Genotoxic effects of the major alkylation damage N7-methylguanine and methyl formamidopyrimidine.

Various alkylating agents are known to preferentially modify guanine in DNA, resulting in the formation of N7-alkylguanine (N7-alkylG) and the imidazole ring opened alkyl-formamidopyrimidine (alkyl-FapyG) lesions. Evaluating the mutagenic effects of N7-alkylG has been challenging due to the instability of the positively charged N7-alkylG. To address this issue, we developed a 2'-fluorine-mediated transition-state destabilization approach, which stabilizes N7-alkylG and prevents spontaneous depurination. We also developed a postsynthetic conversion of 2'-F-N7-alkylG DNA into 2'-F-alkyl-FapyG DNA. Using these methods, we incorporated site-specific N7-methylG and methyl-FapyG into pSP189 plasmid and determined their mutagenic properties in bacterial cells using the supF-based colony screening assay. The mutation frequency of N7-methylG was found to be less than 0.5%. Our crystal structure analysis revealed that N7-methylation did not significantly alter base pairing properties, as evidenced by a correct base pairing between 2'-F-N7-methylG and dCTP in Dpo4 polymerase catalytic site. In contrast, the mutation frequency of methyl-FapyG was 6.3%, highlighting the mutagenic nature of this secondary lesion. Interestingly, all mutations arising from methyl-FapyG in the 5'-GGT(methyl-FapyG)G-3' context were single nucleotide deletions at the 5'-G of the lesion. Overall, our results demonstrate that 2'-fluorination technology is a useful tool for studying the chemically labile N7-alkylG and alkyl-FapyG lesions.

DNA interstrand cross-links induced by the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine.

Oxidative damage to DNA generates 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as two major lesions. Despite the comparable prevalence of these lesions, the biological effects of oxoA remain poorly characterized. Here we report the discovery of a class of DNA interstrand cross-links (ICLs) involving oxidized nucleobases. Under oxidative conditions, oxoA, but not oxoG, readily reacts with an opposite base to produce ICLs, highlighting a latent alkylating nature of oxoA. Reactive halogen species, one-electron oxidants, and the myeloperoxidase/H2O2/Cl- system induce oxoA ICLs, suggesting that oxoA-mediated cross-links may arise endogenously. Nucleobase analog studies suggest C2-oxoA is covalently linked to N2-guanine and N3-adenine for the oxoA-G and oxoA-A ICLs, respectively. The oxoA ICLs presumably form via the oxidative activation of oxoA followed by the nucleophilic attack by an opposite base. Our findings provide insights into oxoA-mediated mutagenesis and contribute towards investigations of oxidative stress-induced ICLs and oxoA-based latent alkylating agents.

Catalytic mechanism of the mismatch-specific DNA glycosylase methyl-CpG-binding domain 4.

Thymine:guanine base pairs are major promutagenic mismatches occurring in DNA metabolism. If left unrepaired, these mispairs can cause C to T transition mutations. In humans, T:G mismatches are repaired in part by mismatch-specific DNA glycosylases such as methyl-CpG-binding domain 4 (hMBD4) and thymine-DNA glycosylase. Unlike lesion-specific DNA glycosylases, T:G-mismatch-specific DNA glycosylases specifically recognize both bases of the mismatch and remove the thymine but only from mispairs with guanine. Despite the advances in biochemical and structural characterizations of hMBD4, the catalytic mechanism of hMBD4 remains elusive. Herein, we report two structures of hMBD4 processing T:G-mismatched DNA. A high-resolution crystal structure of Asp560Asn hMBD4-T:G complex suggests that hMBD4-mediated glycosidic bond cleavage occurs via a general base catalysis mechanism assisted by Asp560. A structure of wild-type hMBD4 encountering T:G-containing DNA shows the generation of an apurinic/apyrimidinic (AP) site bearing the C1'-(S)-OH. The inversion of the stereochemistry at the C1' of the AP-site indicates that a nucleophilic water molecule approaches from the back of the thymine substrate, suggesting a bimolecular displacement mechanism (SN2) for hMBD4-catalyzed thymine excision. The AP-site is stabilized by an extensive hydrogen bond network in the MBD4 catalytic site, highlighting the role of MBD4 in protecting the genotoxic AP-site.

Mutagenesis mechanism of the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine.

