RESUMEN
Proper genome packaging requires coordination of both DNA and histone metabolism. While histone gene transcription and RNA processing adequately provide for scheduled needs, how histone supply adjusts to unexpected changes in demand remains unknown. Here, we reveal that the histone chaperone nuclear autoantigenic sperm protein (NASP) protects a reservoir of soluble histones H3-H4. The importance of NASP is revealed upon histone overload, engagement of the reservoir during acute replication stress, and perturbation of Asf1 activity. The reservoir can be fine-tuned, increasing or decreasing depending on the level of NASP. Our data suggest that NASP does so by balancing the activity of the heat shock proteins Hsc70 and Hsp90 to direct H3-H4 for degradation by chaperone-mediated autophagy. These insights into NASP function and the existence of a tunable reservoir in mammalian cells demonstrate that contingency is integrated into the histone supply chain to respond to unexpected changes in demand.
Asunto(s)
Autoantígenos/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Autofagia , Células HEK293 , Células HeLa , Humanos , Solubilidad , Células Tumorales CultivadasRESUMEN
The heterochromatin protein 1 (HP1)-rich heterochromatin domains next to centromeres are crucial for chromosome segregation during mitosis. This mitotic function requires their faithful reproduction during the preceding S phase, a process whose mechanism and regulation are current puzzles. Here we show that p150, a subunit of chromatin assembly factor 1, has a key role in the replication of pericentric heterochromatin and S-phase progression in mouse cells, independently of its known function in histone deposition. By a combination of depletion and complementation assays in vivo, we link this unique function of p150 to its ability to interact with HP1. Absence of this functional interaction triggers S-phase arrest at the time of replication of pericentromeric heterochromatin, without eliciting known DNA-based checkpoint pathways. Notably, in cells lacking the histone methylases Suv39h, in which pericentric domains do not show HP1 accumulation, p150 is dispensable for S-phase progression.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Heterocromatina/metabolismo , Fase S/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Factor 1 de Ensamblaje de la Cromatina , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN/genética , Ratones , Células 3T3 NIH , Dominios y Motivos de Interacción de Proteínas , ARN Interferente Pequeño/genéticaRESUMEN
Activation-induced cytidine deaminase (AID) protein initiates Ig gene mutation by deaminating cytosines, converting them into uracils. Excision of AID-induced uracils by uracil-N-glycosylase is responsible for most transversion mutations at G:C base pairs. On the other hand, processing of AID-induced G:U mismatches by mismatch repair factors is responsible for most mutation at Ig A:T base pairs. Why mismatch processing should be error prone is unknown. One theory proposes that long patch excision in G1-phase leads to dUTP-incorporation opposite adenines as a result of the higher G1-phase ratio of nuclear dUTP to dTTP. Subsequent base excision at the A:U base pairs produced could then create non-instructional templates leading to permanent mutations at A:T base pairs (1). This compelling theory has remained untested. We have developed a method to rapidly modify DNA repair pathways in mutating mouse B cells in vivo by transducing Ig knock-in splenic mouse B cells with GFP-tagged retroviruses, then adoptively transferring GFP(+) cells, along with appropriate antigen, into primed congenic hosts. We have used this method to show that dUTP-incorporation is unlikely to be the cause of AID-induced mutation of A:T base pairs, and instead propose that A:T mutations might arise as an indirect consequence of nucleotide paucity during AID-induced DNA repair.
Asunto(s)
Adenina/química , Nucleótidos de Desoxiuracil/metabolismo , Genes de Inmunoglobulinas , Mutación , Timina/química , Animales , Emparejamiento Base , Expresión Génica , Centro Germinal/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Pirofosfatasas/metabolismo , Retroviridae/genética , Retroviridae/metabolismoRESUMEN
Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID)-dependent hypermutation of Ig V(D)J rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(D)J regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(D)J hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.
