RESUMEN
The eukaryal single-stranded DNA binding protein replication protein A (RPA) binds short oligonucleotides with high affinity but exhibits low cooperativity in binding longer templates, opposite to prokaryal counterparts. This discrepancy could reflect the smaller size of the replicative template portion availed to RPA. According to current models, this portion accommodates an RNA-DNA primer (RDP) of <40 nt (nested discontinuity) or a several-fold longer Okazaki fragment (initiation zone). Previous in situ UV-crosslinking revealed that RPA also interacts with nascent DNA, especially growing RDPs. Here we compare nascent SV40 DNA chains UV-crosslinked to the middle and large RPA subunits and use the data to re-examine the two models. The middle subunit interacted with the nascent chains after a few DNA residues were added to the RNA primer while the large subunit became accessible after extension by several more. Upon RDP maturation, the middle subunit disengaged while the large subunit remained accessible during further limited extension. A corresponding shift in preference in favor of the large subunit has been reported for purified RPA and synthetic gapped duplexes upon reduction of the gap from 19 to 9 nt. Combined, these facts support the proposal that the mature RDP faces downstream a correspondingly small gap, possibly created by removal of the RNA primer moiety from an adjacent, previously synthesized RDP (nested discontinuity) but insufficient for continuous elongation of the RDP into an Okazaki fragment (initiation zone).
Asunto(s)
Replicación del ADN/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Virus 40 de los Simios/genética , Animales , Unión Competitiva , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/efectos de la radiación , Cartilla de ADN/genética , ADN Viral/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Genoma Viral , Humanos , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Etiquetas de Fotoafinidad , Unión Proteica , Subunidades de Proteína , Proteína de Replicación A , Moldes GenéticosRESUMEN
Replication protein A (RPA) is a heterotrimeric, multi-functional protein that binds single-stranded DNA (ssDNA) and is essential for eukaryotic DNA metabolism. Using heteronuclear NMR methods we have investigated the domain interactions and ssDNA binding of a fragment from the 70 kDa subunit of human RPA (hRPA70). This fragment contains an N-terminal domain (NTD), which is important for hRPA70-protein interactions, connected to a ssDNA-binding domain (SSB1) by a flexible linker (hRPA70(1-326)). Correlation analysis of the amide (1)H and (15)N chemical shifts was used to compare the structure of the NTD and SSB1 in hRPA70(1-326) with two smaller fragments that corresponded to the individual domains. High correlation coefficients verified that the NTD and SSB1 maintained their structures in hRPA70(1-326), indicating weak interdomain coupling. Weak interdomain coupling was also suggested by a comparison of the transverse relaxation rates for hRPA70(1-326) and one of the smaller hRPA70 fragments containing the NTD and the flexible linker (hRPA70(1-168)). We also examined the structure of hRPA70(1-326) after addition of three different ssDNA substrates. Each of these substrates induced specific amide (1)H and/or (15)N chemical shift changes in both the NTD and SSB1. The NTD and SSB1 have similar topologies, leading to the possibility that ssDNA binding induced the chemical shift changes observed for the NTD. To test this hypothesis we monitored the amide (1)H and (15)N chemical shift changes of hRPA70(1-168) after addition of ssDNA. The same amide (1)H and (15)N chemical shift changes were observed for the NTD in hRPA70(1-168) and hRPA70(1-326). The NTD residues with the largest amide (1)H and/or (15)N chemical shift changes were localized to a basic cleft that is important for hRPA70-protein interactions. Based on this relationship, and other available data, we propose a model where binding between the NTD and ssDNA interferes with hRPA70-protein interactions.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Amidas/metabolismo , Secuencias de Aminoácidos , Secuencia Conservada , ADN de Cadena Simple/genética , Humanos , Cinética , Modelos Moleculares , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , Docilidad , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Proteína de Replicación A , RotaciónRESUMEN
Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3'-end of the upstream oligonucleotide or at the 5'-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5'-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3'-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer-template systems containing 5'- or 3'-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.
