inducing-site-specific-replication-blockage-e-coli-using-fluorescent

Inducing a Site Specific Replication Blockage in <i>E. coli</i> Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

University of Newcastle

Although work-related driving is associated with high accident rates, limited research has investigated the factors influencing driving crashes in the work setting. This study explored multilevel influences on self-reported crashes in the workplace by surveying a sample of work-related drivers (n = 380), their workgroup supervisors (n = 88), and fleet managers (n = 47). At the driver level of analysis, safety motivation predicted self-reported crashes. In turn, drivers' perceptions of their fleet managers' safety values (but not drivers' perceptions of their supervisors' safety values), their own attitudes, and their own efficacy beliefs predicted motivation to drive safely. Furthermore, the influence of supervisors and fleet managers interacted such that drivers were more motivated to drive safely if they perceived both their supervisor and fleet manager to value safety. This study also explored the cross-level relationships between supervisors' and fleet managers' perceptions of organizational safety values and drivers' perceptions of managerial safety values and found a relationship between fleet managers' perceptions of organizational safety values and drivers' perceptions of fleet managers' safety values. These results illustrate that perceptions of workplace safety values are transmitted across levels of the organization.

Safety in work vehicles: a multilevel study linking safety values and individual predictors to work-related driving crashes.

We present a method to estimate the spatial distribution of dredged material disposed of at sea. Using both dredged sediments and samples of sea-bed sediment from near the Rame Head disposal site, Plymouth, UK, we applied entropy analysis to the <63 microm sediment fraction and combined the results with the trace metal data in the same fraction, to form a series of groups. We interpret the distribution of sediments in one group (F1) to approximate the distribution of material affected by the disposal site. This distribution includes locations close to the disposal site, and also locations <4 km to the SE and SW, <6 km to the NW and <2 km to the N. This approach demonstrates the feasibility of using trace metal analysis of particular grain size fractions to reduce uncertainty in interpreting the spatial distribution of impacts of dredge disposal.

Estimating the spatial distribution of dredged material disposed of at sea using particle-size distributions and metal concentrations.

In this study, the authors investigated how leader vision influences the change-oriented behaviors of adaptivity and proactivity in the workplace. The authors proposed that leader vision would lead to an increase in adaptivity for employees who were high in openness to work role change. In contrast, they proposed leader vision would be associated with an increase in proactivity when employees were high in role breadth self-efficacy. These propositions were supported in a longitudinal survey of 102 employees who provided self-report data about their leader and their work behaviors. The findings provide insight into the interaction between leaders and followers in responding to a change imperative.

Leader vision and the development of adaptive and proactive performance: a longitudinal study.

This research reports on two field studies which demonstrate that self-leadership training decreases strain via increases in self-efficacy and positive affect. The first, an experimental study, found that strain was reduced in the randomly assigned training group, but not in the control group. The second was a longitudinal study and supported the hypotheses that self-efficacy and positive affect mediated the effect of self-leadership training on strain. Our findings extend both self-leadership and stress management literatures by providing a theoretical framework within which the effects of self-leadership on strain can be understood. Practically speaking, our findings suggest that self-leadership training offers an individual-level preventive approach to stress management.

Help yourself: the mechanisms through which a self-leadership intervention influences strain.

Observations of prenatal movement patterns of mouth and lips essential for feeding could have the potential for an assessment of the readiness to feed after birth. Although there is some research on sucking per se, we know very little about prenatal preparatory movements for sucking, namely, the ability to co-ordinate opening the mouth widely and then pursing the lips as if around a teat or nipple in utero. The purpose of the present study was to test two hypotheses using an adapted version of the Facial Action Coding Scheme: first that mouth stretch (AU 27) will be followed by lip pucker (AU 18), and second that these coordinated movement patterns will increase as a function of gestational age. Fifteen healthy fetuses were scanned four times between 24 and 36 weeks gestation using 4D ultrasound visualization. Results showed a decreased number of mouth stretches with increasing fetal age. Contrary to our expectations, we did not find an increase in movement patterns of mouth stretch followed by lip pucker in preparation for feeding ex utero. The results are discussed in terms of sensory triggers in utero required to elicit preparatory movements for feeding ex utero.

