Cloning, expression, and purification of a catalytic fragment of moloney murine leukemia virus reverse transcriptase: Crystallization of nucleic acid complexes

Sun, Dunming; Jessen, S. M.; Liu, Chunhui; Liu, Xiuping; Najmudin, Shabir; Georgiadis, Millie M.

doi:10.1002/pro.5560070711

Cited by 26 publications

(43 citation statements)

References 29 publications

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“…Crystal structures of Co⅐BLM bound to DNA were determined by using a host-guest system (26)(27)(28)(29) in which the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase (RT), the ''host,'' is bound to the termini of d(ATTAGTTATAACTAAT) 2 (1) or d(ATTTAGT-TAACTAAAT) 2 (2), each containing two preferred sites of drug binding, as the ''guests'' (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

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Crystal structure of DNA-bound Co(III)·bleomycin B ₂ : Insights on intercalation and minor groove binding

Goodwin

Lewis

Long

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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108

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Bleomycins constitute a widely studied class of complex DNA cleaving natural products that are used to treat various cancers. Since their first isolation, the bleomycins have provided a paradigm for the development and discovery of additional DNA-cleaving chemotherapeutic agents. The bleomycins consist of a disaccharide-modified metal-binding domain connected to a bithiazole/Cterminal tail via a methylvalerate-Thr linker and induce DNA damage after oxygen activation through site-selective cleavage of duplex DNA at 5-GT/C sites. Here, we present crystal structures of two different 5-GT containing oligonucleotides in both the presence and absence of bound Co(III)⅐bleomycin B 2. Several findings from our studies impact the current view of bleomycin binding to DNA. First, we report that the bithiazole intercalates in two distinct modes and can do so independently of well ordered minor groove binding of the metal binding/disaccharide domains. Second, the Co(III)-coordinating equatorial ligands in our structure include the imidazole, histidine amide, pyrimidine N1, and the secondary amine of the ␤ aminoalanine, whereas the primary amine acts as an axial ligand. Third, minor groove binding of Co(III)⅐bleomycin involves direct hydrogen bonding interactions of the metal binding domain and disaccharide with the DNA. Finally, modeling of a hydroperoxide ligand coordinated to Co(III) suggests that it is ideally positioned for initiation of C4-H abstraction.antitumor ͉ DNA-binding ͉ HOO-Co(III) bleomycin T he bleomycins are a family of glycopeptide-derived antitumor natural products first isolated from Streptomyces verticillus by Umezawa (1). Bleomycins are used to treat lymphomas (2, 3) and testicular cancers (4), most often in combination therapies. The clinical efficacy of the bleomycins is thought to derive from their ability to mediate single-and double-strand DNA cleavage (5, 6) resulting from Fe 2ϩ and O 2 -dependent (7) C4Ј-H abstraction from pyrimidine nucleotides (8) contained within 5Ј-GT/C dinucleotide sites (9). Of the DNA lesions formed, double-strand DNA cleavage is thought to be most relevant to the cytotoxicity of bleomycin. Although DNA is the generally accepted locus of drug activity, O 2 -activated Fe(II)⅐bleomycin also mediates the selective cleavage of RNA (10), and Cu(I)⅐bleomycin is capable of cleaving DNA (11); however, the in vivo relevance of this alternative nucleic acid target and metal ion remain to be fully determined.The metallobleomycins have served as an important paradigm for nucleic acid-targeted drug design (12,13); and yet, despite extensive study over the past 40 years, fundamental aspects of their activity have yet to be fully defined including the role of particular DNA-binding modes on drug activity and the mechanism of double-strand DNA cleavage (14). Facilitating structural studies, HOO-Co(III)⅐bleomycin ''green'' (Fig. 1) (15) constitutes a stable structural analogue of O 2 activated Fe(II)⅐bleomycin, HOO-Fe(III)⅐bleomycin, that interacts with the same DNA site-selectivity (16-20)...

