preparation-mgm101-recombination-protein-mbp-based-tagging

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

State University of New York Upstate Medical University

RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5' untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 5'-->3' exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5'-UTR to allow rapid 5' to 3' degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.

RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation.

RNase mutants traitement de RNA mitochondrial (RNase MRP) auraient d&ucirc; &ecirc;tre divulgu&eacute;s pr&eacute;senter un d&eacute;faut de sortie-de-mitose provoqu&eacute;e par une augmentation des niveaux d&#39;ARNm CLB2, menant &agrave; accru Clb2p niveaux (B-cycline) et un retard de l&#39;anaphase tardive qui en r&eacute;sulte. Nous d&eacute;crivons ici le d&eacute;faut mol&eacute;culaire derri&egrave;re ce retard. CLB2 ARNm normalement dispara&icirc;t rapidement comme compl&egrave;te mitose de cellules, mais le niveau demeure &eacute;lev&eacute; chez les mutants de la RNase MRP. Ceci est en contradiction directe avec les autres mutants de sortie-de-mitose et est le r&eacute;sultat d&#39;une augmentation de la stabilit&eacute; de l&#39;ARNm CLB2. Nous avons trouv&eacute; que hautement purifi&eacute;e RNase MRP cliv&eacute; la r&eacute;gion 5&#39; non traduite (UTR) de l&#39;ARNm CLB2 &agrave; plusieurs endroits dans un test in vitro. In vivo, nous avons identifi&eacute; des produits de clivage de RNase MRP d&eacute;pendant sur l&#39;ARNm CLB2 correspondant &eacute;troitement &agrave; produits in vitro. &Eacute;limination de ces produits &eacute;tait tributaire de la 5&#39;--&gt; 3&#39; exoribonucl&eacute;ase Xrn1 et pas l&#39;exosome. Nos r&eacute;sultats d&eacute;montrent que l&#39;endoribonucl&eacute;ase RNase MRP s&#39;attache sp&eacute;cifiquement l&#39;ARNm CLB2 dans ses 5&#39;-UTR pour permettre rapide 5&#39; &agrave; 3&#39; la d&eacute;gradation par les nucl&eacute;ases de Xrn1. D&eacute;gradation de l&#39;ARNm CLB2 par l&#39;endonucl&eacute;ase de RNase MRP fournit une nouvelle mani&egrave;re de r&eacute;guler le cycle cellulaire qui compl&egrave;te les m&eacute;canismes de d&eacute;gradation des prot&eacute;ines. En outre, ces r&eacute;sultats indiquent un nouveau m&eacute;canisme de d&eacute;gradation de l&#39;ARNm ne pas vu auparavant dans la levure Saccharomyces cerevisiae.

RNase MRP Clive l&#39;ARNm CLB2 afin de promouvoir la progression du cycle cellulaire : nouvelle m&eacute;thode de d&eacute;gradation de l&#39;ARNm.

RNasi mutanti elaborazione RNA mitocondriale (RNasi MRP) hanno dimostrati di avere un difetto di uscita dalla mitosi che &egrave; causato da un aumento dei livelli di mRNA di CLB2, portando a maggiore Clb2p livelli (B-ciclina) e un conseguente ritardo tardo anafase. Qui descriviamo il difetto molecolare dietro questo ritardo. CLB2 mRNA normalmente scompare rapidamente come le cellule mitosi completo, ma il livello rimane alto in mutanti RNasi MRP. Questo &egrave; in diretto contrasto con altri mutanti uscita dalla mitosi ed &egrave; il risultato di un aumento della stabilit&agrave; del mRNA di CLB2. Abbiamo trovato che altamente purificato RNasi MRP spaccati il 5&#39; untranslated region (UTR) del mRNA CLB2 in parecchi posti in un test in vitro. In vivo, abbiamo identificato prodotti di clivaggio RNasi MRP-dipendente su mRNA CLB2 strettamente corrispondente prodotti in vitro. Lo smaltimento di questi prodotti era dipendente da 5&#39;--&gt; 3&#39; esoribonucleasi Xrn1 e non il exosome. I nostri risultati dimostrano che il cromosoma RNasi MRP specificamente fende il mRNA di CLB2 nel suo 5&#39;-UTR per consentire rapido 5&#39; 3&#39; degrado dalla nucleasi di Xrn1. Degradazione del mRNA CLB2 dall&#39;endonucleasi RNasi MRP fornisce un nuovo modo per regolare il ciclo cellulare che integra il macchinario di degradazione della proteina. Inoltre, questi risultati indicano un nuovo meccanismo di degradazione di mRNA non visto prima nel lievito Saccharomyces cerevisiae.