Prenatal Mouth Movements: Can We Identify Co-Ordinated Fetal Mouth and LIP Actions Necessary for Feeding?

&#x5355;&#x94FE; DNA &#x7ED3;&#x5408;&#x86CB;&#x767D; &#xFF08;&#x529E;&#x5B66;&#x56E2;&#x4F53;) &#x662F;&#x65E0;&#x5904;&#x4E0D;&#x5728;&#x7684;&#x5BE1;&#x805A;&#x86CB;&#x767D;&#xFF0C;&#x7ED1;&#x5B9A;&#x5177;&#x6709;&#x5F88;&#x9AD8;&#x7684;&#x4EB2;&#x548C;&#x529B;&#xFF0C;&#x5BF9;&#x5355;&#x94FE; DNA &#x548C; DNA &#x4EE3;&#x8C22;&#x4E2D;&#x6709;&#x5404;&#x79CD;&#x5404;&#x6837;&#x7684;&#x91CD;&#x8981;&#x89D2;&#x8272;&#x3002;&#x7EB3;&#x5347;&#x7535;&#x55B7;&#x96FE;&#x7535;&#x79BB;&#x8D28;&#x8C31; (nanoESI-MS) &#x7528;&#x6765;&#x76D1;&#x6D4B;&#x5728;&#x5168;&#x957F;&#x548C;&#x622A;&#x65AD;&#x5F62;&#x5F0F; homotetrameric SSB &#x4ECE;&#x5927;&#x80A0;&#x6746;&#x83CC;&#x7684;&#x4E9A;&#x5355;&#x4F4D;&#x4EA4;&#x6D41;&#x3002;&#x4E2D;&#x7684;&#x672C;&#x673A;&#x7684;&#x86CB;&#x767D;&#x8D28;&#x4E9A;&#x5355;&#x4F4D;&#x4EA4;&#x6362;&#x627E;&#x5230;&#x4E86;&#x4E00;&#x6BB5;&#x7684;&#x65F6;&#x95F4;&#xFF0C;&#x6162;&#x6162;&#x5730;&#x53D1;&#x751F;&#xFF0C;&#x4F46;&#x88AB;&#x5927;&#x5927;&#x66F4;&#x8FC5;&#x901F;&#x622A;&#x65AD;&#x7684;&#x5907;&#x9009;&#x6848;&#x6587;&#x7684; SSB &#x4ECE;&#x4E2D;&#x7684;&#x516B;&#x4E2A; C &#x7EC8;&#x7AEF;&#x6B8B;&#x7559;&#x7269;&#x5DF2;&#x88AB;&#x5220;&#x9664;&#x3002;&#x8FD9;&#x79CD;&#x6548;&#x679C;&#x88AB;&#x63D0;&#x8BAE;&#x5BFC;&#x81F4;&#x4ECE; C-&#x603B;&#x7AD9;&#x4ECB;&#x5BFC;&#x7684;&#x7A33;&#x5B9A;&#x7684; SSB &#x56DB;&#x805A;&#x4E19;&#x70EF;&#xFF0C;C &#x603B;&#x7AD9;&#x4E0E;&#x76F8;&#x90BB;&#x7684;&#x4E9A;&#x57FA;&#x7684; DNA &#x7ED3;&#x5408;&#x5185;&#x6838;&#x8FDB;&#x884C;&#x4EA4;&#x4E92;&#x3002;NanoESI MS &#x4E5F;&#x7528;&#x6765;&#x68C0;&#x67E5; DNA &#x7ED1;&#x5B9A;&#x5230; SSB &#x56DB;&#x805A;&#x4E19;&#x70EF;&#x3002;&#x7ED1;&#x5B9A;&#x7684;&#x5355;&#x94FE;&#x5BE1;&#x6838;&#x82F7;&#x9178; [&#x7684; &#xFF08;dT)(70)&#xFF0C;&#xFF08;dT)(35) &#x6216; (dT)(35) &#x7684;&#x4E24;&#x4E2A;&#x5206;&#x5B50;&#x7684;&#x4E00;&#x4E2A;&#x5206;&#x5B50;&#x4E00;&#x4E2A;&#x5206;&#x5B50;] &#x88AB;&#x53D1;&#x73B0;&#xFF0C;&#x9632;&#x6B62; SSB &#x4E9A;&#x5355;&#x4F4D;&#x4EA4;&#x6362;&#x3002;&#x79BB;&#x6563;&#x5BE1;&#x6838;&#x82F7;&#x9178;&#x4E4B;&#x95F4;&#x7684; SSB &#x56DB;&#x805A;&#x7269;&#x72B6;&#x6001;&#x8F6C;&#x79FB;&#x4E5F;&#x6709;&#x4EBA;&#x5E76;&#x7B26;&#x5408;&#x4ECE;&#x89E3;&#x51B3;&#x65B9;&#x6848;&#x9636;&#x6BB5;&#x7684;&#x7814;&#x7A76;&#xFF0C;&#x9884;&#x6D4B;&#x8868;&#x660E; SSB-DNA &#x590D;&#x5408;&#x7269;&#xFF0C;&#x53EF;&#x4EE5;&#x53EF;&#x9760;&#x5730;&#x5206;&#x6790; ESI &#x8D28;&#x8C31;&#x6CD5;&#x3002;

