Targeting BCR/ABL-RAD51 Interaction to Prevent Unfaithful Homeologous Recombination Repair.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3272-3272
Author(s):  
Artur Slupianek ◽  
Yashodhara Dasgupta ◽  
Shuyue Ren ◽  
Kimberly Cramer ◽  
Tomasz Skorski
Keyword(s):  
Amino Acid ◽  
Chromosomal Aberrations ◽  
Genotoxic Stress ◽  
Leukemia Cells ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Abl Kinase ◽  
Recombination Repair ◽  
Resistant Mutants ◽  
Homeologous Recombination

Abstract Abstract 3272 Poster Board III-1 CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) and cell lines transformed by non-mutated BCR/ABL kinase or the tyrosine kinase inhibitor (TKI)-resistant mutants contain numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress. In addition CD34+CD38- CML-CP and CD34+ CML-BC stem cell-enriched populations seem to display more DSBs than normal counterparts. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. We reported that numerous ROS- and radiation- induced DSBs induce chromosomal instability implicating enhanced, but unfaithful repair in BCR/ABL-positive leukemias [Leukemia, 2008]. We show here that BCR/ABL kinase (non-mutated and TKI-resistant mutants) facilitate recombination repair (RR) of DSBs. Although recombination usually represents a faithful mechanism of DSB repair, it may generate chromosomal aberrations when similar (homeologous), but not identical (homologous) templates are employed during the repair. To study unfaithful homeologous recombination repair (HomeoRR) a reporter repair cassette containing I-SceI endonuclease-;inducible DSB site and a repair template displaying 19% divergence sequence relative to the DSB site was integrated into the genome of 32Dcl3 murine hematopoietic cells and BCR/ABL-positive counterparts. BCR/ABL kinase caused about 3-fold increase in HomeoRR activity implicating its role in accumulation of chromosomal aberrations in CML cells. RAD51, a key regulator of recombination repair, forms a complex with BCR/ABL which depends on the proline- rich (PP) regions of RAD51 and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL. SH3+SH2-CD domains of BCR/ABL form a pocket binding the PP regions of RAD51. Single amino acid substitutions in the BCR/ABL SH3+SH2-CD pocket, which disrupted binding to the RAD51 PP regions reduced complex formation with RAD51. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3+SH2-CD pocket mutant displayed slow proliferation rate and responded poorly to genotoxic stress despite intact kinase activity. On the other hand, disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) prevented its interaction with BCR/ABL SH3+SH2-CD pocket. Interestingly, expression of RAD51 PP-LL mutant abrogated the clonogenic capability of BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. BCR/ABL-RAD51 complex results in direct phosphorylation of RAD51 on Y315, which is located in the vicinity of PP motifs in the C-terminal portion (aa 271–339) of RAD51. C-terminal Y315F mutant formed more abundant complex with BCR/ABL that the wild-type form, but it did not restore the lost interaction of the PP/LL mutant. Thus, BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for disassembly of RAD51 from BCR/ABL. In concordance, RAD51[Y315F] mutant remained mostly in the cytoplasm, while the wild-type protein accumulated in the nucleus in BCR/ABL-positive cells in response to DSBs induced by genotoxic treatment. In addition to the regulation of BCR/ABL-RAD51 interaction, phospho-Y315 is located in a critical fragment of RAD51 essential for its filament formation on DSBs, implicating its direct role in recombination. To test this hypothesis we employed a peptide aptamer strategy targeting phospho-Y315 of RAD51. Peptides corresponding to the RAD51 fragment containing phospho-Y315, but not these with Y315F substitution reduced HomeoRR activity by approximately 2-fold in BCR/ABL-positive leukemia cells. Altogether, it appears that PP-regions of RAD51 interact with SH3+SH2-CD niche of BCR/ABL, which leads to phosphorylation of RAD51 on Y315 and disassembly of the complex. Phospho-Y315 stimulates abundant nuclear localization of RAD51 on DSBs, which disrupts the mechanisms responsible for preventing recombination using divergent templates resulting in unfaithful HomeoRR in BCR/ABL-positive leukemia cells. In summary, BCR/ABL-RAD51 interaction promotes survival and accumulation of chromosomal aberrations of CML cells expressing non-mutated and TKI-resistant BCR/ABL kinase. We hypothesize that targeting BCR/ABL-RAD51 interaction may prevent/delay accumulation of secondary chromosomal aberrations and CML-BC progression. Disclosures: No relevant conflicts of interest to declare.

