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1.
Nat Immunol ; 14(11): 1183-1189, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24097111

RESUMEN

The ability of activation-induced cytidine deaminase (AID) to efficiently mediate class-switch recombination (CSR) is dependent on its phosphorylation at Ser38; however, the trigger that induces AID phosphorylation and the mechanism by which phosphorylated AID drives CSR have not been elucidated. Here we found that phosphorylation of AID at Ser38 was induced by DNA breaks. Conversely, in the absence of AID phosphorylation, DNA breaks were not efficiently generated at switch (S) regions in the immunoglobulin heavy-chain locus (Igh), consistent with a failure of AID to interact with the endonuclease APE1. Additionally, deficiency in the DNA-damage sensor ATM impaired the phosphorylation of AID at Ser38 and the interaction of AID with APE1. Our results identify a positive feedback loop for the amplification of DNA breaks at S regions through the phosphorylation- and ATM-dependent interaction of AID with APE1.


Asunto(s)
Linfocitos B/inmunología , Citidina Desaminasa/inmunología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/inmunología , Retroalimentación Fisiológica , Cambio de Clase de Inmunoglobulina , Cadenas Pesadas de Inmunoglobulina/inmunología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Linfocitos B/citología , Citidina Desaminasa/genética , Roturas del ADN de Doble Cadena , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Regulación de la Expresión Génica , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Fosforilación , Unión Proteica , Serina/inmunología , Serina/metabolismo , Transducción de Señal
2.
Mol Cell ; 41(2): 232-42, 2011 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-21255732

RESUMEN

After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.


Asunto(s)
Linfocitos B/enzimología , Citidina Desaminasa/fisiología , Roturas del ADN de Doble Cadena , Secuencias de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/química , Citidina Desaminasa/metabolismo , Proteínas de Unión al ADN , Genes myc , Cambio de Clase de Inmunoglobulina , Activación de Linfocitos , Ratones , Proteínas Nucleares/química , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas Represoras
3.
EMBO J ; 33(15): 1698-712, 2014 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-24966277

RESUMEN

Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways.


Asunto(s)
Proteína BRCA1/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Daño del ADN , Reparación de la Incompatibilidad de ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteína BRCA1/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular/efectos de los fármacos , Aberraciones Cromosómicas , Daño del ADN/efectos de los fármacos , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Ratones , Ratones Mutantes , Mitomicina/farmacología , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas
4.
PLoS Genet ; 11(8): e1005438, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26263206

RESUMEN

Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Citidina Desaminasa/fisiología , Roturas del ADN de Doble Cadena , Proteínas Nucleares/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Inmunoprecipitación de Cromatina , ADN Intergénico/genética , Proteínas de Unión al ADN , Ratones Endogámicos C57BL , Ratones Transgénicos , Unión Proteica , Bazo/citología , Bazo/enzimología , Secuencias Repetidas en Tándem
5.
Proc Natl Acad Sci U S A ; 111(25): 9217-22, 2014 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-24927551

RESUMEN

Somatic hypermutation (SHM) of antibody variable region genes is initiated in germinal center B cells during an immune response by activation-induced cytidine deaminase (AID), which converts cytosines to uracils. During accurate repair in nonmutating cells, uracil is excised by uracil DNA glycosylase (UNG), leaving abasic sites that are incised by AP endonuclease (APE) to create single-strand breaks, and the correct nucleotide is reinserted by DNA polymerase ß. During SHM, for unknown reasons, repair is error prone. There are two APE homologs in mammals and, surprisingly, APE1, in contrast to its high expression in both resting and in vitro-activated splenic B cells, is expressed at very low levels in mouse germinal center B cells where SHM occurs, and APE1 haploinsufficiency has very little effect on SHM. In contrast, the less efficient homolog, APE2, is highly expressed and contributes not only to the frequency of mutations, but also to the generation of mutations at A:T base pair (bp), insertions, and deletions. In the absence of both UNG and APE2, mutations at A:T bp are dramatically reduced. Single-strand breaks generated by APE2 could provide entry points for exonuclease recruited by the mismatch repair proteins Msh2-Msh6, and the known association of APE2 with proliferating cell nuclear antigen could recruit translesion polymerases to create mutations at AID-induced lesions and also at A:T bp. Our data provide new insight into error-prone repair of AID-induced lesions, which we propose is facilitated by down-regulation of APE1 and up-regulation of APE2 expression in germinal center B cells.


