESUMO

Inappropriate repair of UV-induced DNA damage results in human diseases such as Xeroderma pigmentosum (XP), which is associated with an extremely high risk of skin cancer. A variant form of XP is caused by the absence of Polη, which is normally able to bypass UV-induced DNA lesions in an error-free manner. However, Polη is highly error prone when replicating undamaged DNA and, thus, the regulation of the proper targeting of Polη is crucial for the prevention of mutagenesis and UV-induced cancer formation. Spartan is a novel regulator of the damage tolerance pathway, and its association with Ub-PCNA has a role in Polη targeting; however, our knowledge about its function is only rudimentary. Here, we describe a new biochemical property of purified human SPARTAN by showing that it is a DNA-binding protein. Using a DNA binding mutant, we provide in vivo evidence that DNA binding by SPARTAN regulates the targeting of Polη to damage sites after UV exposure, and this function contributes highly to its DNA-damage tolerance function.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , DNA/efeitos da radiação , Células HEK293 , Humanos , Raios Ultravioleta
5.
Nucleic Acids Res ; 44(7): 3176-89, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-26792895

RESUMO

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Reparo de DNA por Recombinação , Motivos de Aminoácidos , DNA/biossíntese , DNA Polimerase III/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Células HEK293 , Humanos , Ubiquitina-Proteína Ligases/fisiologia , Raios Ultravioleta
6.
Nucleic Acids Res ; 43(4): 2116-25, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25662213

RESUMO

Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF483-484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex.


Assuntos
Dano ao DNA , DNA Polimerase III/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Ciclo Celular , Linhagem Celular , Sobrevivência Celular , Humanos , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , Raios Ultravioleta
7.
Nucleic Acids Res ; 42(3): 1711-20, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24198246

RESUMO

Stalling of replication forks at unrepaired DNA lesions can result in discontinuities opposite the damage in the newly synthesized DNA strand. Translesion synthesis or facilitating the copy from the newly synthesized strand of the sister duplex by template switching can overcome such discontinuities. During template switch, a new primer-template junction has to be formed and two mechanisms, including replication fork reversal and D-loop formation have been suggested. Genetic evidence indicates a major role for yeast Rad5 in template switch and that both Rad5 and its human orthologue, Helicase-like transcription factor (HLTF), a potential tumour suppressor can facilitate replication fork reversal. This study demonstrates the ability of HLTF and Rad5 to form a D-loop without requiring ATP binding and/or hydrolysis. We also show that this strand-pairing activity is independent of RAD51 in vitro and is not mechanistically related to that of another member of the SWI/SNF family, RAD54. In addition, the 3'-end of the invading strand in the D-loop can serve as a primer and is extended by DNA polymerase. Our data indicate that HLTF is involved in a RAD51-independent D-loop branch of template switch pathway that can promote repair of gaps formed during replication of damaged DNA.


Assuntos
Dano ao DNA , Replicação do DNA , Fatores de Transcrição Forkhead/metabolismo , Adenosina Trifosfatases/metabolismo , DNA/química , DNA/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , Humanos , Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Moldes Genéticos
8.
DNA Repair (Amst) ; 12(9): 691-8, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23731732

RESUMO

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.


Assuntos
DNA Polimerase Dirigida por DNA/química , Recombinação Homóloga , Antígeno Nuclear de Célula em Proliferação/química , Dano ao DNA , DNA Polimerase III/química , DNA Polimerase III/fisiologia , Replicação do DNA , DNA de Cadeia Simples/biossíntese , DNA Polimerase Dirigida por DNA/fisiologia , Células HeLa , Humanos , Concentração Osmolar , Antígeno Nuclear de Célula em Proliferação/fisiologia , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/fisiologia , Rad51 Recombinase/química , DNA Polimerase iota
9.
EMBO J ; 32(5): 742-55, 2013 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-23395907

RESUMO

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.


Assuntos
DNA Helicases/metabolismo , Reparo do DNA/genética , Recombinação Homóloga , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Reparo do DNA/efeitos da radiação , Replicação do DNA/genética , Replicação do DNA/efeitos da radiação , Instabilidade Genômica , Mutação/genética , Antígeno Nuclear de Célula em Proliferação/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína SUMO-1/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação , Raios Ultravioleta/efeitos adversos
10.
Nucleic Acids Res ; 40(21): 10795-808, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22987070

RESUMO

Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina/metabolismo , Proteínas de Arabidopsis , Linhagem Celular , Replicação do DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Endopeptidases/metabolismo , Humanos , Estrutura Terciária de Proteína , Troca de Cromátide Irmã , Ubiquitina-Proteína Ligases/fisiologia , Proteases Específicas de Ubiquitina
11.
Nucleic Acids Res ; 40(13): 6049-59, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22457066

RESUMO

DNA double-strand breaks (DSBs) can be generated not only by reactive agents but also as a result of replication fork collapse at unrepaired DNA lesions. Whereas ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates damage bypass, modification of yeast PCNA by small ubiquitin-like modifier (SUMO) controls recombination by providing access for the Srs2 helicase to disrupt Rad51 nucleoprotein filaments. However, in human cells, the roles of PCNA SUMOylation have not been explored. Here, we characterize the modification of human PCNA by SUMO in vivo as well as in vitro. We establish that human PCNA can be SUMOylated at multiple sites including its highly conserved K164 residue and that SUMO modification is facilitated by replication factor C (RFC). We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out. Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions. These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.