La RNasi MRP fende il mRNA di CLB2 per promuovere la progressione del ciclo cellulare: nuovo metodo di degradazione di mRNA.

In the yeast Saccharomyces cerevisiae, RNase mitochondrial RNA processing (MRP) is an essential endoribonuclease that consists of one RNA component and at least nine protein components. Characterization of the complex is complicated by the fact that eight of the known protein components are shared with a related endoribonuclease, RNase P. To fully characterize the RNase MRP complex, we purified it to apparent homogeneity in a highly active state using tandem affinity purification. In addition to the nine known protein components, both Rpr2 and a protein encoded by the essential gene YLR145w were present in our preparations of RNase MRP. Precipitation of a tagged version of Ylr145w brought with it the RNase MRP RNA, but not the RNase P RNA. A temperature-sensitive ylr145w mutant was generated and found to exhibit a rRNA processing defect identical to that seen in other RNase MRP mutants, whereas no defect in tRNA processing was observed. Homologues of the Ylr145w protein were found in most yeasts, fungi, and Arabidopsis. Based on this evidence, we propose that YLR145w encodes a novel protein component of RNase MRP, but not RNase P. We recommend that this gene be designated RMP1, for RNase MRP protein 1.

Characterization and purification of Saccharomyces cerevisiae RNase MRP reveals a new unique protein component.

Chez la levure Saccharomyces cerevisiae, traitement de RNA mitochondrial RNase (MRP) est une endoribonucl&eacute;ase essentielle qui se compose d&#39;un composant de RNA et au moins neuf composants de prot&eacute;ine. Caract&eacute;risation du complexe est compliqu&eacute;e par le fait que huit des composants prot&eacute;iques connus sont partag&eacute;s avec une endoribonucl&eacute;ase connexe, RNase P. Pour caract&eacute;riser compl&egrave;tement la RNase MRP complexe, nous avons purifi&eacute; il jusqu&#39;&agrave; homog&eacute;n&eacute;it&eacute; apparente dans un &eacute;tat tr&egrave;s actif &agrave; l&#39;aide de purification d&#39;affinit&eacute; en tandem. Outre les composants prot&eacute;iques connus neuf, Rpr2 tant une prot&eacute;ine cod&eacute;e par le g&egrave;ne essentiel YLR145w &eacute;taient pr&eacute;sents dans nos pr&eacute;paratifs de RNase MRP. Pr&eacute;cipitation d&#39;une version &eacute;tiquet&eacute;e de Ylr145w amena la RNase MRP ARN, mais pas la RNase P RNA. Un mutant sensible &agrave; la temp&eacute;rature ylr145w a &eacute;t&eacute; g&eacute;n&eacute;r&eacute; et a trouv&eacute; que pr&eacute;sentent un d&eacute;faut de traitement des ARNr identique &agrave; celle observ&eacute;e chez les autres mutants de RNase MRP, alors qu&#39;aucune irr&eacute;gularit&eacute; dans le traitement de l&#39;ARNt a &eacute;t&eacute; observ&eacute;e. Les homologues de la prot&eacute;ine de Ylr145w ont &eacute;t&eacute; trouv&eacute;s dans la plupart des levures, des champignons et des Arabidopsis. Apr&egrave;s ce t&eacute;moignage, nous proposons que YLR145w code pour un composant de la nouvelle prot&eacute;ine de RNase MRP, mais pas de RNase P. Nous recommandons que ce g&egrave;ne d&eacute;signer RMP1, pour la prot&eacute;ine 1 de RNase MRP.

Caract&eacute;risation et purification de Saccharomyces cerevisiae RNase MRP r&eacute;v&egrave;le un nouveau composant de prot&eacute;ines uniques.