Reactive oxygen species generate the genotoxic 8-oxoguanine (oxoG) and 8-oxoadenine (oxoA) as major oxidative lesions. The mutagenicity of oxoG is attributed to the lesion's ability to evade the geometric discrimination of DNA polymerases by adopting Hoogsteen base pairing with adenine in a Watson-Crick-like geometry. Compared with oxoG, the mutagenesis mechanism of oxoA, which preferentially induces A-to-C mutations, is poorly understood. In the absence of protein contacts, oxoA:G forms a wobble conformation, the formation of which is suppressed in the catalytic site of most DNA polymerases. Interestingly, human DNA polymerase η (polη) proficiently incorporates dGTP opposite oxoA, suggesting the nascent oxoA:dGTP overcomes the geometric discrimination of polη. To gain insights into oxoA-mediated mutagenesis, we determined crystal structures of polη bypassing oxoA. When paired with dGTP, oxoA adopted a syn-conformation and formed Hoogsteen pairing while in a wobble geometry, which was stabilized by Gln38-mediated minor groove contacts to oxoA:dGTP. Gln38Ala mutation reduced misinsertion efficiency ∼55-fold, indicating oxoA:dGTP misincorporation was promoted by minor groove interactions. Also, the efficiency of oxoA:dGTP insertion by the X-family polß decreased ∼380-fold when Asn279-mediated minor groove contact to dGTP was abolished. Overall, these results suggest that, unlike oxoG, oxoA-mediated mutagenesis is greatly induced by minor groove interactions.

Bypass of the Major Alkylative DNA Lesion by Human DNA Polymerase η.

A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2'-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson-Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson-Crick base pair with dCTP through their keto tautomers.

Promutagenicity of 8-Chloroguanine, A Major Inflammation-Induced Halogenated DNA Lesion.

Chronic inflammation is closely associated with cancer development. One possible mechanism for inflammation-induced carcinogenesis is DNA damage caused by reactive halogen species, such as hypochlorous acid, which is released by myeloperoxidase to kill pathogens. Hypochlorous acid can attack genomic DNA to produce 8-chloro-2'-deoxyguanosine (ClG) as a major lesion. It has been postulated that ClG promotes mutagenic replication using its syn conformer; yet, the structural basis for ClG-induced mutagenesis is unknown. We obtained crystal structures and kinetics data for nucleotide incorporation past a templating ClG using human DNA polymerase ß (polß) as a model enzyme for high-fidelity DNA polymerases. The structures showed that ClG formed base pairs with incoming dCTP and dGTP using its anti and syn conformers, respectively. Kinetic studies showed that polß incorporated dGTP only 15-fold less efficiently than dCTP, suggesting that replication across ClG is promutagenic. Two hydrogen bonds between syn-ClG and anti-dGTP and a water-mediated hydrogen bond appeared to facilitate mutagenic replication opposite the major halogenated guanine lesion. These results suggest that ClG in DNA promotes G to C transversion mutations by forming Hoogsteen base pairing between syn-ClG and anti-G during DNA synthesis.

Mutagenic Replication of the Major Oxidative Adenine Lesion 7,8-Dihydro-8-oxoadenine by Human DNA Polymerases.

Reactive oxygen species attack DNA to produce 7,8-dihyro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as major lesions. The structural basis for the mutagenicity of oxoG, which induces G to T mutations, is well understood. However, the structural basis for the mutagenic potential of oxoA, which induces A to C mutations, remains poorly understood. To gain insight into oxoA-induced mutagenesis, we conducted kinetic studies of human DNA polymerases ß and η replicating across oxoA and structural studies of polß incorporating dTTP/dGTP opposite oxoA. While polη readily bypassed oxoA, it incorporated dGTP opposite oxoA with a catalytic specificity comparable to that of correct insertion, underscoring the promutagenic nature of the major oxidative adenine lesion. Polη and polß incorporated dGTP opposite oxoA ∼170-fold and ∼100-fold more efficiently than that opposite dA, respectively, indicating that the 8-oxo moiety greatly facilitated error-prone replication. Crystal structures of polß showed that, when paired with an incoming dTTP, the templating oxoA adopted an anti conformation and formed Watson-Crick base pair. When paired with dGTP, oxoA adopted a syn conformation and formed a Hoogsteen base pair with Watson-Crick-like geometry, highlighting the dual-coding potential of oxoA. The templating oxoA was stabilized by Lys280-mediated stacking and hydrogen bonds. Overall, these results provide insight into the mutagenic potential and dual-coding nature of the major oxidative adenine lesion.