&#x5927;&#x80A0;&#x57C3;&#x5E0C;&#x6C0F;&#x5927;&#x80A0;&#x6746;&#x83CC;&#x5355;&#x94FE; DNA &#x7ED3;&#x5408;&#x86CB;&#x767D;&#xFF1A; &#x76D0;&#x8C03;&#x5236;&#x7684;&#x4E9A;&#x5355;&#x4F4D;&#x4EA4;&#x6362;&#x548C; DNA &#x7ED1;&#x5B9A;&#x4EA4;&#x6613;&#x7684; nanoESI-&#x8D28;&#x8C31;&#x7814;&#x7A76;&#x3002;

Single-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)(70), one molecule of (dT)(35), or two molecules of (dT)(35)] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.

Escherichia coli single-stranded DNA-binding protein: nanoESI-MS studies of salt-modulated subunit exchange and DNA binding transactions.

Prot&eacute;ines de liaison &agrave; l&#39;ADN simple brin (BSN) sont des prot&eacute;ines oligom&egrave;res omnipr&eacute;sents qui se lient avec une tr&egrave;s forte affinit&eacute; &agrave; l&#39;ADN simple brin et ont une vari&eacute;t&eacute; d&#39;un r&ocirc;le essentiel dans le m&eacute;tabolisme de l&#39;ADN. SPECTROMETRIE ionization mass spectrometry (nanoESI-MS) a &eacute;t&eacute; utilis&eacute; pour surveiller l&#39;&eacute;change de sous-unit&eacute; sous formes de pleine longueur et tronqu&eacute;es de la homot&eacute;tram&egrave;res SSB d&#39;Escherichia coli. &Eacute;change de la sous-unit&eacute; de la prot&eacute;ine native s&#39;est av&eacute;r&eacute; se passent lentement pendant des heures, mais &eacute;tait significativement plus rapide &agrave; une variante tronqu&eacute;e de la BSR dont huit r&eacute;sidus C-terminal ont &eacute;t&eacute; supprim&eacute;s. Cet effet est propos&eacute; &agrave; r&eacute;sulter de l&#39;extr&eacute;mit&eacute; C-terminale m&eacute;di&eacute;e par stabilisation du t&eacute;tram&egrave;re SSB, dont les extr&eacute;mit&eacute;s C-terminales interagissent avec les noyaux de liaison &agrave; l&#39;ADN des sous-unit&eacute;s adjacentes. NanoESI-MS a &eacute;galement permis d&#39;examiner DNA binding pour le t&eacute;tram&egrave;re SSB. Liaison des oligonucl&eacute;otides monocat&eacute;naires [une mol&eacute;cule de (dT)(70), une mol&eacute;cule de (dT)(35), ou deux mol&eacute;cules de (dT)(35)] a &eacute;t&eacute; trouv&eacute;e pour emp&ecirc;cher exchange de sous-unit&eacute; SSB. Transfert de t&eacute;tram&egrave;res SSB entre oligonucl&eacute;otides discrets a &eacute;galement &eacute;t&eacute; observ&eacute; et est conforme aux pr&eacute;dictions d&#39;&eacute;tudes de la phase soluble, ce qui sugg&egrave;re que complexes ADN-SSB peuvent &ecirc;tre analys&eacute;es de mani&egrave;re fiable par spectrom&eacute;trie de masse ESI.