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Section: Resultsmentioning

confidence: 99%

“…The RT fragment (residues 24 -278) (29) and DNA oligonucleotides were purified and RT-DNA crystals were grown as described in ref. 27.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of DNA-bound Co(III)·bleomycin B ₂ : Insights on intercalation and minor groove binding

Goodwin

Lewis

Long

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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108

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“…The equine infectious anemia virus and Rous sarcoma virus RTs are also organized as asymmetric dimers containing one active polymerase site (55). An isolated fragment of the Moloney murine leukemia virus RT is monomeric in the absence of nucleic acid; however, upon addition of DNA, the enzyme forms an asymmetric dimer containing one active polymerase site (58). Since telomerase is most closely related to non-long terminal repeat retroposon RTs, which are thought to form a homodimer upon interaction with their target RNA and DNA (14,63), one might anticipate functional and/or structural similarities between the telomerase RT multimer and this class of RTs (36,46,47).…”

Section: Discussion Htert Multimerization Occurs In Transmentioning

confidence: 99%

Functional Multimerization of the Human Telomerase Reverse Transcriptase

Beattie

Zhou

Robinson

et al. 2001

Molecular and Cellular Biology

130

109

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The telomerase enzyme exists as a large complex (ϳ1,000 kDa) in mammals and at minimum is composed of the telomerase RNA and the catalytic subunit telomerase reverse transcriptase (TERT). In Saccharomyces cerevisiae, telomerase appears to function as an interdependent dimer or multimer in vivo (J. Prescott and E. H. Blackburn, Genes Dev. 11:2790-2800, 1997). However, the requirements for multimerization are not known, and it remained unclear whether telomerase exists as a multimer in other organisms. We show here that human TERT (hTERT) forms a functional multimer in a rabbit reticulocyte lysate reconstitution assay and in human cell extracts. Two separate, catalytically inactive TERT proteins can complement each other in trans to reconstitute catalytic activity. This complementation requires the amino terminus of one hTERT and the reverse transcriptase and C-terminal domains of the second hTERT. The telomerase RNA must associate with only the latter hTERT for reconstitution of telomerase activity to occur. Multimerization of telomerase also facilitates the recognition and elongation of substrates in vitro and in vivo. These data suggest that the catalytic core of human telomerase may exist as a functionally cooperative dimer or multimer in vivo.The catalytic subunit of telomerase, the telomerase reverse transcriptase (TERT), possesses the hallmark amino acid motifs of a reverse transcriptase (RT) (23,29,40,47). However, unlike viral RTs, telomerase is a unique eukaryotic RT that carries an intrinsic RNA template essential for the de novo addition of telomere sequences (reviewed in reference 19). Proteins associated with telomerase activity include TEP1 (22, 48), hsp90/p23 (18, 25), dyskerin (42, 43), L22 (32), and hStau (32) in mammals; the Sm proteins (54) as well as Est1p and Est3p,(26,56) in Saccharomyces cerevisiae; and p80, p95, and p43 in ciliates (1,11,21,35). TEP1 is not essential for telomerase activity in vitro or in vivo (5, 37). A subset of these associated factors are known to serve distinct roles in telomerase assembly and telomere length maintenance (15,16,27,34,41,43,51,54).In S. cerevisiae, different telomerase RNAs can functionally cooperate to form an active telomerase complex in vivo. Prescott and Blackburn demonstrated the presence of at least two primer recognition-elongation sites within S. cerevisiae telomerase (50). In addition, they showed that a mutant telomerase RNA incapable of telomere elongation could nonetheless support elongation in a diploid strain containing one mutant and one wild-type telomerase RNA (50). These results provided the first evidence that telomerase could form an active multimer in vivo that might contain, at minimum, two active sites (50). A recombinant reconstitution assay for human telomerase showed that two separately inactive, nonoverlapping fragments of human telomerase RNA could reconstitute telomerase activity in vitro (59). While consistent with a model of telomerase RNA multimerization, these results are also consistent with reconstitution of a single...

