Biomedical subjects

Jane M Sayer

Publications and source records attributed to Jane M Sayer.

29 records · Page 2Linked to original sources

PubMed2003

Trapping HIV-1 reverse transcriptase before and after translocation on DNA.

A disulfide cross-linking strategy was used to covalently trap as a stable complex (complex N) a short-lived, kinetic intermediate in DNA polymerization. This intermediate corresponds to the product of polymerization prior to translocation. We also prepared the trapped complex that corresponds to the product of polymerization after translocation (complex P). The cross-linking method that we used is a variation of a technique developed by the Verdine and Harrison laboratories. It involves disulfide interchange between an engineered sulfhydryl group of the protein (Q258C mutation) and a disulfide-containing tether attached at the N(2) amino group of a modified dG in either the template or the primer strand of the nucleic acid. We report here a highly efficient synthesis of the precursor, bis(3-aminopropyl)disulfide dihydrochloride, used to introduce this substituent into the oligonucleotide. Efficient cross-linking takes place when the base pair containing the substituent is positioned seven registers from the dNTP-binding site (N site) and the N site is occupied. Complex N, but not complex P, is a substrate for the ATP-based excision reaction that unblocks nucleoside reverse transcriptase inhibitor (NRTI)-terminated primers and causes resistance to several NRTIs, confirming predictions that the excision reaction takes place only when the 3'-end of the primer is bound at the N site. These techniques can be used for biochemical and structural studies of the mechanism of DNA polymerization, translocation, and excision-based resistance of RT to NRTIs. They may also be useful in studying other DNA or RNA polymerases or other enzymes.

Adenosine Triphosphate↗

PubMed2003

Benzo[a]pyrene-dG adduct interference illuminates the interface of vaccinia topoisomerase with the DNA minor groove.

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow in duplex DNA. The enzyme engages the target site within a C-shaped protein clamp. Here we mapped the interface of topoisomerase with the DNA minor groove by introducing chiral C-10 R and S 7,8-diol 9,10-epoxide adducts of benzo[a]pyrene (BP) at single N(2)-deoxyguanosine (dG) positions within the nonscissile DNA strand. These trans opened BPdG adducts fit into the minor groove without perturbing helix conformation or base pairing, and the R and S diastereomers are oriented in opposite directions within the minor groove. We measured the effects of the BPdG adducts on the rate and extent of single-turnover DNA transesterification. We observed a sharp margin of interference effects, whereby +5 and -2 BPdG modifications were well tolerated but +4, +3, and -1 BPdG adducts were severely deleterious. Stereoselective effects at the -1 nucleoside (the R isomer interfered, whereas the S isomer did not) delineated at high resolution the downstream border of the minor groove interface. BPdG inhibition of transesterification is likely caused by steric exclusion of constituents of the topoisomerase from the minor groove. We also applied the BPdG interference method to probe the interactions of exonuclease III with the minor groove. DNAs containing these BPdG adducts were protected from digestion by exonuclease III, which was consistently arrested at positions 2-4 nucleotides prior to the BP-modified guanosine.

Base Sequence↗

PubMed2002

Structures of HIV-1 reverse transcriptase with pre- and post-translocation AZTMP-terminated DNA.

AZT (3'-azido-3'-deoxythymidine) resistance involves the enhanced excision of AZTMP from the end of the primer strand by HIV-1 reverse transcriptase. This reaction can occur when an AZTMP-terminated primer is bound at the nucleotide-binding site (pre-translocation complex N) but not at the 'priming' site (post-translocation complex P). We determined the crystal structures of N and P complexes at 3.0 and 3.1 A resolution. These structures provide insight into the structural basis of AZTMP excision and the mechanism of translocation. Docking of a dNTP in the P complex structure suggests steric crowding in forming a stable ternary complex that should increase the relative amount of the N complex, which is the substrate for excision. Structural differences between complexes N and P suggest that the conserved YMDD loop is involved in translocation, acting as a springboard that helps to propel the primer terminus from the N to the P site after dNMP incorporation.

DNA↗

PubMed2002

Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota.