Prot&eacute;ine de liaison &agrave; l&#39;ADN simple brin Escherichia coli: &eacute;tudes nanoESI-MS de l&#39;&eacute;change de sous-unit&eacute; modul&eacute;s en sel et des op&eacute;rations de liaison de l&#39;ADN.

Single-stranded DNA-binding proteins (SSB) sono onnipresenti oligomerici proteine che legano con affinit&agrave; molto elevata per single-stranded DNA e hanno una variet&agrave; di ruoli essenziali nel metabolismo del DNA. Spettrometria di massa ionizzazione Nanoelectrospray (nanoESI-MS) &egrave; stata utilizzata per monitorare exchange subunit&agrave; in forme full-length e troncate di homotetrameric SSB da Escherichia coli. Scambio di subunit&agrave; della proteina nativa &egrave; stato trovato a verificarsi lentamente in un periodo di ore, ma era significativamente pi&ugrave; rapida in una variante troncata di SSB, da cui sono stati eliminati i otto residui C-terminale. Questo effetto &egrave; proposto come risultato di stabilizzazione C-terminale mediata del tetramero SSB, in cui la C-termini interagiscono con i nuclei di DNA-binding di subunit&agrave; adiacenti. NanoESI-MS &egrave; stato utilizzato anche per esaminare il legame del DNA per il tetramero SSB. Associazione dei oligonucleotides singolo filamento [una molecola di (dT)(70), una molecola di (dT)(35), o due molecole di (dT)(35)] &egrave; stato trovato per evitare scambio di subunit&agrave; SSB. Trasferimento di tetrameri SSB tra oligonucleotidi discreti &egrave; stato osservato anche ed &egrave; coerenza con le previsioni da studi di fase di soluzione, suggerendo che complessi di DNA-SSB possono essere analizzati in maniera affidabile mediante spettrometria di massa ESI.

Escherichia coli single-stranded DNA-binding protein: studi nanoESI-MS exchange sale-modulato subunit&agrave; e transazioni di associazione del DNA.