Asunto(s)
Replicación del ADN , ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Uridina Trifosfato/análogos & derivados , Azidas/química , Secuencia de Bases , Reactivos de Enlaces Cruzados/química , Daño del ADN , Cartilla de ADN/química , Replicación del ADN/efectos de la radiación , ADN de Cadena Simple/efectos de la radiación , Proteínas de Unión al ADN/efectos de la radiación , Humanos , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/efectos de la radiación , Proteína de Replicación A , Rayos Ultravioleta , Uridina Trifosfato/químicaRESUMEN
Replication Protein A (RPA) is required for DNA recombination, repair and replication in all eukaryotes. RPA participation in these pathways is mediated by single-stranded DNA binding and protein interactions. We herein identify a novel protein, Replication Protein Binding Trans-Activator (RBT1), in a yeast two-hybrid assay employing the second subunit of human RPA (RPA32) as bait. RBT1-RPA32 binding was confirmed by glutathione S:-transferase pull-down and co-immunoprecipitation. Fluorescence microscopy indicates that green fluorescence protein-tagged RBT1 is localized to the nucleus in vivo. RBT1 mRNA expression, determined by semi-quantitative RT-PCR, is significantly higher in cancer cell lines MCF-7, ZR-75, SaOS-2 and H661, compared to the cell lines normal non-immortalized human mammary epithelial cells and normal non-immortalized human bronchial epithelial cells. Further, yeast and mammalian one-hybrid analysis shows that RBT1 is a strong transcriptional co-activator. Interestingly, mammalian transactivation data is indicative of significant variance between cell lines; the GAL4-RBT1 fusion protein has significantly higher transcriptional activity in human cancer cells compared to human normal primary non-immortalized epithelial cells. We propose that RBT1 is a novel transcriptional co-activator that interacts with RPA, and has significantly higher activity in transformed cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Clonación Molecular , ADN , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteína de Replicación A , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos HíbridosRESUMEN
ATM is mutated in the human genetic disorder ataxia telangiectasia, which is characterized by ataxia, immune defects, and cancer predisposition. Cells that lack ATM exhibit delayed up-regulation of p53 in response to ionizing radiation. Serine 15 of p53 is phosphorylated in vivo in response to ionizing radiation, and antibodies to ATM immunoprecipitate a protein kinase activity that, in the presence of manganese, phosphorylates p53 at serine 15. Immunoprecipitates of ATM also phosphorylate PHAS-I in a manganese-dependent manner. Here we have purified ATM from human cells using nine chromatographic steps. Highly purified ATM phosphorylated PHAS-I, the 32-kDa subunit of RPA, serine 15 of p53, and Chk2 in vitro. The majority of the ATM phosphorylation sites in Chk2 were located in the amino-terminal 57 amino acids. In each case, phosphorylation was strictly dependent on manganese. ATM protein kinase activity was inhibited by wortmannin with an IC(50) of approximately 100 nM. Phosphorylation of RPA, but not p53, Chk2, or PHAS-I, was stimulated by DNA. The related protein, DNA-dependent protein kinase catalytic subunit, also phosphorylated PHAS-I, RPA, and Chk2 in the presence of manganese, suggesting that the requirement for manganese is a characteristic of this class of enzyme.
Asunto(s)
Ataxia Telangiectasia/enzimología , Placenta/enzimología , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Androstadienos/farmacología , Especificidad de Anticuerpos , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Quinasa de Punto de Control 2 , Reacciones Cruzadas , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Manganeso , Proteínas Nucleares , Fosforilación , Embarazo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Replicación A , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor , WortmaninaRESUMEN
Exposure of mammalian cells to short-wavelength light (UVC) triggers a global response which can either counteract the deleterious effect of DNA damage by enabling DNA repair or lead to apoptosis. Several stress-activated protein kinases participate in this response, making phosphorylation a strong candidate for being involved in regulating the cellular damage response. One factor that is phosphorylated in a UVC-dependent manner is the 32-kDa subunit of the single-stranded DNA-binding replication protein A (RPA32). RPA is required for major cellular processes like DNA replication, and removal of DNA damage by nucleotide excision repair (NER). In this study we examined the signal which triggers RPA32 hyperphosphorylation following UVC irradiation in human cells. Hyperphosphorylation of RPA was observed in cells from patients with either NER or transcription-coupled repair (TCR) deficiency (A, C, and G complementation groups of xeroderma pigmentosum and A and B groups of Cockayne syndrome, respectively). This exclude both NER intermediates and TCR as essential signals for RPA hyperphosphorylation. However, we have observed that UV-sensitive cells deficient in NER and TCR require lower doses of UV irradiation to induce RPA32 hyperphosphorylation than normal cells, indicating that persistent unrepaired lesions contribute to RPA phosphorylation. Finally, the results of UVC irradiation experiments on nonreplicating cells and S-phase-synchronized cells emphasize a major role for DNA replication arrest in the presence of UVC lesions in RPA UVC-induced hyperphosphorylation in mammalian cells.