Asunto(s)
Linfocitos B/metabolismo , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/biosíntesis , Endonucleasas/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Centro Germinal/metabolismo , Mutación , Hipermutación Somática de Inmunoglobulina/fisiología , Animales , Linfocitos B/citología , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Centro Germinal/citología , Ratones , Ratones Noqueados , Enzimas Multifuncionales , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo
6.
J Immunol ; 193(11): 5370-8, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25411432

RESUMEN

IgH class switching occurs rapidly after activation of mature naive B cells, resulting in a switch from expression of IgM and IgD to expression of IgG, IgE, or IgA; this switch improves the ability of Abs to remove the pathogen that induces the humoral immune response. Class switching occurs by a deletional recombination between two switch regions, each of which is associated with a H chain constant region gene. Class switch recombination (CSR) is instigated by activation-induced cytidine deaminase, which converts cytosines in switch regions to uracils. The uracils are subsequently removed by two DNA-repair pathways, resulting in mutations, single-strand DNA breaks, and the double-strand breaks required for CSR. We discuss several aspects of CSR, including how CSR is induced, CSR in B cell progenitors, the roles of transcription and chromosomal looping in CSR, and the roles of certain DNA-repair enzymes in CSR.


Asunto(s)
Linfocitos B/fisiología , Citidina Desaminasa/metabolismo , Cambio de Clase de Inmunoglobulina , Cadenas Pesadas de Inmunoglobulina/genética , Animales , Reparación del ADN , Humanos , Activación de Linfocitos
7.
J Immunol ; 192(10): 4887-96, 2014 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-24729610

RESUMEN

Activation-induced cytidine deaminase (AID) initiates Ab class-switch recombination (CSR) in activated B cells resulting in exchanging the IgH C region and improved Ab effector function. During CSR, AID instigates DNA double-strand break (DSB) formation in switch (S) regions located upstream of C region genes. DSBs are necessary for CSR, but improper regulation of DSBs can lead to chromosomal translocations that can result in B cell lymphoma. The protein kinase ataxia telangiectasia mutated (ATM) is an important proximal regulator of the DNA damage response (DDR), and translocations involving S regions are increased in its absence. ATM phosphorylates H2AX, which recruits other DNA damage response (DDR) proteins, including mediator of DNA damage checkpoint 1 (Mdc1) and p53 binding protein 1 (53BP1), to sites of DNA damage. As these DDR proteins all function to promote repair and recombination of DSBs during CSR, we examined whether mouse splenic B cells deficient in these proteins would show alterations in S region DSBs when undergoing CSR. We find that in atm(-/-) cells Sµ DSBs are increased, whereas DSBs in downstream Sγ regions are decreased. We also find that mutations in the unrearranged Sγ3 segment are reduced in atm(-/-) cells. Our data suggest that ATM increases AID targeting and activity at downstream acceptor S regions during CSR and that in atm(-/-) cells Sµ DSBs accumulate as they lack a recombination partner.