Human DNA polymerase iota (poliota) is a Y-family polymerase whose cellular function is presently unknown. Here, we report on the ability of poliota to bypass various stereoisomers of benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts in vitro. We find that the BaP DE dG adducts pose a strong block to poliota-dependent replication and result in a high frequency of base misincorporations. In contrast, misincorporations opposite BaP DE and BcPh DE dA adducts generally occurred with a frequency ranging between 2 x 10(-3) and 6 x 10(-4). Although dTMP was inserted efficiently opposite all dA adducts, further extension was relatively poor, with one exception (a cis opened adduct derived from BcPh DE) where up to 58% extension past the lesion was observed. Interestingly, another human Y-family polymerase, polkappa, was able to extend dTMP inserted opposite a BaP DE dA adduct. We suggest that poliota might therefore participate in the error-free bypass of DE-adducted dA in vivo by predominantly incorporating dTMP opposite the damaged base. In many cases, elongation would, however, require the participation of another polymerase more specialized in extension, such as polkappa.

Base Sequence↗

PubMed2002

Benzo[a]pyrene diol epoxide-deoxyguanosine adducts are accurately bypassed by yeast DNA polymerase zeta in vitro.

The possible role of bypass DNA polymerase zeta in mutagenic translesion synthesis past benzo[a]pyrene (BP) 7,8-diol-9,10-epoxide (DE) N(2)-deoxyguanosine (dG) adducts has been examined. We prepared 59-mer DNA templates containing dG adducts derived from trans opening of enantiomers of BP DE-2, in which the 7-hydroxyl group and epoxide oxygen are trans. The 10S-BP DE-dG and 10R-BP DE-dG adducts derive from the (+)- and (-)-DE-2 enantiomers, respectively. The adducted dG is located at a site identified as a G-->T mutational hotspot in random mutagenesis studies of (+)-BP DE-2 in Chinese hamster V-79 cells. Yeast pol zeta (complex of Gst-Rev3p and Rev7p) formed extension products (total of all lengths) of 71, 74 and 88% of a primer annealed to the 10S-BP DE-dG, 10R-BP DE-dG and non-adducted 59-mer templates, respectively. However, only 18 and 19% of the primer was extended to the full-length product on 10S-BP DE-dG and 10R-BP DE-dG adducted templates compared to 55% of the primer on the non-adducted template. A major 34-mer product corresponding to primer elongation up to and including the base before the adduct indicated that nucleotide incorporation opposite both adducts was strongly blocked. Full-length products were isolated from gels and subjected to PCR amplification and cloning. Sequence analysis of more than 300 clones of these full-length products on each template showed that only the correct dCMP was incorporated opposite both the adducted and non-adducted G-hotspot in the template. This corresponds to a probability of mutation lower than 0.3%, the limit of detection, and demonstrates the remarkable fidelity of yeast pol zeta in translesion synthesis past these BP DB-dG lesions in vitro.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2002

Novel stereoselective control over cis vs trans opening of benzo[c]phenanthrene 3,4-diol 1,2-epoxides by the exocyclic N(2)-amino group of deoxyguanosine in the presence of hexafluoropropan-2-ol.

We describe a novel and efficient synthesis (62-84% yields) of the eight possible, diastereomerically pure, cis and trans, R and S O(6)-allyl-protected N(2)-dGuo phosphoramidite building blocks derived through cis and trans opening of (+/-)-3alpha,4beta-dihydroxy-1beta,2beta-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene [BcPh DE-1 (1)] and (+/-)-3alpha,4beta-dihydroxy-1alpha,2alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene [BcPh DE-2 (2)] by hexafluoropropan-2-ol (HFP)-mediated addition of O(6)-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (3) at C-1 of the epoxides. Simply changing the relative amount of HFP used in the reaction mixture can achieve a wide ratio of cis/trans addition products. Thus, the observed cis/trans adduct ratio for the reaction of DE-1 (1) in the presence of 5 equiv of 3 varied from 17/83 to 91/9 over the range of 5-532 equiv of HFP. The corresponding ratios for DE-2 (2) varied from 2/98 to 61/39 under the same set of conditions. When 1 or 2 was fused with a 20-fold excess of 3 at 140 degrees C in the absence of solvent HFP, almost exclusive trans addition (>95%) was observed for the both DEs. Through the use of varying amounts of HFP in the reaction mixture as described above, each of the eight possible phosphoramidite oligonucleotide building blocks (DE-1/DE-2, cis/trans, R/S) of the BcPh DE N(2)-dGuo adducts can be prepared in an efficient fashion. To rationalize the varying cis-to-trans ratio, we propose that the addition of 3 to 1 or 2 in the absence of solvent or in the presence of small amounts of HFP proceeds primarily via an S(N)2 mechanism to produce mainly trans-opened adducts. In contrast, increasing amounts of HFP promote increased participation of an S(N)1 mechanism involving a relatively stable carbocation with two possible conformations. One of these conformations reacts with 3 to give mostly trans adduct, while the other conformation reacts with 3 to give mostly cis adduct.