&#x4E00;&#x672C;&#x9396; DNA &#x7D50;&#x5408;&#x86CB;&#x767D;&#x8CEA; (SSBs)&#x3001;&#x3055;&#x307E;&#x3056;&#x307E;&#x306A;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x306E; DNA &#x4EE3;&#x8B1D;&#x306E;&#x4E00;&#x672C;&#x9396; DNA &#x306B;&#x975E;&#x5E38;&#x306B;&#x9AD8;&#x3044;&#x89AA;&#x548C;&#x6027;&#x3092;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x3057;&#x3066;&#x30E6;&#x30D3;&#x30AD;&#x30BF;&#x30B9;&#x306E;&#x30AA;&#x30EA;&#x30B4;&#x30DE;&#x30FC;&#x86CB;&#x767D;&#x8CEA;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;Nanoelectrospray &#x30A4;&#x30AA;&#x30F3;&#x5316;&#x8CEA;&#x91CF;&#x5206;&#x6790; &#xFF08;&#x5B89;&#x4FA1;&#xFF09; &#x5927;&#x8178;&#x83CC;&#x7531;&#x6765; homotetrameric SSB &#x306E;&#x30D5;&#x30EB;&#x306E;&#x9577;&#x3055;&#x3068;&#x5207;&#x308A;&#x6368;&#x3066;&#x3089;&#x308C;&#x305F;&#x5F62;&#x3067;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8; exchange &#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x86CB;&#x767D;&#x8CEA;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x4EA4;&#x63DB;&#x3086;&#x3063;&#x304F;&#x308A;&#x3068;&#x6642;&#x9593;&#x306E;&#x671F;&#x9593;&#x306B;&#x308F;&#x305F;&#x3063;&#x3066;&#x767A;&#x751F;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x308F;&#x304B;&#x3063;&#x305F;&#x304C;&#x3001;&#x5207;&#x308A;&#x6368;&#x3066;&#x3089;&#x308C;&#x305F;&#x4E00;&#x7A2E;&#x306E; 8 &#x306E; C &#x672B;&#x7AEF;&#x30A2;&#x30DF;&#x30CE;&#x9178;&#x6B8B;&#x57FA;&#x304C;&#x524A;&#x9664;&#x3055;&#x308C;&#x305F; SSB &#x3067;&#x975E;&#x5E38;&#x306B;&#x8FC5;&#x901F;&#x3060;&#x3063;&#x305F;&#x3002;&#x3053;&#x306E;&#x52B9;&#x679C;&#x304C; C &#x30C6;&#x30EB;&#x30DF;&#x30CB;&#x99C5;&#x304C;&#x96A3;&#x63A5;&#x3059;&#x308B;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E; DNA &#x7D50;&#x5408;&#x30B3;&#x30A2;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3001;SSB 4 &#x91CF;&#x4F53;&#x306E; C &#x672B;&#x7AEF;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x5B89;&#x5B9A;&#x5316;&#x306B;&#x8D77;&#x56E0;&#x3059;&#x308B;. &#x3057;&#x307E;&#x3059;&#x3002;&#x5B89;&#x4FA1; MS &#x3082; DNA &#x7D50;&#x5408; SSB 4 &#x91CF;&#x4F53;&#x3092;&#x8ABF;&#x67FB;&#x3059;&#x308B;&#x3053;&#x3068;&#x3060;&#x3063;&#x305F;&#x3002;&#x4E00;&#x672C;&#x9396;&#x30AA;&#x30EA;&#x30B4;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9; [&#xFF08;dT)(70)&#x3001;1 &#x3064;&#x306E;&#x5206;&#x5B50;&#x306E; dT)(35)&#x3001;(dT)(35) &#x306E; 2 &#x3064;&#x306E;&#x5206;&#x5B50;&#x306E; 1 &#x5206;&#x5B50;] &#x306E;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x306F; SSB &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x4EA4;&#x63DB;&#x3092;&#x9632;&#x3050;&#x305F;&#x3081;&#x306B;&#x767A;&#x898B;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;SSB &#x30C6;&#x30C8;&#x30E9;&#x30DE;&#x30FC;&#x96E2;&#x6563;&#x30AA;&#x30EA;&#x30B4;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x9593;&#x8EE2;&#x9001;&#x3082;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x305F;&#x3042;&#x308A; ESI &#x8CEA;&#x91CF;&#x5206;&#x6790;&#x6CD5;&#x306B;&#x3088;&#x308B; SSB DNA &#x8907;&#x5408;&#x4F53;&#x3092;&#x78BA;&#x5B9F;&#x306B;&#x5206;&#x6790;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x4E00;&#x8CAB;&#x6027;&#x306E;&#x3042;&#x308B;&#x4E88;&#x6E2C;&#x30BD;&#x30EA;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3; &#x30D5;&#x30A7;&#x30FC;&#x30BA;&#x306E;&#x7814;&#x7A76;&#x304B;&#x3089;&#x3002;

&#x5927;&#x8178;&#x83CC;&#x5927;&#x8178;&#x83CC;&#x4E00;&#x672C;&#x9396; DNA &#x7D50;&#x5408;&#x86CB;&#x767D;&#x8CEA;: &#x5869;&#x5909;&#x8ABF;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x4EA4;&#x63DB;&#x3068; DNA &#x7D50;&#x5408;&#x30C8;&#x30E9;&#x30F3;&#x30B6;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x5B89;&#x4FA1; MS &#x7814;&#x7A76;&#x3002;