Asunto(s)
Citidina Desaminasa/inmunología , Reordenamiento Génico de Linfocito B/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/inmunología , Citidina Desaminasa/genética , Daño del ADN/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Reordenamiento Génico de Linfocito B/genética , Histonas/genética , Histonas/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Ratones , Ratones Noqueados , Fosforilación/genética , Fosforilación/inmunología , Proteína 1 de Unión al Supresor Tumoral P53
8.
J Immunol ; 193(3): 1440-50, 2014 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-24973444

RESUMEN

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The AID C terminus is required for CSR, but not for S-region DNA double-strand breaks (DSBs) during CSR, and it is not required for SHM. AID lacking the C terminus (ΔAID) is a dominant negative (DN) mutant, because human patients heterozygous for this mutant fail to undergo CSR. In agreement, we show that ΔAID is a DN mutant when expressed in AID-sufficient mouse splenic B cells. To have DN function, ΔAID must have deaminase activity, suggesting that its ability to induce DSBs is important for the DN function. Supporting this hypothesis, Msh2-Msh6 have been shown to contribute to DSB formation in S regions, and we find in this study that Msh2 is required for the DN activity, because ΔAID is not a DN mutant in msh2(-/-) cells. Our results suggest that the DNA DSBs induced by ΔAID are unable to participate in CSR and might interfere with the ability of full-length AID to participate in CSR. We propose that ΔAID is impaired in its ability to recruit nonhomologous end joining repair factors, resulting in accumulation of DSBs that undergo aberrant resection. Supporting this hypothesis, we find that the S-S junctions induced by ΔAID have longer microhomologies than do those induced by full-length AID. In addition, our data suggest that AID binds Sµ regions in vivo as a monomer.


Asunto(s)
Citidina Desaminasa/fisiología , Reparación de la Incompatibilidad de ADN/inmunología , Reordenamiento Génico/inmunología , Animales , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Reparación de la Incompatibilidad de ADN/genética , Eliminación de Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fragmentos de Péptidos/genética , Cultivo Primario de Células
9.
Trends Immunol ; 32(5): 194-201, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21493144

RESUMEN

Activation-induced cytidine deaminase (AID) instigates mutations and DNA breaks in Ig genes that undergo somatic hypermutation and class switch recombination during B cell activation in response to immunization and infection. This review discusses how AID expression and activity are regulated, including recent discoveries of AID-interacting proteins that might recruit AID to Ig genes, and allow it to target both DNA strands. Also discussed is the accumulating evidence that AID binds to, mutates, and creates breaks at numerous non-Ig sites in the genome, which initiates cell transformation and malignancies.


Asunto(s)
Linfocitos B/enzimología , Citidina Desaminasa/metabolismo , Regulación Enzimológica de la Expresión Génica , Activación de Linfocitos/inmunología , Animales , Linfocitos B/inmunología , Citidina Desaminasa/genética , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Humanos , Cambio de Clase de Inmunoglobulina , Ratones , Estabilidad del ARN , Hipermutación Somática de Inmunoglobulina
10.
J Immunol ; 189(5): 2374-82, 2012 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-22826323

RESUMEN

Activation-induced cytidine deaminase (AID) initiates DNA double-strand breaks (DSBs) in the IgH gene (Igh) to stimulate isotype class switch recombination (CSR), and widespread breaks in non-Igh (off-target) loci throughout the genome. Because the DSBs that initiate class switching occur during the G1 phase of the cell cycle, and are repaired via end joining, CSR is considered a predominantly G1 reaction. By contrast, AID-induced non-Igh DSBs are repaired by homologous recombination. Although little is known about the connection between the cell cycle and either induction or resolution of AID-mediated non-Igh DSBs, their repair by homologous recombination implicates post-G1 phases. Coordination of DNA breakage and repair during the cell cycle is critical to promote normal class switching and prevent genomic instability. To understand how AID-mediated events are regulated through the cell cycle, we have investigated G1-to-S control in AID-dependent genome-wide DSBs. We find that AID-mediated off-target DSBs, like those induced in the Igh locus, are generated during G1. These data suggest that AID-mediated DSBs can evade G1/S checkpoint activation and persist beyond G1, becoming resolved during S phase. Interestingly, DSB resolution during S phase can promote not only non-Igh break repair, but also Ig CSR. Our results reveal novel cell cycle dynamics in response to AID-initiated DSBs, and suggest that the regulation of the repair of these DSBs through the cell cycle may ensure proper class switching while preventing AID-induced genomic instability.