Nel lievito Saccharomyces cerevisiae, elaborazione di RNA mitocondriale RNAsi (MRP) &egrave; un cromosoma essenziale che &egrave; costituito da una componente di RNA e almeno nove componenti proteiche. Caratterizzazione del complesso &egrave; complicata dal fatto che otto dei componenti proteine conosciute sono condivisi con un cromosoma correlato, RNasi P. Per caratterizzare la RNasi MRP complessi, abbiamo purificato e di apparente omogeneit&agrave; in uno stato altamente attivo utilizzando la purificazione di affinit&agrave; tandem. Oltre ai nove componenti proteine conosciute, sia Rpr2 che una proteina codificata dal gene essenziale YLR145w erano presenti nelle nostre preparazioni di RNAsi MRP. Precipitazione di una tag versione del Ylr145w ha portato con s&eacute; la RNasi MRP RNA, ma non la RNasi P RNA. Un mutante ylr145w sensibile alla temperatura &egrave; stato generato e trovato ad per esibire un difetto di elaborazione rRNA identico a quello visto in altri mutanti RNasi MRP, considerando che &egrave; stato osservato alcun difetto nell&#39;elaborazione dei tRNA. Omologhi della proteina Ylr145w sono stati trovati nella maggior parte dei lieviti, funghi e Arabidopsis. Basata su questa prova, proponiamo che YLR145w codifica una componente proteica romanzo di RNAsi MRP, ma non la RNasi P. Si raccomanda che questo gene essere designato RMP1, proteina RNasi MRP 1.

Caratterizzazione e purificazione di Saccharomyces cerevisiae RNasi MRP rivela un nuovo componente proteica unica.

RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components.

RNase MRP est une endoribonucl&eacute;ase ribonucl&eacute;oprot&eacute;ique trouv&eacute;e dans trois endroits cellulaires o&ugrave; sont trait&eacute;es les substrats distincts : les mitochondries, le nucl&eacute;ole et le cytoplasme. Cytoplasmique RNase MRP est l&#39;enzyme nucl&eacute;olaire est transitoirement relocalis&eacute;e au cours de la mitose. Nucl&eacute;olaire RNase MRP (NuMRP) a &eacute;t&eacute; purifi&eacute;e jusqu&#39;&agrave; homog&eacute;n&eacute;it&eacute;, et nous avons purifi&eacute; intensivement le mitochondrial RNase MRP (MtMRP) &agrave; un seul composant de RNA identique &agrave; l&#39;ARN NuMRP. Les constituants prot&eacute;iques de la NuMRP ont &eacute;t&eacute; identifi&eacute;s par spectrom&eacute;trie de masse avec succ&egrave;s, aucune des prot&eacute;ines connues NuMRP trouv&eacute;es dans la pr&eacute;paration de MtMRP. Que des traces du noyau NuMRP prot&eacute;ine, Pop4, ont &eacute;t&eacute; d&eacute;tect&eacute;s en MtMRP par Western blot. Activit&eacute; in vitro de deux enzymes a &eacute;t&eacute; compar&eacute;e. MtMRP s&#39;est fendue seulement mitochondrial ORI5 substrat, tandis que NuMRP cliv&eacute;e tous les trois substrats. Cependant, l&#39;enzyme NuMRP cliv&eacute; le substrat ORI5 aux sites autre que l&#39;enzyme MtMRP. En outre, les diff&eacute;rences enzymatiques de force ionique pr&eacute;f&eacute;r&eacute;e confirment ces enzymes comme des entit&eacute;s distinctes. Magn&eacute;sium s&#39;est av&eacute;r&eacute; essentiel pour les deux enzymes. Nous avons test&eacute; un certain nombre d&#39;inhibiteurs signal&eacute;s notamment la puromycine, pentamidine, lithium et pAp. Inhibition de la puromycine a sugg&eacute;r&eacute; qu&#39;il lie directement &agrave; l&#39;ARN de MRP, r&eacute;affirmant le r&ocirc;le de la composante du RNA dans la catalyse. En conclusion, notre &eacute;tude confirme que les enzymes NuMRP et MtMRP sont des entit&eacute;s distinctes avec diff&eacute;rentes activit&eacute;s et constituants prot&eacute;iques mais une sous-unit&eacute; de RNA commune, ce qui sugg&egrave;re que l&#39;ARN doit jouer un r&ocirc;le crucial dans l&#39;activit&eacute; catalytique.

Comparaison des mitochondries et des nucl&eacute;oles RNase MRP r&eacute;v&egrave;le des composants de RNA identiques avec des activit&eacute;s enzymatiques distinctes et constituants prot&eacute;iques.