Asunto(s)
Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/genética , Línea Celular Transformada , Proteínas de Unión al ADN/metabolismo , Fibroblastos , Humanos , Luz , Fosforilación , Proteína de Replicación ARESUMEN
Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (subunits of 70, 32, and 14 kDa) that is required for cellular DNA metabolism. RPA has been reported to interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examined the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the double helix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that this specific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDa subunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of the damage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest that RPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.
Asunto(s)
Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Secuencia Conservada , ADN/química , Reparación del ADN , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Humanos , Unión Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteína de Replicación A , Proteína de la Xerodermia Pigmentosa del Grupo A , Dedos de ZincRESUMEN
The protein-tyrosine kinase substrate annexin II is a growth regulated gene whose expression is increased in several human cancers. While the precise function of this protein is not understood, annexin II is proposed to be involved in multiple physiological activities, including DNA synthesis and cell proliferation. Targeted disruption of the annexin II gene affects calcium signaling, tyrosine phosphorylation and apoptosis, indicating the important physiological role of this protein. We used a transient co-transfection assay to regulate annexin II expression in human HeLa, 293 and 293T cells, and measured the effects of annexin II down regulation on DNA synthesis and proliferation. Transfection of cells with an antisense annexin II vector results in inhibition of cell division and proliferation, with concomitant reduction in annexin II message and protein levels. Cellular DNA synthesis is significantly reduced in antisense transfected cells. Replication extracts made from antisense transfected cells have significantly reduced efficiency to support SV40 in vitro DNA replication, while the extracts made from sense transfected cells are fully capable of replication. Our results indicate an important role of annexin II in cellular DNA synthesis and cell proliferation.
Asunto(s)
Anexina A2/genética , Anexina A2/metabolismo , División Celular/genética , Células Cultivadas , ADN/biosíntesis , Replicación del ADN , ADN sin Sentido/genética , Regulación hacia Abajo , Regulación de la Expresión Génica , Células HeLa/metabolismo , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Virus 40 de los Simios/genéticaRESUMEN
Human XPA is an essential component in the multienzyme nucleotide excision repair (NER) pathway. The solution structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219) was recently determined [Buchko et al. (1998) Nucleic Acids Res. 26, 2779-2788, Ikegami et al. (1998) Nat. Struct. Biol. 5, 701-706] and shown to consist of a compact zinc-binding core and a loop-rich C-terminal subdomain connected by a linker sequence. Here, the solution structure of XPA-MBD was further refined using an entirely new class of restraints based on pseudocontact shifts measured in cobalt-substituted XPA-MBD. Using this structure, the surface of XPA-MBD which interacts with DNA and a fragment of the largest subunit of replication protein A (RPA70 Delta C327: M1-Y326) was determined using chemical shift mapping. DNA binding in XPA-MBD was highly localized in the loop-rich subdomain for DNA with or without a lesion [dihydrothymidine (dhT) or 6-4-thymidine-cytidine (64TC)], or with DNA in single- or double-stranded form, indicating that the character of the lesion itself is not the driving force for XPA binding DNA. RPA70 Delta C327 was found to contact regions in both the zinc-binding and loop-rich subdomains. Some overlap of the DNA and RPA70 Delta C327 binding regions was observed in the loop-rich subdomain, indicating a possible cooperative DNA-binding mode between XPA and RPA70 Delta C327. To complement the chemical shift mapping data, the backbone dynamics of free XPA-MBD and XPA-MBD bound to DNA oligomers containing dhT or 64TC lesions were investigated using 15N NMR relaxation data. The dynamic analyses for the XPA-MBD complexes with DNA revealed localized increases and decreases in S2 and an increase in the global correlation time. Regions of XPA-MBD with the largest increases in S2 overlapped regions having the largest chemical shifts changes upon binding DNA, indicating that the loop-rich subdomain becomes more rigid upon binding DNA. Interestingly, S2 decreased for some residues in the zinc-binding core upon DNA association, indicating a possible concerted structural rearrangement on binding DNA.