Selective Anti-Leukemia Targeting of the Interaction Between BCR/ABL and Mammalian RecA Homologs

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 195-195
Author(s):  
Artur Slupianek ◽  
Shuyue Ren ◽  
Tomasz Skorski
Keyword(s):  
Amino Acid ◽  
Chromosomal Instability ◽  
Chronic Phase ◽  
Direct Interaction ◽  
Genotoxic Stress ◽  
Single Amino Acid ◽  
Leukemia Cells ◽  
Amino Acid Substitutions ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract We showed before that cells transformed by BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2 and TEL/PDGFR, inducing chronic myeloproliferative disorders (MPDs), and CD34+ chronic myeloid leukemia (CML) stem/ progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain an excess of DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress [Blood, 2005; Cell Cycle, 2006; DNA Repair, 2006; Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cellenriched populations seem to display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Elevated levels of DSBs were also observed in leukemia cells expressing imatinib-resistant BCR/ABL kinase mutants. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. Numerous ROS- and radiation- induced DSBs are not lethal for BCR/ABL-positive leukemia cells; instead, they induce chromosomal instability implicating enhanced, but unfaithful repair [Leukemia, 2008]. The previous report [Mol. Cell, 2001] and ongoing studies demonstrated that BCR/ABL kinase (non-mutated and imatinib-resistant mutants) modulates expression of the mammalian RecA homologs RAD51, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which are responsible for homologous recombination repair (HRR) of DSBs. RAD51 plays a key role in HRR in cells transformed by BCR/ ABL and other FTKs [Mol. Cell, 2001; Mol. Cell. Biol., 2002]. BCR/ABL stimulates the expression of, interacts with and phosphorylates RAD51 on Y315, which is located in a critical fragment of RAD51 essential for its filament formation on DNA. Accordingly, our recent results indicated that BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for nuclear RAD51 foci formation in response to DNA damage. In addition to RAD51, BCR/ABL interacts directly with and phosphorylates RAD51B and XRCC2, but not other RecA homologs. Altogether, it appears that BCR/ABL can deregulate the expression and phosphorylation of some RecA homologs, which may have a significant impact on the efficiency and fidelity of DSB repair resulting in protection from apoptosis and chromosomal instability. Therefore, disassembly of BCR/ABL from RecA homologs should reduce the capability of CML cells to repair numerous ROS-induced DSBs and eventually trigger apoptosis. Based on this hypothesis we investigated the mechanisms of association between BCR/ABL and RecA homologs. Interactions between BCR/ ABL and RAD51 or RAD51B depend on the proline- rich (PP) regions of RAD51 and RAD51B, and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL, which form a pocket binding the PP regions. Disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) abrogated direct interaction with the BCR/ABL SH3-SH2-CD pocket. On the other hand, single amino acid substitutions in the BCR/ABL SH3-SH2-CD pocket, which eliminated its capability of binding the PP regions, prevented complex formation with RAD51 and RAD51B. In addition, RAD51 and RAD51B may interact with members of the BCR/ABL proteome such as Grb2 and Shc (RAD51 and RAD51B), and c-CrkL (only RAD51B), but not Gab2 and c-Cbl. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3-SH2-CD pocket mutant, where single amino acid substitutions disrupted its direct interaction with RAD51, displayed a slower proliferation rate and responded poorly to genotoxic stress despite intact kinase activity in comparison to cells transformed with non-mutated BCR/ABL. Interestingly, expression of RAD51 PP-LL mutant eliminated BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. These results suggest that the interaction between BCR/ABL and RAD51 may be targeted for selective elimination of leukemia cells and/or suppression of genomic instability. To test this hypothesis we are employing the peptide aptamer strategy targeting RAD51 PP regions in CD34+ cells obtained from imatinib-sensitive and imatinib-resistant CML patients and healthy volunteers in vivo and in vitro. In summary, we hypothesize that mechanisms regulating the association of BCR/ABL with RAD51 and other mammalian RecA homologs may be explored for the planning of more effective anti-tumor modalities.