Structural basis for the bypass of the major oxaliplatin-DNA adducts by human DNA polymerase η.

Oxaliplatin, together with cisplatin, is among the most important drugs used in cancer chemotherapy. Oxaliplatin, which contains a bulky diaminocyclohexane (DACH) moiety, kills cancer cells mainly by producing (DACH)Pt-GpG intrastrand cross-links that impede transcription. The Pt-GpG tolerance by translesion DNA synthesis (TLS) polymerases contributes to the resistance of tumors to platinum-based chemotherapy. In particular, human DNA polymerase η (Polη) readily bypasses Pt-GpG adducts. While many structural studies have addressed how TLS polymerases interact with cisplatin-DNA adducts, a structure of DNA polymerase in complex with oxaliplatin-DNA adducts has not been reported, limiting our understanding of bypass of the bulky (DACH)Pt-GpG lesion by TLS polymerases. Herein, we report the first structure of DNA polymerase bound to oxaliplatinated DNA. We determined a crystal structure of Polη incorporating dCTP opposite the 3'G of the (DACH)Pt-GpG, which provides insights into accurate, efficient bypass of the oxaliplatin-GpG adducts by TLS polymerases. In the catalytic site of Polη, the 3'G of the (DACH)Pt-GpG formed three Watson-Crick hydrogen bonds with incoming dCTP and the primer terminus 3'-OH was optimally positioned for nucleotidyl transfer. To accommodate the bulky (DACH)Pt-GpG lesion, the Val59-Trp64 loop in the finger domain of Polη shifted from the positions observed in the corresponding Polη-cisplatin-GpG and undamaged structures, suggesting that the flexibility of the Val59-Trp64 loop allows the enzyme's bypass of the (DACH)Pt-GpG adducts. Overall, the Polη-oxaliplatin-GpG structure provides a structural basis for TLS-mediated bypass of the major oxaliplatin-DNA adducts and insights into resistance to platinum-based chemotherapy in humans.

Structural and Kinetic Studies of the Effect of Guanine N7 Alkylation and Metal Cofactors on DNA Replication.

A wide variety of endogenous and exogenous alkylating agents attack DNA to preferentially generate N7-alkylguanine (N7-alkylG) adducts. Studies of the effect of N7-alkylG lesions on biological processes have been difficult in part because of complications arising from the chemical lability of the positively charged N7-alkylG, which can readily produce secondary lesions. To assess the effect of bulky N7-alkylG on DNA replication, we prepared chemically stable N7-benzylguanine (N7bnG)-containing DNA and evaluated nucleotide incorporation opposite the lesion by human DNA polymerase ß (polß), a model enzyme for high-fidelity DNA polymerases. Kinetic studies showed that the N7-benzyl-G lesion greatly inhibited dCTP incorporation by polß. The crystal structure of polß incorporating dCTP opposite N7bnG showed a Watson-Crick N7bnG:dCTP structure. The polß-N7bnG:dCTP structure showed an open protein conformation, a relatively disordered dCTP, and a lack of catalytic metal, which explained the inefficient nucleotide incorporation opposite N7bnG. This indicates that polß is sensitive to major groove adducts in the templating base side and deters nucleotide incorporation opposite bulky N7-alkylG adducts by adopting a catalytically incompetent conformation. Substituting Mg2+ for Mn2+ induced an open-to-closed conformational change due to the presence of catalytic metal and stably bound dCTP and increased the catalytic efficiency by ∼10-fold, highlighting the effect of binding of the incoming nucleotide and catalytic metal on protein conformation and nucleotidyl transfer reaction. Overall, these results suggest that, although bulky alkyl groups at guanine-N7 may not alter base pairing properties of guanine, the major groove-positioned lesions in the template could impede nucleotidyl transfer by some DNA polymerases.

Insights into the effect of minor groove interactions and metal cofactors on mutagenic replication by human DNA polymerase ß.