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/química , ADN/química , Proteínas de Unión al ARN/química , Sitios de Unión , ADN/metabolismo , Replicación del ADN , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Proteínas de Unión al ARN/metabolismo , Proteína de Replicación A , Soluciones , Termodinámica , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
Human Replication Protein A (hsRPA) is required for multiple cellular processes in DNA metabolism including DNA repair, replication and recombination. It binds single-stranded DNA with high affinity and interacts specifically with multiple proteins. hsRPA forms a heterotrimeric complex composed of 70-, 32- and 14-kDa subunits (henceforth RPA70, RPA32, and RPA14). The N-terminal 168 residues of RPA70 form a structurally distinct domain that stimulates DNA polymerase alpha activity, interacts with several transcriptional activators including tumor suppressor p53, and during the cell cycle it signals escape from the DNA damage induced G2/M checkpoint. We have solved the global fold of the fragment corresponding to this domain (RPA70 delta 169) and we find residues 8-108 of the N-terminal domain are structured. The remaining C-terminal residues are unstructured and may form a flexible linker to the DNA-binding domain of RPA70. The globular region forms a five-stranded anti-parallel beta-barrel. The ends of the barrel are capped by short helices. Two loops on one side of the barrel form a large basic cleft which is a likely site for binding the acidic motifs of transcriptional activators. Many lethal or conditional lethal yeast point mutants map to this cleft, whereas no mutations with severe phenotype have been found in the linker region.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/química , Pliegue de Proteína , Secuencia de Aminoácidos , Proteínas de Unión al ADN/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación Proteica , Proteína de Replicación ARESUMEN
Cytoplasmic extracts from early-passage (young), late-passage (senescent) normal human fibroblast (HF) cultures and immortalized human cell lines (HeLa, HT-1080, and MANCA) were analyzed for their ability to support semiconservative DNA synthesis in an in vitro SV40-ori DNA replication system. Unsupplemented extracts from the three permanent cell lines were demonstrated to be active in this system; whereas young HF extracts were observed to be minimally active, and no activity could be detected in the senescent HF extracts. The activity of these extracts was compared after supplementation with three recombinant human replication factors: (1) the catalytic subunit of DNA polymerase alpha (DNA pol-alpha-cat), (2) the three subunits of replication protein A (RPA), and (3) DNA topoisomerase I (Topo I). The addition of all three recombinant proteins is required for optimum activity in the young and senescent HF extracts; the order of the level of activity is: transformed > young HF > senescent HF. Young HF extracts supplemented with RPA alone are able to support significant replicative activity but not senescent extracts which require both RPA and DNA pol-alpha-cat for any detectable activity. The necessary requirement for these factors is confirmed by the failure of unsupplemented young and senescent extracts to activate MANCA extracts that have been immunodepleted of DNA pol-alpha-cat or RPA. Immunocytochemical studies revealed that RPA, DNA pol-alpha, PCNA, and topo I levels are higher in the immortal cell types used in these studies. In the HF cells, levels of DNA pol-alpha-cat and PCNA are higher (per mg protein) in the low-passage than in the senescent cells. By contrast, RPA levels, as determined by immunocytochemical or Western blot studies, were observed to be similar in both young and senescent cell nuclei. Taken together, these results indicate that the low to undetectable activity of young HF extracts in this system is due mainly to reduced intracellular levels of RPA, while the senescent HF extracts are relatively deficient in DNA polymerase alpha and probably some other essential replication factors, as well as RPA. Moreover, the retention of RPA in the senescent HF nuclei contributes to the low level of this factor in the cytoplasmic extracts from these cells.