Targeting Phosphotyrosine-315 In RAD51 Recombinase to Prevent BCR-ABL1 - Mediated Unfaithful Homeologous Recombination Repair.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1190-1190
Author(s):  
Artur Slupianek ◽  
Yashodhara Dasgupta ◽  
Shu-yue Ren ◽  
Kimberly Cramer ◽  
Tomasz Skorski
Keyword(s):  
Chromosomal Aberrations ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Chronic Phase ◽  
Sh3 Domain ◽  
Leukemia Cells ◽  
Dsb Repair ◽  
Direct Role ◽  
Recombination Repair ◽  
Homeologous Recombination

Abstract Abstract 1190 Background: CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast phase (CML-BP) and cell lines transformed by non-mutated BCR-ABL1 kinase or tyrosine kinase inhibitor (TKI)-resistant mutants contain numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) (Nowicki et al., Blood, 2006; Cramer et al., Cancer Res., 2008). DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. We reported that numerous ROS and radiation induced DSBs generated chromosomal aberrations in BCR-ABL1-positive leukemia cells (Koptyra et al., Leukemia, 2008), which may contribute to the malignant progression to CML-BP. We also showed that homologous recombination repair (HomoRR) driven by RAD51 recombinase is one of the major DSB repair mechanisms, which is stimulated by BCR-ABL1 (Slupianek et al., Molecular Cell, 2001). Although recombination usually represents a faithful mechanism of DSB repair, it may generate chromosomal aberrations when similar (homeologous), but not identical (homologous) templates are employed during the repair. Here we investigated if BCR-ABL1 can modulate RAD51 recombinase to corrupt the fidelity of recombination and if this process can be targeted to prevent genomic instability in leukemia cells. Result: To study unfaithful homeologous recombination repair (HomeoRR) a reporter repair cassette containing I-SceI endonuclease–inducible DSB site and a repair template displaying 1% sequence divergence relative to the DSB site was integrated into the genome of 32Dcl3 murine hematopoietic cells, BCR-ABL1-32Dcl3 cells and BCR-ABL1(T315I)-32Dcl3 cells. BCR-ABL1 and BCR-ABL1(T315I) kinase caused about a 3-fold increase in HomeoRR activity implicating its role in the accumulation of chromosomal aberrations in CML cells. The magnitude of HomeoRR stimulation depended on BCR-ABL1 expression levels. RAD51 recombinase, a key regulator of recombination repair, forms a complex with BCR-ABL1 which depends on the proline- rich (PP) regions of RAD51 and the SH3 domain of BCR-ABL1, but does not depend on its kinase activity. In fact, BCR-ABL1(K1172R) kinase-dead mutant formed complexes more abundantly with RAD51 than the kinase-active BCR-ABL1. BCR-ABL1-RAD51 complex formation resulted in direct phosphorylation of RAD51 on Y315 [RAD51(phosphoY315)], which is located in the vicinity of PP motifs in the C-terminal portion of RAD51. Phosphorylation-less C-terminal RAD51(Y315F) mutant formed a stronger complex with BCR-ABL1 than the wild-type form. Altogether, it appears that RAD51 PP bind to the SH3 domain of BCR-ABL1 kinase followed by quick phosphorylation of RAD51 on Y315 and disassembly of RAD51(phosphoY315) from the complex. Y315 is located in a critical fragment of RAD51 essential for its filament formation on DSBs, implicating its direct role in recombination. In fact RAD51(phosphoY315) was found in the nuclei of BCR-ABL1 leukemia cells, but not of parental cells and formed numerous foci on DSBs. Phosphorylation-less RAD51(Y315F) mutant abrogated foci formation and inhibited HomeoRR. Thus, BCR-ABL1 – RAD51(phosphoY315) pathway appears to promote unfaithful HomeoRR and chromosomal instability. To test if RAD51(phosphoY315) can be targeted to prevent unfaithful HomeoRR in leukemia cells, a peptide aptamer strategy was applied. Aptamer mimicking the RAD51(phosphoY315) fragment, but not that with the Y315F phosphorylation-less substitution inhibited RAD51 foci formation and HomeoRR activity in BCR-ABL1 leukemia cells. Conclusion: In summary, the BCR-ABL1-RAD51 axis may promote accumulation of chromosomal aberrations of CML cells expressing non-mutated and TKI-resistant BCR-ABL1 kinase. We hypothesize that targeting BCR-ABL1-RAD51 interaction may prevent/delay accumulation of secondary chromosomal aberrations and CML-BP progression. Furthermore, RAD51 and recombination is also affected by other oncogenic tyrosine kinases (OTKs) such as TEL-ABL1, TEL-PDGFR, ZNF198-FGFR1, TEL-JAK2, JAK2[V617F], NPM-ALK, IGF-1R, EGFR and FLT3-ITD suggesting that abrogation of OTK-mediated aberrant modulation of RAD51 may inhibit genetic instability and tumor progression. Disclosures: No relevant conflicts of interest to declare.