&#xB2E8;&#xC77C; &#xAC00;&#xB2E5; DNA &#xC758;&#xBB34;&#xC801;&#xC778; &#xB2E8;&#xBC31;&#xC9C8; (SSBs) &#xB2E8;&#xC77C; &#xAC00;&#xB2E5; DNA &#xB9E4;&#xC6B0; &#xB192;&#xC740; &#xC120;&#xD638;&#xB3C4;&#xC640; &#xBC14;&#xC778;&#xB529; &#xBC0F; DNA &#xBB3C;&#xC9C8; &#xB300;&#xC0AC;&#xC5D0; &#xC788;&#xB294; &#xD544;&#xC218; &#xC5ED;&#xD560;&#xC758; &#xB2E4;&#xC591; &#xD55C; &#xC720;&#xBE44; &#xCFFC;&#xD130; &#xC2A4; oligomeric &#xB2E8;&#xBC31;&#xC9C8;&#xC740;. Nanoelectrospray &#xC774;&#xC628;&#xD654; &#xC9C8;&#xB7C9; &#xBD84;&#xC11D; (MS nanoESI)&#xB294; &#xB300;&#xC7A5;&#xADE0;&#xC5D0;&#xC11C; SSB homotetrameric&#xC758; &#xC804;&#xCCB4; &#xAE38;&#xC774; &#xBC0F; &#xC798;&#xB9B0; &#xD615;&#xD0DC;&#xC5D0;&#xC11C; &#xC18C; &#xB2E8;&#xC704; exchange&#xB97C; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9; &#xB418;&#xC5C8;&#xB2E4;. &#xAE30;&#xBCF8; &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0;&#xC11C; &#xC18C; &#xB2E8;&#xC704; &#xAD50;&#xD658; &#xC2DC;&#xAC04;&#xC758; &#xAE30;&#xAC04; &#xB3D9;&#xC548; &#xCC9C;&#xCC9C;&#xD788; &#xBC1C;&#xC0DD;&#xC744; &#xBC1C;&#xACAC; &#xD588;&#xC9C0;&#xB9CC; SSB 8 C &#xD130;&#xBBF8;&#xB110; &#xC794;&#xB958;&#xBB3C; &#xD588;&#xC9C0;&#xB9CC; &#xC0AD;&#xC81C;&#xC758; &#xC798;&#xB9B0;&#xB41C; &#xBCC0;&#xC885;&#xC5D0; &#xD6E8;&#xC52C; &#xB354; &#xBE60;&#xB978;. &#xC774; &#xD6A8;&#xACFC; C-&#xB9D0;&#xB2E8; &#xC911;&#xC7AC; &#xC548;&#xC815;&#xD654; SSB tetramer, C-&#xD14C;&#xB974;&#xBBF8;&#xB2C8; &#xC778;&#xC811; subunits&#xC758; DNA &#xBB36;&#xB294; &#xCF54;&#xC5B4;&#xC640; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC758; &#xACB0;&#xACFC;&#xB85C; &#xC81C;&#xC548; &#xD569;&#xB2C8;&#xB2E4;. NanoESI MS SSB tetramer &#xBC14;&#xC778;&#xB529;&#xD560; DNA &#xAC80;&#xC0AC;&#xC5D0; &#xC0AC;&#xC6A9; &#xB418;&#xC5C8;&#xB2E4;. &#xB2E8;&#xC77C; &#xAC00;&#xB2E5; oligonucleotides [&#xD55C; &#xBD84;&#xC790; (dT)(70), (dT)(35), &#xB610;&#xB294; (Dt)(35)&#xC758; 2 &#xAC1C;&#xC758; &#xBD84;&#xC790;&#xC5D0;&#xC758; &#xD55C; &#xBD84;&#xC790;&#xC758;]&#xC758; &#xBC14;&#xC778;&#xB529; SSB &#xC18C; &#xB2E8;&#xC704; &#xAD50;&#xD658; &#xBC29;&#xC9C0;&#xB97C; &#xBC1C;&#xACAC;. &#xC774;&#xC0B0; oligonucleotides &#xC0AC;&#xC774; SSB tetramers&#xC758; &#xC804;&#xC1A1; &#xB610;&#xD55C; &#xAD00;&#xCC30; &#xB418;&#xC5C8;&#xB2E4; &#xC774;&#xBA70; &#xC608;&#xCE21; &#xC194;&#xB8E8;&#xC158; &#xB2E8;&#xACC4; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xC77C;&#xAD00; &#xB41C; &#xC81C;&#xC548; SSB DNA &#xB2E8;&#xC9C0; &#xC548;&#xC815;&#xC801;&#xC73C;&#xB85C; ESI &#xC9C8;&#xB7C9; &#xBD84;&#xC11D; &#xD558; &#xC5EC; &#xBD84;&#xC11D; &#xB420; &#xC218; &#xC788;&#xB2E4;.