Asunto(s)
Anticuerpos/genética , Citidina Desaminasa/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Conversión Génica , Animales , Línea Celular , Pollos , Células Clonales , Citometría de Flujo , Región de Cambio de la Inmunoglobulina , Mutación , Uracil-ADN Glicosidasa/metabolismoAsunto(s)
Imagenología Tridimensional , Melanoma Experimental/diagnóstico por imagen , Ingeniería Metabólica/métodos , Neoplasias Cutáneas/diagnóstico por imagen , Animales , Vías Biosintéticas/genética , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Melaninas/biosíntesis , Melanoma Experimental/patología , Ratones , Neoplasias Cutáneas/patología , Microambiente TumoralRESUMEN
Heterochromatin protein 1 (HP1), a major component of constitutive heterochromatin, is recruited to DNA damage sites. However, the mechanism involved in this recruitment and its functional importance during DNA repair remain major unresolved issues. Here, by characterizing HP1α dynamics at laser-induced damage sites in mammalian cells, we show that the de novo accumulation of HP1α occurs within both euchromatin and heterochromatin as a rapid and transient event after DNA damage. This recruitment is strictly dependent on p150CAF-1, the largest subunit of chromatin assembly factor 1 (CAF-1), and its ability to interact with HP1α. We find that HP1α depletion severely compromises the recruitment of the DNA damage response (DDR) proteins 53BP1 and RAD51. Moreover, HP1α depletion leads to defects in homologous recombination-mediated repair and reduces cell survival after DNA damage. Collectively, our data reveal that HP1α recruitment at early stages of the DDR involves p150CAF-1 and is critical for proper DNA damage signaling and repair.
Asunto(s)
Factor 1 de Ensamblaje de la Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Animales , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Daño del ADN , Reparación del ADN , Humanos , Ratones , Células 3T3 NIHRESUMEN
Immunoglobulin (Ig) class switch recombination (CSR) involves the breakage and subsequent repair of two DNA sequences, known as switch (S) regions, which flank IgH constant region exons. The resolution of CSR-associated breaks is thought to require the nonhomologous end-joining (NHEJ) DNA repair pathway, but the role of the NHEJ factor DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in this process has been unclear. A new study, in which broken IgH-containing chromosomes in switching B cells were visualized directly, clearly demonstrated that DNA-PKcs and, unexpectedly, the nuclease Artemis are involved in the resolution of switch breaks.
Asunto(s)
Roturas del ADN , Reparación del ADN/genética , Reparación del ADN/inmunología , Cambio de Clase de Inmunoglobulina , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Rotura Cromosómica , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Hibridación Fluorescente in Situ , Ratones , Modelos Genéticos , Modelos Inmunológicos , Proteínas Nucleares/metabolismo , Recombinación GenéticaRESUMEN
DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone chaperone Asf1 and MCM2-7, the putative replicative helicase, are connected through a histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork progression and histone supply and demand.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , ADN/metabolismo , Histonas/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , ADN de Cadena Simple/metabolismo , Células HeLa , Humanos , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Modelos Biológicos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Interferencia de ARN , Fase SRESUMEN
Deoxyribonucleic acid double-stranded breaks act as intermediates in Ig V(D)J recombination and probably perform a similar function in class switch recombination between IgH C genes. In SCID mice, V(D)J recombination is blocked because the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein is defective. We show in this study that switching to all isotypes examined was detectable when the SCID mutation was introduced into anti-hen egg lysozyme transgenic B cells capable of undergoing class switch recombination, but switching was significantly reduced in comparison with control B cells of the same specificity lacking the RAG1 gene. Thus, DNA-PKcs is involved in switching to all isotypes, but plays a lesser role in the switching process than it does in V(D)J-coding joint formation. The higher level of switching observed by us in SCID B cells compared with that observed by others in DNA-PKcs(null) cells raises the possibility that kinase-deficient DNA-PKcs can function in switching. Point mutation of G:C base pairs with cytidines on the sense strand was greatly reduced in recombined switch regions from SCID cells compared with control RAG1(-/-) B cells. The preferential loss of sense strand cytidine mutations from hybrid S regions in SCID cells suggests the possibility that nicks might form in S regions of activated B cells on the template strand independently of activation-induced cytidine deaminase and are converted to double-strand breaks when activation-induced cytidine deaminase deaminates the non-template strand.