La RNasi MRP &egrave; un cromosoma ribonucleoproteina trovato in tre posizioni cellulare dove vengono elaborati distinti substrati: i mitocondri, il nucleolo e citoplasma. Citoplasmatica RNasi MRP &egrave; l&#39;enzima nucleolare &egrave; transitoriamente relocalized durante la mitosi. Nucleolare RNasi MRP (NuMRP) &egrave; stato purificato di omogeneit&agrave;, e abbiamo ampiamente purificato mitocondriale RNasi MRP (MtMRP) per un singolo componente di RNA identici al RNA NuMRP. Sebbene le componenti proteiche del NuMRP sono stati individuati dalla spettrometria di massa con successo, nessuna delle proteine NuMRP note sono stata trovata nella preparazione MtMRP. Solo tracce del nucleo NuMRP della proteina, Pop4, sono state rilevate nel MtMRP di Western blot. Attivit&agrave; in vitro dei due enzimi &egrave; stato confrontato. MtMRP spaccati solo mitocondriale ORI5 substrato, mentre NuMRP spaccati tutti tre substrati. Tuttavia, l&#39;enzima NuMRP spaccati al substrato, ORI5 a siti differenti rispetto l&#39;enzima MtMRP. Inoltre, enzimatiche differenze di forza ionica preferita confermano questi enzimi come entit&agrave; distinte. Magnesio &egrave; stato trovato per essere essenziale per entrambi gli enzimi. Abbiamo testato una serie di inibitori segnalati con puromicina, pentamidina, litio e pAp. Inibizione con puromicina sugger&igrave; che si lega direttamente al RNA MRP, riaffermando il ruolo della componente RNA nella catalisi. In conclusione, il nostro studio conferma che gli enzimi NuMRP e MtMRP sono entit&agrave; distinte con differenti attivit&agrave; e componenti proteiche ma una subunit&agrave; di RNA comune, suggerendo che il RNA deve giocare un ruolo cruciale nell&#39;attivit&agrave; catalitica.

Confronto di mitocondriale e nucleolare RNasi MRP rivela identici componenti di RNA con attivit&agrave; enzimatica distinte e i componenti delle proteine.

RNase mitochondrial RNA processing (MRP) is an essential, evolutionarily conserved endoribonuclease composed of 10 different protein subunits and a single RNA. RNase MRP has established roles in multiple pathways including ribosome biogenesis, cell cycle regulation, and mitochondrial DNA replication. Although each of these functions is important to cell growth, additional functions may exist given the essential nature of the complex. To identify novel RNase MRP substrates, we utilized RNA immunoprecipitation and microarray chip analysis to identify RNA that physically associates with RNase MRP. We identified several new potential substrates for RNase MRP including a cell cycle-regulated transcript, CTS1; the yeast homolog of the mammalian p27(Kip1), SIC1; and the U2 RNA component of the spliceosome. In addition, we found RNase MRP to be involved in the regulation of the Ty1 transposon RNA. These results reinforce and broaden the role of RNase MRP in cell cycle regulation and help to identify new roles of this endoribonuclease.

Global identification of new substrates for the yeast endoribonuclease, RNase mitochondrial RNA processing (MRP).

Traitement de RNA mitochondrial RNase (MRP) est qu&#39;une endoribonucl&eacute;ase essentiel, &eacute;volutivement conserv&eacute; compos&eacute; de 10 sous-unit&eacute;s prot&eacute;iques et un ARN simple. RNase MRP a cr&eacute;&eacute; des r&ocirc;les dans des voies multiples, y compris la biog&eacute;n&egrave;se des ribosomes, la r&eacute;gulation du cycle cellulaire et la r&eacute;plication de l&#39;ADN mitochondriale. Bien que chacune de ces fonctions est importante pour la croissance des cellules, des fonctions suppl&eacute;mentaires peuvent exister compte tenu du caract&egrave;re essentiel du complexe. Pour identifier de nouveaux substrats de RNase MRP, nous avons utilis&eacute; des RNA immunopr&eacute;cipitation et microarray chip analyse pour identifier les ARN qui associe physiquement de RNase MRP. Nous avons identifi&eacute; plusieurs nouveaux substrats potentiels pour RNase MRP, y compris une transcription cycle cellulaire r&eacute;glement&eacute;s, CTS1 ; l&#39;homologue de la levure de la p27(Kip1) chez les mammif&egrave;res, SIC1 ; et la composante ARN U2 du splic&eacute;osome. En outre, nous avons trouv&eacute; RNase MRP &agrave; &ecirc;tre impliqu&eacute; dans la r&eacute;gulation du transposon Ty1 RNA. Ces r&eacute;sultats renforcent et &eacute;largir le r&ocirc;le de RNase MRP dans la r&eacute;gulation du cycle cellulaire et aident &agrave; identifier les nouveaux r&ocirc;les de cette endoribonucl&eacute;ase.