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

scholarly journals Mechanism of Darunavir (DRV)’s High Genetic Barrier to HIV-1 Resistance: A Key V32I Substitution in Protease Rarely Occurs, but Once It Occurs, It Predisposes HIV-1 To Develop DRV Resistance

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Manabu Aoki ◽  
Debananda Das ◽  
Hironori Hayashi ◽  
Hiromi Aoki-Ogata ◽  
Yuki Takamatsu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Amino Acid ◽  
Critical Role ◽  
Viral Fitness ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Genetic Barrier ◽  
High Level ◽  
Hiv 1

ABSTRACTDarunavir (DRV) has bimodal activity against HIV-1 protease, enzymatic inhibition and protease dimerization inhibition, and has an extremely high genetic barrier against development of drug resistance. We previously generated a highly DRV-resistant HIV-1 variant (HIVDRVRP51). We also reported that four amino acid substitutions (V32I, L33F, I54M, and I84V) identified in the protease of HIVDRVRP51are largely responsible for its high-level resistance to DRV. Here, we attempted to elucidate the role of each of the four amino acid substitutions in the development of DRV resistance. We found that V32I is a key substitution, which rarely occurs, but once it occurs, it predisposes HIV-1 to develop high-level DRV resistance. When two infectious recombinant HIV-1 clones carrying I54M and I84V (rHIVI54Mand rHIVI84V, respectively) were selected in the presence of DRV, V32I emerged, and the virus rapidly developed high-level DRV resistance. rHIVV32Ialso developed high-level DRV resistance. However, wild-type HIVNL4-3(rHIVWT) failed to acquire V32I and did not develop DRV resistance. Compared to rHIVWT, rHIVV32Iwas highly susceptible to DRV and had significantly reduced fitness, explaining why V32I did not emerge upon selection of rHIVWTwith DRV. When the only substitution is at residue 32, structural analysis revealed much stronger van der Waals interactions between DRV and I-32 than between DRV and V-32. These results suggest that V32I is a critical amino acid substitution in multiple pathways toward HIV-1’s DRV resistance development and elucidate, at least in part, a mechanism of DRV’s high genetic barrier to development of drug resistance. The results also show that attention should be paid to the initiation or continuation of DRV-containing regimens in people with HIV-1 containing the V32I substitution.IMPORTANCEDarunavir (DRV) is the only protease inhibitor (PI) recommended as a first-line therapeutic and represents the most widely used PI for treating HIV-1-infected individuals. DRV possesses a high genetic barrier to development of HIV-1’s drug resistance. However, the mechanism(s) of the DRV’s high genetic barrier remains unclear. Here, we show that the preexistence of certain single amino acid substitutions such as V32I, I54M, A71V, and I84V in HIV-1 protease facilitates the development of high-level DRV resistance. Interestingly, allin vitro-selected highly DRV-resistant HIV-1 variants acquired V32I but never emerged in wild-type HIV (HIVWT), and V32I itself rendered HIV-1 more sensitive to DRV and reduced viral fitness compared to HIVWT, strongly suggesting that the emergence of V32I plays a critical role in the development of HIV-1’s resistance to DRV. Our results would be of benefit in the treatment of HIV-1-infected patients receiving DRV-containing regimens.