Asunto(s)
Citidina Desaminasa/fisiología , Roturas del ADN de Doble Cadena , Cambio de Clase de Inmunoglobulina/genética , Isotipos de Inmunoglobulinas/genética , Fase S/genética , Fase S/inmunología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células Cultivadas , Citidina Desaminasa/deficiencia , Citidina Desaminasa/genética , Reparación del ADN/genética , Reparación del ADN/inmunología , Fase G1/genética , Fase G1/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
11.
J Exp Med ; 204(7): 1677-89, 2007 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-17591858

RESUMEN

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), which converts cytosines to uracils in switch (S) regions. Subsequent excision of dU by uracil DNA glycosylase (UNG) of the base excision repair (BER) pathway is required to obtain double-strand break (DSB) intermediates for CSR. Since UNG normally initiates faithful repair, it is unclear how the AID-instigated S region lesions are converted into DSBs rather than correctly repaired by BER. Normally, DNA polymerase beta (Polbeta) would replace the dC deaminated by AID, leading to correct repair of the single-strand break, thereby preventing CSR. We address the question of whether Polbeta might be specifically down-regulated during CSR or inhibited from accessing the AID-instigated lesions, or whether the numerous AID-initiated S region lesions might simply overwhelm the BER capacity. We find that nuclear Polbeta levels are induced upon activation of splenic B cells to undergo CSR. When Polbeta(-/-) B cells are activated to switch in culture, they switch slightly better to IgG2a, IgG2b, and IgG3 and have more S region DSBs and mutations than wild-type controls. We conclude that Polbeta attempts to faithfully repair S region lesions but fails to repair them all.


Asunto(s)
Linfocitos B/inmunología , Citidina Desaminasa/metabolismo , ADN Polimerasa beta/fisiología , Región de Cambio de la Inmunoglobulina/genética , Recombinación Genética , Uracil-ADN Glicosidasa/metabolismo , Animales , Linfocitos B/enzimología , Citosina/metabolismo , ADN Polimerasa beta/deficiencia , ADN Polimerasa beta/genética , Cartilla de ADN , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Región de Cambio de la Inmunoglobulina/inmunología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Uracilo/metabolismo
12.
J Immunol ; 187(5): 2464-75, 2011 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-21804017

RESUMEN

Activation-induced cytidine deaminase (AID) is induced in B cells during an immune response and is essential for both class-switch recombination (CSR) and somatic hypermutation of Ab genes. The C-terminal 10 aa of AID are required for CSR but not for somatic hypermutation, although their role in CSR is unknown. Using retroviral transduction into mouse splenic B cells, we show that the C terminus is not required for switch (S) region double-strand breaks (DSBs) and therefore functions downstream of DSBs. Using chromatin immunoprecipitation, we show that AID binds cooperatively with UNG and the mismatch repair proteins Msh2-Msh6 to Ig Sµ and Sγ3 regions, and this depends on the C terminus and the deaminase activity of AID. We also show that mismatch repair does not contribute to the efficiency of CSR in the absence of the AID C terminus. Although it has been demonstrated that both UNG and Msh2-Msh6 are important for introduction of S region DSBs, our data suggest that the ability of AID to recruit these proteins is important for DSB resolution, perhaps by directing the S region DSBs toward accurate and efficient CSR via nonhomologous end joining.