Asunto(s)
Senescencia Celular , Replicación del ADN , Western Blotting , Línea Celular Transformada , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Recién Nacido , Masculino , Virus 40 de los Simios/fisiologíaRESUMEN
Human replication protein A (RPA) is a multiple subunit single-stranded DNA-binding protein that is required for multiple processes in cellular DNA metabolism. This complex, composed of subunits of 70, 32, and 14 kDa, binds to single-stranded DNA (ssDNA) with high affinity and participates in multiple protein-protein interactions. The 70-kDa subunit of RPA is known to be composed of multiple domains: an N-terminal domain that participates in protein interactions, a central DNA-binding domain (composed of two copies of a ssDNA-binding motif), a putative (C-X2-C-X13-C-X2-C) zinc finger, and a C-terminal intersubunit interaction domain. A series of mutant forms of RPA were used to elucidate the roles of these domains in RPA function. The central DNA-binding domain was necessary and sufficient for interactions with ssDNA; however, adjacent sequences, including the zinc-finger domain and part of the N-terminal domain, were needed for optimal ssDNA-binding activity. The role of aromatic residues in RPA-DNA interactions was examined. Mutation of any one of the four aromatic residues shown to interact with ssDNA had minimal effects on RPA activity, indicating that individually these residues are not critical for RPA activity. Mutation of the zinc-finger domain altered the structure of the RPA complex, reduced ssDNA-binding activity, and eliminated activity in DNA replication.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Dedos de Zinc , Antígenos Virales de Tumores/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Proteína de Replicación A , Virus 40 de los Simios/inmunología , Xerodermia Pigmentosa/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo A , Dedos de Zinc/genéticaRESUMEN
Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. RPA is required for multiple processes in cellular DNA metabolism. RPA has been reported to (1) bind with high affinity to single-stranded DNA (ssDNA), (2) bind specifically to certain double-stranded DNA (dsDNA) sequences, and (3) have DNA helix-destabilizing ("unwinding") activity. We have characterized both dsDNA binding and helix destabilization. The affinity of RPA for dsDNA was lower than that of ssDNA and precisely correlated with the melting temperature of the DNA fragment. The rates of helix destabilization and dsDNA binding were similar, and both were slow relative to the rate of binding ssDNA. We have previously mapped the regions required for ssDNA binding [Walther et al. (1999) Biochemistry 38, 3963-3973]. Here, we show that both helix-destabilization and dsDNA-binding activities map to the central DNA-binding domain of the 70-kDa subunit and that other domains of RPA are needed for optimal activity. We conclude that all types of RPA binding are manifestations of RPA ssDNA-binding activity and that dsDNA binding occurs when RPA destabilizes a region of dsDNA and binds to the resulting ssDNA. The 70-kDa subunit of all RPA homologues contains a highly conserved putative (C-X2-C-X13-C-X2-C) zinc finger. This motif directly interacts with DNA and contributes to dsDNA-binding/unwinding activity. Evidence is presented that a metal ion is required for the function of the zinc-finger motif.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Dedos de Zinc , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Calor , Humanos , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Unión Proteica/genética , Proteína de Replicación A , Dedos de Zinc/genéticaRESUMEN
UNLABELLED: Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation. DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism. KEYWORDS: camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation
Asunto(s)
Antígenos Nucleares , Camptotecina/farmacología , Daño del ADN/genética , ADN Helicasas , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas Serina-Treonina Quinasas/genética , Alcaloides/farmacología , Androstadienos/farmacología , Ataxia Telangiectasia/genética , Ciclo Celular/genética , Reparación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Proteína Quinasa Activada por ADN , Etopósido/farmacología , Humanos , Autoantígeno Ku , Proteínas Nucleares/genética , Fosforilación , Proteína de Replicación A , Estaurosporina/análogos & derivados , Células Tumorales Cultivadas , WortmaninaRESUMEN
Studies to elucidate the reactions that occur at the eukaryotic replication fork have been limited by the model systems available. We have established a method for isolating and characterizing Simian Virus 40 (SV40) replication complexes. SV40 rolling circle complexes are isolated using paramagnetic beads and then incubated under replication conditions to obtain continued elongation. In rolling circle replication, the normal mechanism for termination of SV40 replication does not occur and the elongation phase of replication is prolonged. Thus, using this assay system, elongation phase reactions can be examined in the absence of initiation or termination. We show that the protein requirements for elongation of SV40 rolling circles are equivalent to complete SV40 replication reactions. The DNA produced by SV40 rolling circles is double-stranded, unmethylated and with a much longer length than the template DNA. These properties are similar to those of physiological replication forks. We show that proteins associated with the isolated rolling circles, including SV40 T antigen, DNA polymerase alpha, replication protein A (RPA) and RF-C, are necessary for continued DNA synthesis. PCNA is also required but is not associated with the isolated complexes. We present evidence suggesting that synthesis of the leading and lagging strands are co-ordinated in SV40 rolling circle replication. We have used this system to show that both RPA-protein and RPA-DNA interactions are important for RPA's function in elongation.