Assuntos
Quebras de DNA de Cadeia Dupla , Antígeno Nuclear de Célula em Proliferação/metabolismo , Sumoilação , Replicação do DNA , Histonas/metabolismo , Recombinação Homóloga , Humanos , Mutação , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Proteína SUMO-1/metabolismo
12.
DNA Repair (Amst) ; 10(6): 567-76, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21565563

RESUMO

The error-free repair of double-strand DNA breaks by homologous recombination (HR) ensures genomic stability using undamaged homologous sequence to copy genetic information. While some of the aspects of the initial steps of HR are understood, the molecular mechanisms underlying events downstream of the D-loop formation remain unclear. Therefore, we have reconstituted D-loop-based in vitro recombination-associated DNA repair synthesis assay and tested the efficacy of polymerases Pol δ and Pol η to extend invaded primer, and the ability of three helicases (Mph1, Srs2 and Sgs1) to displace this extended primer. Both Pol δ and Pol η extended up to 50% of the D-loop substrate, but differed in product length and dependency on proliferating cell nuclear antigen (PCNA). Mph1, but not Srs2 or Sgs1, displaced the extended primer very efficiently, supporting putative role of Mph1 in promoting the synthesis-dependent strand-annealing pathway. The experimental system described here can be employed to increase our understanding of HR events following D-loop formation, as well as the regulatory mechanisms involved.


Assuntos
DNA Helicases/metabolismo , DNA Polimerase III/metabolismo , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Genética , Especificidade por Substrato
13.
Nucleic Acids Res ; 37(13): 4247-55, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19443450

RESUMO

Human Ape2 protein has 3' phosphodiesterase activity for processing 3'-damaged DNA termini, 3'-5' exonuclease activity that supports removal of mismatched nucleotides from the 3'-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3'-5' exonuclease and 3' phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3'-damaged termini but also by removing 3'-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis.


Assuntos
Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Exodesoxirribonucleases/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Adenina/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/análise , Endonucleases , Humanos , Peróxido de Hidrogênio/farmacologia , Enzimas Multifuncionais , Oxirredução , Antígeno Nuclear de Célula em Proliferação/análise
14.
Nucleic Acids Res ; 34(9): 2508-15, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16687656

RESUMO

DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.


Assuntos
Pareamento Incorreto de Bases , Exodesoxirribonucleases/metabolismo , Sítios de Ligação , DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Endonucleases , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/isolamento & purificação , Humanos , Enzimas Multifuncionais , Mutação , Diester Fosfórico Hidrolases/metabolismo , Especificidade por Substrato
15.
Brain Res Mol Brain Res ; 139(1): 103-14, 2005 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-15992958

RESUMO

We describe the structure of the rat importin 9 gene, together with its transcripts and the encoded protein with its putative functional domains. The importin 9 gene contains 24 exons in a genomic region spanning >52,000 bp. It is transcribed into two mRNAs, generated by means of alternative polyadenylation site usage arranged in tandem. Both transcripts possess the same noncanonical polyadenylation signal (AGUAAA) in rat, this hexamer being conserved in all vertebrates examined. Additionally, intron 8 is bordered by AT-AC dinucleotides. Importin 9 is expressed throughout adult rat tissues, but the 114-kDa Importin 9 protein was detected only in the brain. The localization of the Importin 9 protein was examined by immunohistochemistry in both adult rat tissues and primary hippocampal cell cultures. The strongest labeling was detected in vivo in areas populated by neurons in high density and also in the dendritic processes emanating from these cells. This protein was clearly concentrated in the nuclei of these cells, although their cytoplasms too were heavily labeled. Strong cytoplasmic and very strong nuclear staining was found in a vast majority of the cells with neuronal morphology in vitro. Cultured cells with glial morphology generally exhibited a weaker cytoplasmic labeling. In these cells, the signal decorated the nuclear envelope without nuclear staining and gradually dwindled toward the cell periphery. These results hint at the cell- or tissue-type specific functions of this type of importin protein.


Assuntos
Carioferinas/genética , Carioferinas/metabolismo , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Hipocampo/citologia , Humanos , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Neurônios/citologia , Ratos , Distribuição Tecidual
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