Neurogenic and antineurogenic effects from modifications at the Notch locus

Development ◽  
1990 ◽  
Vol 109 (1) ◽  
pp. 167-175 ◽  
Author(s):  
J. Palka ◽  
M. Schubiger ◽  
H. Schwaninger
Keyword(s):  
Nervous System ◽  
Amino Acid ◽  
Distinctive Feature ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Significant Extent ◽  
Notch Protein ◽  
Notch Locus ◽  
Mother Cells

The best studied mutations at the Notch locus produce a neurogenic phenotype, with a massive overgrowth of the nervous system at the expense of epidermis. We report here that, in the development of the adult peripheral nervous system, the Abruptex alleles of Notch have the opposite phenotype, namely an underproduction of sensory organs or sensilla. This arises primarily not from an arrest of the lineages that produce sensilla, from the degeneration of sensillar cells, or from the transformation into neurons of cells that normally secrete the cuticular components of a sensillum (as can happen in Notch alleles). Rather, our evidence argues strongly that the sensillar mother cells never form. This implies that the Notch protein plays a role in the process that first generates a difference between sensillar mother cells and ordinary epidermal cells. The number of sensilla formed on the wing of flies carrying multiple doses of Notch+ is virtually the same as that of wild type, i.e. the Abruptex phenotype is not reproduced to any significant extent. This suggests that the single amino acid substitutions that occur in Abruptex mutants confer on the protein some functionally distinctive feature, possibly more powerful intermolecular binding or altered stability.

BCR/ABL Regulates the Expression and Interacts with Werner Syndrome Helicase/Exonuclease To Modulate Its Biochemical Properties.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2873-2873
Author(s):  
Artur Slupianek ◽  
Stanislaw Jozwiakowski ◽  
Ewa Gurdek ◽  
Michal O. Nowicki ◽  
Tomasz Skorski
Keyword(s):  
Werner Syndrome ◽  
Genotoxic Stress ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Premature Aging ◽  
Leukemia Cells ◽  
Dna Double Strand Breaks ◽  
Abl Kinase ◽  
A Genome ◽  
Transformed Cell Lines

Abstract A genome-wide screen suggested that BCR/ABL kinase might stimulate WRN, a member of the RecQ-like DNA helicases family. The Werner syndrome protein (WRN) exerts DNA helicase and 3′-5′ exonuclease activities. Inactivating mutations in the WRN gene causes Werner syndrome, characterized by premature aging, genomic instability and cancer predisposition. The WRN helicase unwinds unusual DNA structures, which can occur physiologically, or can be accidentally generated during DNA repair (double-stranded DNA with mismatched tails, bimolecular G4 quartets and Holliday junctions). In addition, WRN physically interacts with components of two major systems for DNA double-strand breaks (DSBs) repair: non-homologous end-joining (NHEJ) and homologous recombination (HR). Here we demonstrated that BCR/ABL regulates the expression of WRN mRNA and protein in CML primary cells and BCR/ABL-transformed cell lines. BCR/ABL kinase-induced WRN expression is mediated by c-MYC, but not STAT5 - dependent transcription as well as by inhibition of caspases-dependent cleavage. In addition, immunoprecipitation and pull-down studies indicated that BCR/ABL interacts directly with WRN resulting in its tyrosine phosphorylation. Mutation analysis revealed that multiple domains/amino acid residues of BCR/ABL and WRN are involved in the interaction. BCR/ABL-positive leukemia cells exerted an enhanced WRN-dependent helicase activity. In addition, immunoprecipitation and double-immunofluorescence co-localization studies demonstrated an elevated interaction between WRN and RAD51 in BCR/ABL cells undergoing genotoxic stress in comparison to parental counterparts. Altogether, it is likely that WRN is involved in DSBs repair by HR in leukemia cells. More detailed studies are underway to pinpoint the role of WRN in DNA damage response in BCR/ABL-transformed cells.

scholarly journals Effects of the Δ67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision

2004 ◽  
Vol 78 (18) ◽  
pp. 9987-9997 ◽  
Author(s):  
Paul L. Boyer ◽  
Tomozumi Imamichi ◽  
Stefan G. Sarafianos ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Coding Region ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Long-term use of combination therapy against human immunodeficiency virus type (HIV-1) provides strong selective pressure on the virus, and HIV-1 variants that are resistant to multiple inhibitors have been isolated. HIV-1 variants containing amino acid substitutions within the coding region of HIV-1 reverse transcriptase (RT), such as the 3′-azido-3′-deoxythymidine (AZT)-resistant variant AZT-R (M41L/D67N/K70R/T215Y/K219Q) and a variant containing an insertion in the fingers domain (S69SGR70/T215Y), are resistant to the nucleoside RT inhibitor (NRTI) AZT because of an increase in the level of excision of AZT monophosphate (AZTMP) from the primer. While rare, variants have also been isolated which contain deletions in the RT coding region. One such virus, described by Imamichi et al. (J. Virol 74:10958-10964, 2000; J. Virol. 74:1023-1028, 2000; J. Virol. 75:3988-3992, 2001), contains numerous amino acid substitutions and a deletion of codon 67, which we have designated the Δ67 complex of mutations. We have expressed and purified HIV-1 RT containing these mutations. We compared the polymerase and pyrophosphorolysis (excision) activity of an RT with the Δ67 complex of mutations to wild-type RT and the two other AZT-resistant variants described above. All of the AZT-resistant variants we tested excise AZTMP and 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA [tenofovir]) from the end of a primer more efficiently than wild-type RT. Although the variant RTs excised d4TMP less efficiently than AZTMP and PMPA, they were able to excise d4TMP more efficiently than wild-type RT. HIV-1 RT containing the Δ67 complex of mutations was not able to excise as broad a range of NRTIs as the fingers insertion variant SSGR/T215Y, but it was able to polymerize efficiently with low concentrations of deoxynucleoside triphosphates and seems to be able to excise AZTMP and PMPA at lower ATP concentrations than AZT-R or SSGR/T215Y, suggesting that a virus containing the Δ67 complex of mutations would replicate reasonably well in quiescent cells, even in the presence of AZT.

scholarly journals Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways

2002 ◽  
Vol 46 (9) ◽  
pp. 3035-3038 ◽  
Author(s):  
Barry G. Hall
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Dna Shuffling ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Single Mutation ◽  
Wild Type ◽  
In Vitro Evolution ◽  
Evolutionary Pathway

ABSTRACT In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 β-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

2001 ◽  
Vol 47 (12) ◽  
pp. 1088-1094 ◽  
Author(s):  
Yew-Loom Chen ◽  
Tsung-Yin Tang ◽  
Kuo-Joan Cheng
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Synergistic Effects ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Neocallimastix Patriciarum

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10 128 µmol glucose·min–1·(mg protein)–1 at pH 6.0. It was more thermostable at 60°C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.Key words: xylanase, Neocallimastix patriciarum, alkalophilicity, random mutagenesis, directed evolution.

scholarly journals Amino Acid Substitutions in GyrA of Burkholderia glumae Are Implicated in Not Only Oxolinic Acid Resistance but Also Fitness on Rice Plants

2006 ◽  
Vol 73 (4) ◽  
pp. 1114-1119 ◽  
Author(s):  
Yukiko Maeda ◽  
Akinori Kiba ◽  
Kouhei Ohnishi ◽  
Yasufumi Hikichi
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Oxolinic Acid ◽  
The Other ◽  
Amino Acid Substitutions ◽  
Burkholderia Glumae ◽  
Field Isolates ◽  
Rice Plants ◽  
Resistant Mutants

ABSTRACT Oxolinic acid (OA) resistance in field isolates of Burkholderia glumae, a causal agent of bacterial grain rot, is dependent on an amino acid substitution at position 83 in GyrA (GyrA83). In the present study, among spontaneous in vitro mutants from the OA-sensitive B. glumae strain Pg-10, we selected OA-resistant mutants that emerged at a rate of 5.7 � 10−10. Nucleotide sequence analysis of the quinolone resistance-determining region in GyrA showed that Gly81Cys, Gly81Asp, Asp82Gly, Ser83Arg, Asp87Gly, and Asp87Asn are observed in these OA-resistant mutants. The introduction of each amino acid substitution into Pg-10 resulted in OA resistance, similar to what was observed for mutants with the responsible amino acid substitution. In vitro growth of recombinants with Asp82Gly was delayed significantly compared to that of Pg-10; however, that of the other recombinants did not differ significantly. The inoculation of each recombinant into rice spikelets did not result in disease. In inoculated rice spikelets, recombinants with Ser83Arg grew less than Pg-10 during flowering, and growth of the other recombinants was reduced significantly. On the other hand, the reduced growth of recombinants with Ser83Arg in spikelets was compensated for under OA treatment, resulting in disease. These results suggest that amino acid substitutions in GyrA of B. glumae are implicated in not only OA resistance but also fitness on rice plants. Therefore, GyrA83 substitution is thought to be responsible for OA resistance in B. glumae field isolates.

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