&#xBCD1;&#xC6D0; &#xC131; &#xB300;&#xC7A5;&#xADE0; &#xB2E8;&#xC77C; &#xAC00;&#xB2E5; DNA &#xC758;&#xBB34;&#xC801;&#xC778; &#xB2E8;&#xBC31;&#xC9C8;: nanoESI MS &#xC5F0;&#xAD6C; &#xC18C;&#xAE08; &#xBCC0;&#xC870; &#xC18C; &#xB2E8;&#xC704; &#xAD50;&#xD658; &#xBC0F; DNA &#xBC14;&#xC778;&#xB529; &#xAC70;&#xB798;&#xC758;.

Prote&iacute;nas de uni&oacute;n al ADN monocatenario (SSBs) son prote&iacute;nas oligom&eacute;ricas omnipresentes que se unen con gran afinidad a ADN monocatenario y tienen una variedad de roles esenciales en el metabolismo del ADN. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) fue utilizada para supervisar el intercambio de la subunidad en formas truncadas y larga duraci&oacute;n de la homotetrameric SSB de Escherichia coli. Intercambio de la subunidad de la prote&iacute;na nativa fue encontrado para ocurrir lentamente durante un per&iacute;odo de horas, pero fue significativamente m&aacute;s r&aacute;pida en una variante truncada de SSB desde que se eliminaron los ocho residuos c-terminal. Este efecto se propone como resultado de la estabilizaci&oacute;n del c-terminal mediada de la tetr&aacute;mero SSB, en el que los t&eacute;rminos C interact&uacute;an con los corazones de uni&oacute;n al ADN de subunidades adyacentes. NanoESI-MS tambi&eacute;n fue utilizado para examinar la DNA obligatorio para el tetr&aacute;mero SSB. Enlace de monocatenario oligonucle&oacute;tidos [una mol&eacute;cula de (dT)(70), una mol&eacute;cula de (dT)(35), o dos mol&eacute;culas de (dT)(35)] fue encontrado para evitar intercambio de subunidad SSB. Transferencia de tetr&aacute;meros SSB entre oligonucle&oacute;tidos discretos tambi&eacute;n se observ&oacute; y es consistente con las predicciones de los estudios de la fase de soluci&oacute;n, lo que sugiere que complejos de SSB-DNA pueden analizarse confiablemente por espectrometr&iacute;a de masas ESI.

Prote&iacute;na de uni&oacute;n a ADN de cadena simple de Escherichia coli: nanoESI-MS estudios de intercambio de subunidad modulada de sal y las operaciones de uni&oacute;n de ADN.