Journal Article↗

PubMed2002

Different effects on human topoisomerase I by minor groove and intercalated deoxyguanosine adducts derived from two polycyclic aromatic hydrocarbon diol epoxides at or near a normal cleavage site.

Topoisomerase I (top1) relieves supercoiling in DNA by forming transient covalent cleavage complexes. These cleavage complexes can accumulate in the presence of damaged DNA or anticancer drugs that either intercalate or lie in the minor groove. Recently we reported that covalent diol epoxide (DE) adducts of benzo[a]pyrene (BaP) at the exocyclic amino group of G(+1) block cleavage at a preferred cleavage site ( approximately CTT-G(+1)G(+2)A approximately ) and cause accumulation of cleavage products at remote sites. In the present study, we have found that the 10S G(+2) adduct of BaP DE, which lies toward the scissile bond in the minor groove, blocks normal cleavage, whereas the 10R isomer, which orients away from this bond, allows normal cleavage but blocks religation. In contrast to BaP, the pair of benzo[c] phenanthrene (BcPh) DE adducts at G(+2), which intercalate from the minor groove either between G(+1)/G(+2) or between G(+2)/A, allow normal cleavage but block religation. Both intercalated BcPh DE adducts at G(+1) suppress normal cleavage, as do both groove bound BaP DE adducts at this position. These studies demonstrate that these DE adducts provide a novel set of tools to study DNA topoisomerases and emphasize the importance of contacts between the minor groove and top1's catalytic site.

Base Sequence↗

PubMed2002

Human topoisomerase I inhibition: docking camptothecin and derivatives into a structure-based active site model.

Human topoisomerase I (top1) is an important target for anti-cancer drugs, which include camptothecin (CPT) and its derivatives. To elucidate top1 inhibition in vitro, we made a series of duplex DNA substrates containing a deoxyadenosine stereospecifically modified by a covalent adduct of benzo[a]pyrene (BaP) diol epoxide [Pommier, Y., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 10739-10744]. The known orientation of the hydrocarbon adduct in the DNA duplex relative to the top1 cleavage site, in combination with a top1/DNA crystal structure [Redinbo, M. R., et al. (1998) Science 279, 1504-1513], was used to construct a structure-based model to explain the in vitro top1 inhibition results obtained with adducted DNA duplexes. Here we experimentally determined that the lactone form of CPT was stabilized by an irreversible top1/DNA covalent complex. We removed the BaP moiety from the DNA in the published model, and docked the lactone forms of CPT and derivatives into the top1/DNA active site cavity. The docked ligands were minimized, and interaction energy scores between the ligands and the top1/DNA complex were determined. CPT docks perpendicular to the DNA backbone, projects outward from the major groove, and makes a network of potential H-bonds with the active site DNA and top1 residues, including Arg364, Lys532, and Asn722. The results are consistent with the known structure-activity relationships of CPT and derivatives. In addition, the model proposed a novel top1/N352A "resistance" mutation for 10-OH derivatives of CPT. The in vitro biochemical characterization of the top1/N352A mutant supported the model.

Binding Sites↗

PubMed2002

Preferential misincorporation of purine nucleotides by human DNA polymerase eta opposite benzo[a]pyrene 7,8-diol 9,10-epoxide deoxyguanosine adducts.