Asunto(s)
ADN Helicasas , Replicación del ADN , ADN Viral/biosíntesis , Desoxirribonucleoproteínas/aislamiento & purificación , Proteínas de Homeodominio , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Virus 40 de los Simios/genética , Transactivadores , Antígenos Virales de Tumores/aislamiento & purificación , ADN Polimerasa I/aislamiento & purificación , ADN Circular , Proteínas de Unión al ADN/aislamiento & purificación , Células Eucariotas , Células HeLa , Humanos , Separación Inmunomagnética , Sustancias Macromoleculares , Antígenos de Histocompatibilidad Menor , Conformación de Ácido Nucleico , Unión Proteica , Proteína de Replicación C , Factores de Tiempo , Replicación ViralRESUMEN
Replication protein A (RPA), a heterotrimeric single-stranded DNA binding protein, is required for recombination, and stimulates homologous pairing and DNA strand exchange promoted in vitro by human recombination protein HsRad51. Co-immunoprecipitation revealed that purified RPA interacts physically with HsRad51, as well as with HsDmc1, the homolog that is expressed specifically in meiosis. The interaction with HsRad51 was mediated by the 70 kDa subunit of RPA, and according to experiments with deletion mutants, this interaction required amino acid residues 169-326. In exponentially growing mammalian cells, 22% of nuclei showed foci of RPA protein and 1-2% showed foci of Rad51. After gamma-irradiation, the percentage of cells with RPA foci increased to approximately 50%, and those with Rad51 foci to 30%. All of the cells with foci of Rad51 had foci of RPA, and in those cells the two proteins co-localized in a high fraction of foci. The interactions of human RPA with Rad51, replication proteins and DNA are suited to the linking of recombination to replication.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Recombinación Genética , Animales , Células Cultivadas , Proteínas de Unión al ADN/aislamiento & purificación , Fibroblastos/metabolismo , Rayos gamma , Humanos , Ratones , Peso Molecular , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Pruebas de Precipitina , Recombinasa Rad51 , Ratas , Proteína de Replicación ARESUMEN
Replication protein A (RPA) is a multisubunit single-stranded DNA-binding (ssDNA) protein that is required for cellular DNA metabolism. RPA homologues have been identified in all eukaryotes examined. All homologues are heterotrimeric complexes with subunits of approximately 70, approximately 32, and approximately 14 kDa. While RPA homologues are evolutionarily conserved, they are not functionally equivalent. To gain a better understanding of the functional differences between RPA homologues, we analyzed the DNA-binding parameters of RPA from human cells and the budding yeast Saccharomyces cerevisiae (hRPA and scRPA, respectively). Both yeast and human RPA bind ssDNA with high affinity and low cooperativity. However, scRPA has a larger occluded binding site (45 nucleotides versus 34 nucleotides) and a higher affinity for oligothymidine than hRPA. Mutant forms of hRPA and scRPA containing the high-affinity DNA-binding domain from the 70-kDa subunit had nearly identical DNA binding properties. In contrast, subcomplexes of the 32- and 14-kDa subunits from both yeast and human RPA had weak ssDNA binding activity. However, the binding constants for the yeast and human subcomplexes were 3 and greater than 6 orders of magnitude lower than those for the RPA heterotrimer, respectively. We conclude that differences in the activity of the 32- and 14-kDa subunits of RPA are responsible for variations in the ssDNA-binding properties of scRPA and hRPA. These data also indicate that hRPA and scRPA have different modes of binding to ssDNA, which may contribute to the functional disparities between the two proteins.