&#x845B;&#x7531; &alpha;&#x25B;&theta; &#x6838;&#x5FC3;&#x7684;&#x5927;&#x80A0;&#x57C3;&#x5E0C;&#x6C0F;&#x5927;&#x80A0;&#x6746;&#x83CC; Pol III &#x590D;&#x5236;&#x9176;&#x7684; DNA &#x5408;&#x6210;&#xFF0C;&#x9700;&#x8981;&#x901A;&#x8FC7;&#x5728; &alpha; &#x805A;&#x5408;&#x9176;&#x4E9A;&#x5355;&#x4F4D;&#x7684;&#x7F51;&#x7AD9;&#x7ED1;&#x5B9A;&#x5230; &beta;(2) &#x5939;&#x3002;&#x4EE5;&#x524D;&#x4E0D;&#x7406;&#x89E3;&#x7684; &alpha; &#x662F;&#x6821;&#x5BF9;&#x5916;&#x5207;&#x4E9A;&#x57FA; &#x25B; &#x5982;&#x4F55;&#x5F71;&#x54CD; DNA &#x5408;&#x6210;&#x3002;&#x5728;&#x8FD9;&#x9879;&#x5DE5;&#x4F5C;&#xFF0C;&#x6279;&#x91CF;&#x68C0;&#x6D4B; DNA &#x7684;&#x590D;&#x5236;&#x88AB;&#x7528;&#x6765;&#x63ED;&#x9732;&#x975E;&#x6821;&#x5BF9;&#x6D3B;&#x52A8;&#x7684; &#x25B;&#x3002; &#x8BF1;&#x53D8;&#x751F;&#x7269;&#x7269;&#x7406;&#x7814;&#x7A76;&#x548C;&#x5355;&#x5206;&#x5B50;&#x9886;&#x5148;&#x7EDE;&#x7EBF;&#x590D;&#x5236;&#x68C0;&#x5B9A;&#x6CD5;&#x4E0E;&#x7EC4;&#x5408;&#x8FFD;&#x6EAF;&#x5230;&#x65B0;&#x578B; &beta;-&#x7ED1;&#x5B9A;&#x7AD9;&#x70B9; &#x25B;&#xFF0C;&#x7ED3;&#x5408;&#x5728; &alpha;&#xFF0C;&#x7F51;&#x7AD9;&#x7EF4;&#x62A4; &alpha;&#x25B;&theta;-&beta;(2) &#x590D;&#x5236;&#x9176;&#x5728; DNA &#x5408;&#x6210;&#x7684;&#x805A;&#x5408;&#x6A21;&#x5F0F;&#x5173;&#x95ED;&#x7684;&#x72B6;&#x6001;&#x5728;&#x6B64;&#x6D3B;&#x52A8;&#x3002;&#x190;-&beta; &#x76F8;&#x4E92;&#x4F5C;&#x7528;&#xFF0C;&#x5728;&#x8FDB;&#x5316;&#x8FC7;&#x7A0B;&#x4E2D;&#x8981;&#x5F31;&#xFF0C;&#x56E0;&#x6B64;&#x9002;&#x5408;&#x7528;&#x4E8E;&#x77AC;&#x6001;&#x4E2D;&#x65AD;&#x542F;&#x7528;&#x5907;&#x7528;&#x5F02;&#x6027;&#x7B80;&#x5217;&#x548C;&#x7ED3;&#x5408;&#x86CB;&#x767D;&#xFF0C;&#x5176;&#x4ED6;&#x5939;&#x5177;&#x7684;&#x8BBF;&#x95EE;&#x6240;&#x9009;&#x56E0;&#x6B64;&#x4F7F;&#x5F97;&#x5FAE;&#x8C03;&#x7A33;&#x5B9A;&#x6027;&#x7684;&#x5404;&#x79CD;&#x6784;&#x8C61;&#x72B6;&#x6001;&#x7684; DNA &#x6A21;&#x677F;&#x4E0A;&#x590D;&#x5236;&#x9176;&#x7684;&#x86CB;&#x767D;&#x8D28;-&#x86CB;&#x767D;&#x8D28;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7684;&#x7F51;&#x7EDC;&#x6240;&#x4F5C;&#x51FA;&#x7684;&#x91CD;&#x8981;&#x8D21;&#x732E;&#x3002;

&#x76F4;&#x63A5;&#x6821;&#x5BF9;&#x5458;&#x5939;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7A33;&#x5B9A; Pol III &#x4E2D;&#x7684;&#x805A;&#x5408;&#x6A21;&#x5F0F;&#x590D;&#x5236;&#x9176;&#x3002;

Processive DNA synthesis by the Î±É›Î¸ core of the Escherichia coli Pol III replicase requires it to be bound to the Î²(2) clamp via a site in the Î± polymerase subunit. How the É› proofreading exonuclease subunit influences DNA synthesis by Î± was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of É›. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel Î²-binding site in É› that, in conjunction with the site in Î±, maintains a closed state of the Î±É›Î¸-Î²(2) replicase in the polymerization mode of DNA synthesis. The É›-Î² interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode.

Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼ 64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron density of the C-peptide. Here we show that SSB forms a monomer at pH 3.4, which is suitable for studies by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The OB-domain retains its 3D structure in the monomer, and the C-peptide is shown by nuclear Overhauser effects and lanthanide-induced pseudocontact shifts to bind to the OB-domain at a site that harbors ssDNA in the crystal structure of the SSB-ssDNA complex. (15)N relaxation data demonstrate high flexibility of the polypeptide segment linking the C-peptide to the OB-domain and somewhat increased flexibility of the C-peptide compared with the OB-domain, suggesting that the C-peptide either retains high mobility in the bound state or is in a fast equilibrium with an unbound state.

Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy.

Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

Claire Mason

School of Environmental and Life Sciences

University of Newcastle

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Claire Mason

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Environmental and Life Sciences

University of Newcastle

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

School of Environmental and Life Sciences