Human DNA polymerase eta was used to copy four stereoisomeric deoxyguanosine (dG) adducts derived from benzo[a]pyrene 7,8-diol 9,10-epoxide (diastereomer with the 7-hydroxyl group and epoxide oxygen trans (BaP DE-2)). The adducts, formed by either cis or trans epoxide ring opening of each enantiomer of BaP DE-2 by N(2) of dG, were placed at the fourth nucleotide from the 5'-end in two 16-mer sequence contexts, 5' approximately CG*A approximately and 5' approximately GG*T. poleta was remarkably error prone at all four diol epoxide adducts, preferring to misincorporate G and A at frequencies 3- to more than 50-fold greater than the frequencies for T or the correct C, although the highest rates were 60-fold below the rate of incorporation of C opposite a non-adducted G. Anti to syn rotation of the adducted base, consistent with previous NMR data for a BaP DE-2 dG adduct placed just beyond a primer terminus, provides a rationale for preferring purine misincorporation. Extension of purine misincorporations occurred preferentially, but extension beyond the adduct site was weak with V(max)/K(m) values generally 10-fold less than for misincorporation. Mostly A was incorporated opposite (+)-BaP DE-2 dG adducts, which correlates with published observations that G --> T is the most common type of mutation that (+)-BaP DE-2 induces in mammalian cells.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2002

Site-specific mutagenesis in Escherichia coli by N2-deoxyguanosine adducts derived from the highly carcinogenic fjord-region benzo[c]phenanthrene 3,4-diol 1,2-epoxides.

Although there have been numerous studies of site-specific mutagenesis by dGuo adducts of benzo[a]pyrene diol epoxides (B[a]P DEs), the present study represents the first example of site-specific mutagenesis by dGuo adducts of the highly carcinogenic benzo[c]phenanthrene 3,4-diol 1,2-epoxides (B[c]Ph DEs). The eight adducts that would result from cis- and trans-opening at C-1 of four optically active isomers of B[c]Ph DEs by the N(2)-amino group of dGuo were incorporated into 5'-TTCGAATCCTTCCCCC (context III) and 5'-GGGGTTCCCGAGCGGC (context IV) at the underlined site. These modified oligonucleotides along with unmodified controls were ligated into single-stranded M13mp7L2, which were then used to transfect SOS-induced Escherichia coli. Upon replication of the lesions in each of the two sequence contexts, mutational analysis of the progeny was performed by differential hybridization. For the 16 adducts, the mutation frequencies varied over 2 orders of magnitude with a reasonably even distribution (0.4-1% for three adducts, 1-2% for six adducts, 3-7.4% for five adducts, and one adduct each at 11 and 39%). For all but this last adduct, the mutation frequency for a given B[c]Ph DE adduct was less than for its B[a]P analogue with the same stereochemistry in the same sequence. For the vectors containing adducts with S configuration at the site of attachment of the hydrocarbon to the dGuo base, the main base substitution was G --> T followed by G --> A. In contrast, for the vectors containing adducts with R configuration, the main base substitution was G --> A. The most notable observation in the present study is the low frequency of mutations induced by the B[c]Ph DE-dGuo adducts relative to their B[a]P counterparts. A possible structural basis for this difference is proposed.

Base Sequence↗

PubMed2001

Efficiency and accuracy of SOS-induced DNA polymerases replicating benzo[a]pyrene-7,8-diol 9,10-epoxide A and G adducts.

Nucleotide incorporation fidelity, mismatch extension, and translesion DNA synthesis efficiencies were determined using SOS-induced Escherichia coli DNA polymerases (pol) II, IV, and V to copy 10R and 10S isomers of trans-opened benzo[a]pyrene-7,8-diol 9,10-epoxide (BaP DE) A and G adducts. A-BaP DE adducts were bypassed by pol V with moderate accuracy and considerably higher efficiency than by pol II or IV. Error-prone pol V copied G-BaP DE-adducted DNA poorly, forming A*G-BaP DE-S and -R mismatches over C*G-BaP DE-S and -R correct matches by factors of approximately 350- and 130-fold, respectively, even favoring G*G-BaP DE mismatches over correct matches by factors of 2-4-fold. In contrast, pol IV bypassed G-BaP DE adducts with the highest efficiency and fidelity, making misincorporations with a frequency of 10(-2) to 10(-4) depending on sequence context. G-BaP DE-S-adducted M13 DNA yielded 4-fold fewer plaques when transfected into SOS-induced DeltadinB (pol IV-deficient) mutant cells compared with the isogenic wild-type E. coli strain, consistent with the in vitro data showing that pol IV was most effective by far at copying the G-BaP DE-S adduct. SOS polymerases are adept at copying a variety of lesions, but the relative contribution of each SOS polymerase to copying damaged DNA appears to be determined by the lesion's identity.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