Asunto(s)
Replicación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , ADN Helicasas/química , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Holoenzimas/química , Holoenzimas/genética , Humanos , Peso Molecular , Fragmentos de Péptidos/química , Unión Proteica/genética , Proteína de Replicación A , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Especificidad de la EspecieRESUMEN
Homologs of Escherichia coli RecA recombination protein, which have been found throughout the living kingdom, promote homologous pairing and strand exchange. The nucleoprotein filament, within which strand exchange occurs, has been conserved through evolution, but conservation of the polarity of exchange and the significance of that directionality has not been settled. Using oligonucleotides as substrates, and assays based on fluorescence resonance energy transfer (FRET), we distinguished the biased formation of homologous joints at either end of duplex DNA from the subsequent directionality of strand exchange. As with E. coli RecA protein, the homologous Rad51 proteins from both Homo sapiens (HsRad51) and Saccharomyces cerevisiae (ScRad51) propagated DNA strand exchange preferentially in the 5' to 3' direction. The data suggest that 5' to 3' polarity is a conserved intrinsic property of recombination filaments.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/genética , ADN/metabolismo , Proteínas Fúngicas/metabolismo , Rec A Recombinasas/metabolismo , Recombinación Genética , Composición de Base , Secuencia de Bases , ADN/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Modelos Biológicos , Nucleoproteínas/metabolismo , Recombinasa Rad51 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMEN
Human replication protein A (huRPA) is a multisubunit protein which is involved in DNA replication, repair and recombination processes. It exists as a stable heterotrimer consisting of p70, p32 and p14 subunits. To understand the contribution of huRPA subunits to DNA binding we applied the photoaffinity labeling technique. The photoreactive oligonucleotide was synthesized in situ by DNA polymerases. 5-[N-(2-nitro-5-azidobenzoyl)-trans -3-aminopropenyl-1]deoxyuridine-5'-triphosphate (NABdUTP) was used as substrate for elongation of a radiolabeled primer logical ortemplate either by human DNA polymerase alpha primase (polalpha), human DNA polymerase beta (polbeta) or Klenow fragment of Escherichia coli DNA polymerase I (KF). The polymerase was incubated with NABdUTP and radiolabeled primer-template in the presence or absence of huRPA. The reaction mixtures were then irradiated with monochromatic UV light (315 nm) and the crosslinked products were separated by SDS-PAGE. The results clearly demonstrate crosslinking of the huRPA p70 and p32 subunits with DNA. The p70 subunit appears to bind to the single-stranded part of the DNA duplex, the p32 subunit locates near the 3'-end of the primer, while the p14 subunit locates relatively far from the 3'-end of the primer. This approach opens new possibilities for analysis of huRPA loading on DNA in the course of DNA replication and DNA repair.
Asunto(s)
Azidas/química , Reactivos de Enlaces Cruzados , Proteínas de Unión al ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Etiquetas de Fotoafinidad/química , Uridina Trifosfato/análogos & derivados , Azidas/metabolismo , ADN/química , ADN Polimerasa I/metabolismo , ADN Polimerasa beta/metabolismo , ADN de Cadena Simple/química , Escherichia coli/enzimología , Humanos , Estructura Molecular , Etiquetas de Fotoafinidad/síntesis química , Fotoquímica , Proteína de Replicación A , Moldes Genéticos , Rayos Ultravioleta , Uridina Trifosfato/química , Uridina Trifosfato/metabolismoRESUMEN
Replication Protein A (RPA) from human cells is a stable complex of 70-, 32-, and 14-kDa subunits that is required for multiple processes in DNA metabolism. RPA binds with high affinity to single-stranded DNA and interacts with multiple proteins, including proteins required for the initiation of SV40 DNA replication, DNA polymerase alpha and SV40 large T antigen. We have used a series of mutant derivatives of RPA to map the regions of RPA required for specific protein-protein interactions and have examined the roles of these interactions in DNA replication. T antigen, DNA polymerase alpha and the activation domain of VP16 all have overlapping sites of interaction in the N-terminal half (residues 1-327) of the 70-kDa subunit of RPA. In addition, the interaction site for DNA polymerase alpha is composed of two functionally distinct regions, one (residues 1- approximately 170) which stimulates polymerase activity and a second (residues approximately 170-327) which increases polymerase processivity. In the latter, both the direct protein-protein interaction and ssDNA-binding activities of RPA were needed for RPA to modulate polymerase processivity. We also found that SV40 T antigen inhibited the ability of RPA to increase processivity of DNA polymerase alpha, suggesting that this activity of RPA may be important for elongation but not during the initiation of DNA replication. DNA polymerase alpha, but not T antigen also interacted with the 32- and/or 14-kDa subunits of RPA, but these interactions did not seem to effect polymerase activity.