Asunto(s)
Citidina Desaminasa/metabolismo , Proteínas de Unión al ADN/metabolismo , Cambio de Clase de Inmunoglobulina/fisiología , Región de Cambio de la Inmunoglobulina/fisiología , Proteína 2 Homóloga a MutS/metabolismo , Uracil-ADN Glicosidasa/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Separación Celular , Inmunoprecipitación de Cromatina , Citidina Desaminasa/química , Citometría de Flujo , Inmunoglobulina G , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
14.
J Immunol ; 184(11): 6177-87, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-20483782

RESUMEN

Ig class switch recombination (CSR) occurs in activated mature B cells, and causes an exchange of the IgM isotype for IgG, IgE, or IgA isotypes, which increases the effectiveness of the humoral immune response. DNA ds breaks in recombining switch (S) regions, where CSR occurs, are required for recombination. Activation-induced cytidine deaminase initiates DNA ds break formation by deamination of cytosines in S regions. This reaction requires reactive oxygen species (ROS) intermediates, such as hydroxyl radicals. In this study we show that the ROS scavenger N-acetylcysteine inhibits CSR. We also demonstrate that IFN-gamma treatment, which is used to induce IgG2a switching, increases intracellular ROS levels, and activates p53 in switching B cells, and show that p53 inhibits IgG2a class switching through its antioxidant-regulating function. Finally, we show that p53 inhibits DNA breaks and mutations in S regions in B cells undergoing CSR, suggesting that p53 inhibits the activity of activation-induced cytidine deaminase.


Asunto(s)
Antioxidantes/metabolismo , Cambio de Clase de Inmunoglobulina/genética , Inmunoglobulina G/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Western Blotting , Separación Celular , Citidina Desaminasa/metabolismo , Roturas del ADN de Doble Cadena , Citometría de Flujo , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/inmunología
15.
J Exp Med ; 201(12): 1885-90, 2005 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-15955838

RESUMEN

The mechanisms that target class switch recombination (CSR) to antibody gene switch (S) regions are unknown. Analyses of switch site locations in wild-type mice and in mice that lack the Smu tandem repeats show shifts indicating that a 4-5-kb DNA domain (bounded upstream by the Imu promoter) is accessible for switching independent of Smu sequences. This CSR-accessible domain is reminiscent of the promoter-defined domains that target somatic hypermutation. Within the 4-5-kb CSR domain, the targeting of S site locations also depends on the Msh2 mismatch repair protein because Msh2-deficient mice show an increased focus of sites to the Smu tandem repeat region. We propose that Msh2 affects S site location because sequences with few activation-induced cytidine deaminase targets generate mostly switch DNA cleavages that require Msh2-directed processing to allow CSR joining.


Asunto(s)
Diversidad de Anticuerpos/genética , Proteínas de Unión al ADN/metabolismo , Cambio de Clase de Inmunoglobulina/inmunología , Región de Cambio de la Inmunoglobulina/genética , Modelos Genéticos , Proteínas Proto-Oncogénicas/metabolismo , Animales , Cartilla de ADN , Proteínas de Unión al ADN/genética , Cambio de Clase de Inmunoglobulina/genética , Ratones , Ratones Noqueados , Proteína 2 Homóloga a MutS , Reacción en Cadena de la Polimerasa/métodos , Proteínas Proto-Oncogénicas/genética , Secuencias Repetidas en Tándem/genética
16.
J Exp Med ; 202(4): 561-8, 2005 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-16103411

RESUMEN

Class switch recombination (CSR) occurs by an intrachromosomal deletion whereby the IgM constant region gene (Cmu) is replaced by a downstream constant region gene. This unique recombination event involves formation of double-strand breaks (DSBs) in immunoglobulin switch (S) regions, and requires activation-induced cytidine deaminase (AID), which converts cytosines to uracils. Repair of the uracils is proposed to lead to DNA breaks required for recombination. Uracil DNA glycosylase (UNG) is required for most CSR activity although its role is disputed. Here we use ligation-mediated PCR to detect DSBs in S regions in splenic B cells undergoing CSR. We find that the kinetics of DSB induction corresponds with AID expression, and that DSBs are AID- and UNG-dependent and occur preferentially at G:C basepairs in WRC/GYW AID hotspots. Our results indicate that AID attacks cytosines on both DNA strands, and staggered breaks are processed to blunt DSBs at the initiating ss break sites. We propose a model to explain the types of end-processing events observed.