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“…However, dimerization is apparently not important for all retrovirus RTs since previous experiments performed with the RTs from mouse mammary tumor virus and bovine leukemia virus indicate that these enzymes might be active as monomers (25,34). The RT from murine leukemia virus is a monomeric ϳ80-kDa protein that was suggested to dimerize upon binding to nucleic acid (10,33,FIG. 6.…”

Section: Discussionmentioning

confidence: 99%

Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

Werner

Wöhrl

2000

J Virol

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The genes encoding the ␣ (63-kDa) and ␤ (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV ␣␤ and ␣Pol RTs within which one subunit was selectively mutated. When ␣␤ heterodimers contained the Asp 1813Asn mutation in their ␤ subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their ␣ subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in ␣␤ heterodimers mutated in the ␤ subunit but was lost when the mutation was present in the ␣ subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the ␣ subunit of the heterodimeric RSV RT ␣␤. Reverse transcriptase (RT) of Rous sarcoma virus (RSV) is a component of the Gag-Pol precursor protein.Pol is composed of polymerase, RNase H, and integrase domains and an additional short 4.1-kDa protein located at the C terminus of the protein. Pol is processed into polypeptides of various lengths by the viral protease; its ␣ polypeptide (63 kDa) contains the polymerase and RNase H domains, and its ␤ polypeptide (95 kDa) consists of the polymerase, RNase H, and integrase domains but lacks the C-terminal 4.1-kDa protein (1,7,8,17,28,30). In addition, the integrase domain (32 kDa) is also present and active as a separate enzyme (9, 30). Three forms of RT have been isolated from avian sarcoma and leukosis viruses (ASLV): ␣, ␤, and ␣␤, with the major form being the heterodimer (8,11,16). We have shown previously that the different forms of RSV RT can be expressed and purified from insect cells using the baculovirus expression system (37). In order to examine the subunit organization of RSV RT, the technique of subunit-selective mutagenesis was used (13,21) to analyze the effect of RSV RTs carrying a mutation in only one of the two subunits constituting the ␣␤ or ␣Pol heterodimer.It has been shown previously by biochemical and crystallographic data that heterodimeric human immunodeficiency virus type 1 (HIV-1) RT p66-p51 reveals an asymmetric subunit organization. The polymerase active site is present only in the larger p66 subunit of the heterodimer (13,14,19,36), while the p51 subunit, which is identical in sequence to p66 but lacks the RNase H domain, is not directly involved in catalysis (21). However, p51 has to fulfill an important stabilizing function since the monomeric subunits are inactive (26,27). Recent kinetic analyses we performed with homodimeric p51 RT from equine infectious anemia virus (EIAV) lacking the RNase H domain indicated an asymmetric subunit organization similar to that of heterodimeric p66-p51 RT, with the polymera...

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Cloning, expression, and purification of a catalytic fragment of moloney murine leukemia virus reverse transcriptase: Crystallization of nucleic acid complexes

Cited by 26 publications

References 29 publications

Crystal structure of DNA-bound Co(III)·bleomycin B ₂ : Insights on intercalation and minor groove binding

Crystal structure of DNA-bound Co(III)·bleomycin B ₂ : Insights on intercalation and minor groove binding

Functional Multimerization of the Human Telomerase Reverse Transcriptase

Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

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Cloning, expression, and purification of a catalytic fragment of moloney murine leukemia virus reverse transcriptase: Crystallization of nucleic acid complexes

Cited by 26 publications

References 29 publications

Crystal structure of DNA-bound Co(III)·bleomycin B 2 : Insights on intercalation and minor groove binding

Crystal structure of DNA-bound Co(III)·bleomycin B 2 : Insights on intercalation and minor groove binding

Functional Multimerization of the Human Telomerase Reverse Transcriptase

Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

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Product

Resources

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Crystal structure of DNA-bound Co(III)·bleomycin B ₂ : Insights on intercalation and minor groove binding

Crystal structure of DNA-bound Co(III)·bleomycin B ₂ : Insights on intercalation and minor groove binding