Identification globale de nouveaux substrats pour l&#39;endoribonucl&eacute;ase de levure, RNase ARN mitochondrial (MRP).

Elaborazione di RNA mitocondriale RNAsi (MRP) &egrave; che un cromosoma essenziale, evolutivamente conservato, composto di 10 subunit&agrave; proteiche differenti e un unico RNA. RNasi MRP ha stabilito i ruoli in pi&ugrave; percorsi tra cui biogenesi del ribosoma, regolazione del ciclo cellulare e la replicazione del DNA mitocondriale. Anche se ciascuna di queste funzioni &egrave; importante per la crescita delle cellule, funzioni aggiuntive possono esistere data la natura essenziale del complesso. Per identificare i substrati RNasi MRP romanzo, abbiamo utilizzato RNA immunoprecipitazione e microarray del chip analisi per identificare gli RNA che associa fisicamente con RNasi MRP. Abbiamo individuato diversi nuovi potenziali substrati per RNasi MRP compresa una trascrizione regolata dal ciclo cellulare, CTS1; l&#39;omologo di lievito del p27(Kip1) dei mammiferi, SIC1; e il componente di RNA U2 della spliceosoma. Inoltre, abbiamo trovato la RNasi MRP di essere coinvolto nella regolazione del transposon Ty1 RNA. Questi risultati rafforzano e ampliano il ruolo delle RNasi MRP nella regolazione del ciclo cellulare e contribuiscono a individuare nuovi ruoli di questo cromosoma.

Identificazione globale di nuovi substrati per il cromosoma di lievito, RNase mitochondrial RNA processing (MRP).

Mgm101 is a Rad52-type single-stranded annealing protein (SSAP) required for mitochondrial DNA (mtDNA) repair and maintenance. Structurally, Mgm101 forms large oligomeric rings. Here we determine the function(s) of a 32-amino acid carboxyl-terminal tail (Mgm101(238-269)) conserved in the Mgm101 family of proteins. Mutagenic analysis shows that Lys-253, Trp-257, Arg-259, and Tyr-268 are essential for mtDNA maintenance. Mutations in Lys-251, Arg-252, Lys-260, and Tyr-266 affect mtDNA stability at 37Â°C and under oxidative stress. The Y268A mutation severely affects single-stranded DNA (ssDNA) binding without altering the ring structure. Mutations in the Lys-251-Arg-252-Lys-253 positive triad also affect ssDNA binding. Moreover, the C-tail alone is sufficient to mediate ssDNA binding. Finally, we find that the W257A and R259A mutations dramatically affect the conformation and oligomeric state of Mgm101. These structural alterations correlate with protein degradation in vivo. The data thus indicate that the C-tail of Mgm101, likely displayed on the ring surface, is required for ssDNA binding, higher-order structural organization, and protein stability. We speculate that an initial electrostatic and base-stacking interaction with ssDNA could remodel ring organization. This may facilitate the formation of nucleoprotein filaments competent for mtDNA repair. These findings could have broad implications for understanding how SSAPs promote DNA repair and genome maintenance.

A short carboxyl-terminal tail is required for single-stranded DNA binding, higher-order structural organization, and stability of the mitochondrial single-stranded annealing protein Mgm101.

Mgm101 est une Rad52 de type simple brin recuite prot&eacute;ine (SSAP) n&eacute;cessaire &agrave; l&#39;entretien et la r&eacute;paration de l&#39;ADN (ADN mitochondrial) mitochondriale. Structurellement, Mgm101 forme de grands anneaux oligom&egrave;re. Ici, nous d&eacute;terminons l&#39;ou les fonctions d&#39;une queue de carboxyl terminal acide amine 32 (Mgm101(238-269)) conserv&eacute; dans la famille de Mgm101 des prot&eacute;ines. Mutag&egrave;ne analyse montre que les Lys-253, Trp-257, Arg-259 et Tyr-268 sont essentiels pour l&#39;entretien de l&#39;ADNmt. Des mutations dans Lys-251, 252-Arg, Lys-260 et Tyr-266 affectent la stabilit&eacute; de l&#39;ADN mitochondrial &agrave; 37&deg; C et sous stress oxydatif. La mutation de Y268A affecte gravement les ADN simple brin (ssDNA) contraignant sans modifier la structure de l&#39;anneau. Mutations dans la triade positive Lys-251-Arg-252-Lys-253 affectent &eacute;galement liaison ADN simple brin. Par ailleurs, la queue C seul suffit &agrave; la m&eacute;diation obligatoire d&#39;ADN simple brin. Enfin, nous trouvons que les mutations W257A et R259A un effet dramatique sur la conformation et l&#39;&eacute;tat oligom&egrave;re de Mgm101. Ces alt&eacute;rations structurales sont en corr&eacute;lation avec la d&eacute;gradation des prot&eacute;ines in vivo. Par cons&eacute;quent, les donn&eacute;es indiquent que le C-queue de Mgm101, affich&eacute;e, sur la surface de l&#39;anneau, est requise pour la liaison de l&#39;ADN simple brin, organisation structurelle d&#39;ordre sup&eacute;rieur et stabilit&eacute; des prot&eacute;ines. Nous croyons qu&#39;une interaction &eacute;lectrostatique et empilement base initiale avec l&#39;ADN simple brin pourrait remodeler organisation d&#39;anneau. Cela peut faciliter la formation de filaments de nucl&eacute;oprot&eacute;ine comp&eacute;tentes pour la r&eacute;paration de l&#39;ADN mitochondrial. Ces r&eacute;sultats pourraient avoir des r&eacute;percussions g&eacute;n&eacute;rales permettant de comprendre comment la SSAP promotion entretien r&eacute;paration et g&eacute;nome d&#39;ADN.