Asunto(s)
Linfocitos B/inmunología , Citidina Desaminasa/genética , ADN Glicosilasas/genética , Reordenamiento Génico de Linfocito B/genética , Cadenas mu de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina/genética , Animales , Linfocitos B/citología , Citidina Desaminasa/inmunología , ADN/genética , ADN/inmunología , ADN Glicosilasas/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Reordenamiento Génico de Linfocito B/inmunología , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Cadenas mu de Inmunoglobulina/inmunología , Ratones , Ratones Mutantes , Recombinación Genética/genética , Recombinación Genética/inmunología , Hipermutación Somática de Inmunoglobulina/inmunología , Bazo/citología , Bazo/inmunología , Uracil-ADN Glicosidasa
17.
J Immunol ; 183(2): 1222-8, 2009 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-19553545

RESUMEN

The Msh2 mismatch repair (MMR) protein is critical for class switch recombination (CSR) events that occur in mice that lack the Smu tandem repeat (SmuTR) region (SmuTR(-/-) mice). The pattern of microhomology among switch junction sites in Msh2-deficient mice is also dependent on the presence or absence of SmuTR sequences. It is not known whether these CSR effects reflect an individual function of Msh2 or the function of Msh2 within the MMR machinery. In the absence of the SmuTR sequences, Msh2 deficiency nearly ablates CSR. We now show that Mlh1 or Exo1 deficiencies also eliminate CSR in the absence of the SmuTR. Furthermore, in SmuTR(-/-) mice, deficiencies of Mlh1 or Exo1 result in increased switch junction microhomology as has also been seen with Msh2 deficiency. These results are consistent with a CSR model in which the MMR machinery is important in processing DNA nicks to produce double-stranded breaks, particularly in sequences where nicks are infrequent. We propose that double-stranded break paucity in MMR-deficient mice leads to increased use of an alternative joining pathway where microhomologies are important for CSR break ligation. Interestingly, when the SmuTR region is present, deficiency of Msh2 does not lead to the increased microhomology seen with Mlh1 or Exo1 deficiencies, suggesting that Msh2 might have an additional function in CSR. It is also possible that the inability to initiate MMR in the absence of Msh2 results in CSR junctions with less microhomology than joinings that occur when MMR is initiated but then proceeds abnormally due to Mlh1 or Exo1 deficiencies.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Exodesoxirribonucleasas/deficiencia , Cambio de Clase de Inmunoglobulina/genética , Región de Cambio de la Inmunoglobulina , Proteína 2 Homóloga a MutS/deficiencia , Proteínas Nucleares/deficiencia , Secuencias Repetidas en Tándem , Animales , Linfocitos B/inmunología , Células Cultivadas , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Reparación del ADN , Región de Cambio de la Inmunoglobulina/genética , Ratones , Ratones Noqueados , Homólogo 1 de la Proteína MutL
18.
J Exp Med ; 197(10): 1377-83, 2003 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-12743174

RESUMEN

Mismatch repair proteins participate in antibody class switch recombination, although their roles are unknown. Previous nucleotide sequence analyses of switch recombination junctions indicated that the roles of Msh2 and the MutL homologues, Mlh1 and Pms2, differ. We now asked if Msh2 and Mlh1 function in the same pathway during switch recombination. Splenic B cells from mice deficient in both these proteins were induced to undergo switching in culture. The frequency of switching is reduced, similarly to that of B cells singly deficient in Msh2 or Mlh1. However, the nucleotide sequences of the Smu-Sgamma3 junctions resemble junctions from Mlh1- but not from Msh2-deficient cells, suggesting Mlh1 functions either independently of or before Msh2. The substitution mutations within S regions that are known to accompany switch recombination are increased in Msh2- and Mlh1 single-deficient cells and further increased in the double-deficient cells, again suggesting these proteins function independently in class switch recombination. The finding that MMR functions to reduce mutations in switch regions is unexpected since MMR proteins have been shown to contribute to somatic hypermutation of antibody variable region genes.