Une courte queue carboxyl terminal est n&eacute;cessaire pour la fixation &agrave; l&#39;ADN simple brin, organisation structurelle d&#39;ordre sup&eacute;rieur et la stabilit&eacute; de la prot&eacute;ine mitochondriale de recuit de simple brin Mgm101.

Mgm101 &egrave; una Rad52-tipo single-stranded ricottura (SSAP) per proteina mitocondriale del DNA (mtDNA) riparazione e manutenzione. Strutturalmente, Mgm101 forma grandi anelli oligomerici. Qui noi determinare la funzione di una 32-amino acida carbossilico-terminale coda (Mgm101(238-269)) conservato nella famiglia Mgm101 di proteine. Mutageni analisi dimostra che la Lys-253, Trp-257, 259-Arg e Tyr-268 sono essenziali per il mantenimento del mtDNA. Mutazioni Lys-251, 252-Arg, Lys-260 e Tyr-266 possono influenzare la stabilit&agrave; del DNA mitocondriale a 37&deg; C e sotto stress ossidativo. La mutazione Y268A colpisce gravemente single-stranded DNA (ssDNA) associazione senza alterare la struttura ad anello. Mutazioni nella triade Lys-251-Arg-252-Lys-253 positiva anche interessano ssDNA binding. Inoltre, la coda-C da sola &egrave; sufficiente per mediare ssDNA binding. Infine, troviamo che le mutazioni W257A e R259A possono influenzare drasticamente la conformazione e lo stato oligomerica della Mgm101. Queste alterazioni strutturali si correlano con la degradazione delle proteine in vivo. I dati indicano quindi che la C-coda di Mgm101, probabilmente visualizzata sulla superficie dell&#39;anello, &egrave; necessaria per ssDNA binding, organizzazione strutturale pi&ugrave; alto ordine e stabilit&agrave; proteica. Si ipotizzano che un&#39;interazione elettrostatica e accatastamento di base iniziale con ssDNA potrebbe rimodellare organizzazione anello. Ci&ograve; pu&ograve; facilitare la formazione di filamenti di nucleoproteine competente per la riparazione del DNA mitocondriale. Queste scoperte potrebbero avere ampie implicazioni per comprendere come SSAP promuovere manutenzione riparazione e del genoma del DNA.

Una breve coda carbossi-terminale &egrave; necessaria per single-stranded DNA binding, organizzazione strutturale pi&ugrave; alto ordine e stabilit&agrave; della single-stranded mitocondriale ricottura proteina Mgm101.

Article	Year
RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation. Molecular and cellular biology\| PubMed ID: 14729943	2004
Characterization and purification of Saccharomyces cerevisiae RNase MRP reveals a new unique protein component. The Journal of biological chemistry\| PubMed ID: 15637077	2005
Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components. RNA (New York, N.Y.)\| PubMed ID: 20086051	2010
Global identification of new substrates for the yeast endoribonuclease, RNase mitochondrial RNA processing (MRP). The Journal of biological chemistry\| PubMed ID: 22977255	2012
A short carboxyl-terminal tail is required for single-stranded DNA binding, higher-order structural organization, and stability of the mitochondrial single-stranded annealing protein Mgm101. Molecular biology of the cell\| PubMed ID: 23536705	2013

Sara Wierzbicki

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