Asunto(s)
Enzimas Reparadoras del ADN , Cambio de Clase de Inmunoglobulina/genética , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas/fisiología , Recombinación Genética , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfatasas/fisiología , Animales , Disparidad de Par Base , Secuencia de Bases , Proteínas Portadoras , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Ratones , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Datos de Secuencia Molecular , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas Nucleares
19.
J Exp Med ; 195(3): 367-73, 2002 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-11828012

RESUMEN

B cells from mice deficient in mismatch repair (MMR) proteins show decreased ability to undergo class switch recombination in vitro and in vivo. The deficit is not accompanied by any reduction in cell viability or alterations in the cell cycle in B cells cultured in vitro. To assess the role of MMR in switching we examined the nucleotide sequences of Smicro-Sgamma3 recombination junctions in splenic B cells induced in culture to switch to IgG3. The data demonstrate clear differences in the sequences of switch junctions in wild-type B cells in comparison with Msh2-, Mlh1-, and Pms2-deficient B cells. Sequences of switch junctions from Msh2-deficient cells showed decreased lengths of microhomology between Smicro and Sgamma3 relative to junctions from wild-type cells and an increase in insertions, i.e., nucleotides which do not appear to be derived from either the Smicro or Sgamma3 parental sequence. By contrast, 23% of junctions from Mlh1- and Pms2-deficient cells occurred at unusually long stretches of microhomology. The data indicate that MMR proteins are directly involved in class switching and that the role of Msh2 differs from that of Mlh1 and Pms2.


Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/inmunología , Disparidad de Par Base , Enzimas Reparadoras del ADN , Reparación del ADN/genética , Reparación del ADN/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Cambio de Clase de Inmunoglobulina , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfatasas/deficiencia , Animales , Linfocitos B/inmunología , Secuencia de Bases , Proteínas Portadoras , ADN/genética , Proteínas de Unión al ADN/deficiencia , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Datos de Secuencia Molecular , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/deficiencia , Proteínas Nucleares , Proteínas Proto-Oncogénicas/deficiencia , Recombinación Genética , Homología de Secuencia de Ácido Nucleico
20.
J Exp Med ; 200(3): 321-30, 2004 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-15280420

RESUMEN

The structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3' single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a mismatch repair (MMR) protein involved in class switch recombination (CSR). Therefore, ERCC1-XPF has abilities that might be useful for antibody CSR. We tested whether ERCC1 is involved in CSR and found that Ercc1(-)(/)(-) splenic B cells show moderately reduced CSR in vitro, demonstrating that ERCC1-XPF participates in, but is not required for, CSR. To investigate the role of ERCC1 in CSR, the nucleotide sequences of switch (S) regions were determined. The mutation frequency in germline Smicro segments and recombined Smicro-Sgamma3 segments cloned from Ercc1(-)(/)(-) splenic B cells induced to switch in culture was identical to that of wild-type (WT) littermates. However, Ercc1(-)(/)(-) cells show increased targeting of the mutations to G:C bp in RGYW/WRCY hotspots and mutations occur at sites more distant from the S-S junctions compared with WT mice. The results indicate that ERCC1 is not epistatic with MMR and suggest that ERCC1 might be involved in processing or repair of DNA lesions in S regions during CSR.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Endonucleasas/fisiología , Cambio de Clase de Inmunoglobulina , Recombinación Genética , Animales , Disparidad de Par Base , Reparación del ADN , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Ratones , Ratones Endogámicos C57BL , Proteína 2 Homóloga a MutS , Mutación , Proteínas Proto-Oncogénicas/fisiología
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