DNA polymerases accommodate various base-pair conformations in the event of incorrect insertions. In particular, Watson-Crick-like dG:dTTP base pair has been observed at the insertion site of human DNA polymerase ß (pol ß). A potential factor contributing to the diverse conformations of base-pair mismatches is minor groove interactions. To gain insights into the effect of minor groove interactions on base-pair conformations, we generated an Asn279Ala polß mutant that cannot make minor groove contacts with an incoming nucleotide. We conducted structural and kinetic studies of Asn279Ala polß in complex with incoming dTTP and templating dG or O6-methyl-dG. The crystal structure of the Asn279Ala polß-G:T complex showed a wobble dG:dTTP base pair, indicating that the previously observed Watson-Crick-like dG:dTTP conformation was induced by the minor groove contact. In contrast, O6-methyl-dG, an analog of the enol tautomer of guanine, formed a Watson-Crick-like base pair with dTTP in the absence of the minor groove contact. These results suggest that the Watson-Crick-like G:T base pair at the insertion site is formed by the rare enol tautomers of G or T, whose population is increased by the minor groove hydrogen bond with Asn279. Kinetic studies showed that Asn279Ala mutation decreased dG:dTTP misincorporation rate six-fold in the presence of Mg2+ but increased the rate three-fold in the presence of Mn2+, highlighting the effect of minor groove interactions and metal ions on promutagenic replication by polß.

Synthesis, structure, and biological evaluation of a platinum-carbazole conjugate.

Cisplatin resistance is caused, in part, by the efficient removal of the helix-distorting cisplatin 1,2-intrastrand cross-links by nucleotide excision repair (NER) machinery. To make a platinum-DNA adduct that causes less helical distortion than the cisplatin 1,2-intrastrand adduct, we designed and synthesized a monofunctional platinum-carbazole conjugate (carbazoplatin). The 2.5 Å crystal structure of carbazoplatin-DNA adduct revealed both the monoplatination of the N7 of a guanine (G) base and the intercalation into two G:C base pairs, while causing a minor distortion of the DNA helix. A 50-mer dsDNA containing a single carbazoplatin lesion was poorly processed by UvrABC endonuclease, the prokaryotic NER machinery that detects helical distortion and performs dual incision around the lesion. Our cell viability assay indicated that the cytotoxic pathways of carbazoplatin might be different from those of cisplatin; carbazoplatin was 5-8 times more cytotoxic than cisplatin against PANC-1 and MDA-MB-231 cancer cell lines.

N7 methylation alters hydrogen-bonding patterns of guanine in duplex DNA.

N7-Alkyl-2'-deoxyguanosines are major adducts in DNA that are generated by various alkylating mutagens and drugs. However, the effect of the N7 alkylation on the hydrogen-bonding patterns of the guanine remains poorly understood. We prepared N7-methyl-2'-deoxyguanosine (N7mdG)-containing DNA using a transition-state destabilization strategy, developed a novel polß-host-guest complex system, and determined eight crystal structures of N7mdG or dG paired with dC, dT, dG, and dA. The structures of N7mdG:dC and N7mdG:dG are very similar to those of dG:dC and dG:dG, respectively, indicating the involvement of the keto tautomeric form of N7mdG in the base pairings with dC and dG. On the other hand, the structure of N7mdG:dT shows that the mispair forms three hydrogen bonds and adopts a Watson-Crick-like geometry rather than a wobble geometry, suggesting that the enol tautomeric form of N7mdG involves in its base pairing with dT. In addition, N7mdG:dA adopts a novel shifted anti:syn base pair presumably via the enol tautomeric form of N7mdG. The polß-host-guest complex structures reveal that guanine-N7 methylation changes the hydrogen-bonding patterns of the guanine when paired with dT or dA and suggest that N7 alkylation may alter the base pairing patterns of guanine by promoting the formation of the rare enol tautomeric form of guanine.

Structural basis for the inefficient nucleotide incorporation opposite cisplatin-DNA lesion by human DNA polymerase ß.

Human DNA polymerase ß (polß) has been suggested to play a role in cisplatin resistance, especially in polß-overexpressing cancer cells. Polß has been shown to accurately albeit slowly bypass the cisplatin-1,2-d(GpG) (Pt-GG) intramolecular cross-link in vitro. Currently, the structural basis for the inefficient Pt-GG bypass mechanism of polß is unknown. To gain structural insights into the mechanism, we determined two ternary structures of polß incorporating dCTP opposite the templating Pt-GG lesion in the presence of the active site Mg(2+) or Mn(2+). The Mg(2+)-bound structure shows that the bulky Pt-GG adduct is accommodated in the polß active site without any steric hindrance. In addition, both guanines of the Pt-GG lesion form Watson-Crick base pairing with the primer terminus dC and the incoming dCTP, providing the structural basis for the accurate bypass of the Pt-GG adduct by polß. The Mn(2+)-bound structure shows that polß adopts a catalytically suboptimal semiclosed conformation during the insertion of dCTP opposite the templating Pt-GG, explaining the inefficient replication across the Pt-GG lesion by polß. Overall, our studies provide the first structural insights into the mechanism of the potential polß-mediated cisplatin resistance.

The spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase ß.

To provide molecular-level insights into the spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase ß (polß), we report four crystal structures of polß complexed with dG•dTTP and dA•dCTP mismatches in the presence of Mg2+ or Mn2+. The Mg(2+)-bound ground-state structures show that the dA•dCTP-Mg2+ complex adopts an 'intermediate' protein conformation while the dG•dTTP-Mg2+ complex adopts an open protein conformation. The Mn(2+)-bound 'pre-chemistry-state' structures show that the dA•dCTP-Mn2+ complex is structurally very similar to the dA•dCTP-Mg2+ complex, whereas the dG•dTTP-Mn2+ complex undergoes a large-scale conformational change to adopt a Watson-Crick-like dG•dTTP base pair and a closed protein conformation. These structural differences, together with our molecular dynamics simulation studies, suggest that polß increases replication fidelity via a two-stage mismatch discrimination mechanism, where one is in the ground state and the other in the closed conformation state. In the closed conformation state, polß appears to allow only a Watson-Crick-like conformation for purine•pyrimidine base pairs, thereby discriminating the mismatched base pairs based on their ability to form the Watson-Crick-like conformation. Overall, the present studies provide new insights into the spontaneous replication error and the replication fidelity mechanisms of polß.

Transition-state destabilization reveals how human DNA polymerase ß proceeds across the chemically unstable lesion N7-methylguanine.

N7-Methyl-2'-deoxyguanosine (m7dG) is the predominant lesion formed by methylating agents. A systematic investigation on the effect of m7dG on DNA replication has been difficult due to the chemical instability of m7dG. To gain insights into the m7dG effect, we employed a 2'-fluorine-mediated transition-state destabilzation strategy. Specifically, we determined kinetic parameters for dCTP insertion opposite a chemically stable m7dG analogue, 2'-fluoro-m7dG (Fm7dG), by human DNA polymerase ß (polß) and solved three X-ray structures of polß in complex with the templating Fm7dG paired with incoming dCTP or dTTP analogues. The kinetic studies reveal that the templating Fm7dG slows polß catalysis ∼ 300-fold, suggesting that m7dG in genomic DNA may impede replication by some DNA polymerases. The structural analysis reveals that Fm7dG forms a canonical Watson-Crick base pair with dCTP, but metal ion coordination is suboptimal for catalysis in the polß-Fm7dG:dCTP complex, which partially explains the slow insertion of dCTP opposite Fm7dG by polß. In addition, the polß-Fm7dG:dTTP structure shows open protein conformations and staggered base pair conformations, indicating that N7-methylation of dG does not promote a promutagenic replication. Overall, the first systematic studies on the effect of m7dG on DNA replication reveal that polß catalysis across m7dG is slow, yet highly accurate.

Streamlining bottom-up protein identification based on selective ultraviolet photodissociation (UVPD) of chromophore-tagged histidine- and tyrosine-containing peptides.

We report a fast and highly efficient diazonium reaction that couples a nitroazobenzene chromophore to tyrosine and histidine residues, thus endowing peptides with high photoabsorption cross sections at 351 nm in the gas phase. Only the tagged peptides undergo ultraviolet photodissociation (UVPD) at 351 nm, as demonstrated for several Tyr- and His-containing peptides from protein digests. Additional selectivity is achieved by the integration of the UVPD-MS method with an in silico database search restricted to Tyr- and His-containing peptides. A modified MassMatrix algorithm condenses analysis by filtering the input database file to include Tyr/His-containing peptides only, thus reducing the search space and increasing confidence. In summary, derivatization of specific amino acid residues in conjunction with selective activation of the derivatized peptides provides a streamlined approach to shotgun proteomics.

Metal-dependent conformational activation explains highly promutagenic replication across O6-methylguanine by human DNA polymerase ß.

Human DNA polymerase ß (polß) inserts, albeit slowly, T opposite the carcinogenic lesion O6-methylguanine (O6MeG) ∼30-fold more frequently than C. To gain insight into this promutagenic process, we solved four ternary structures of polß with an incoming dCTP or dTTP analogue base-paired with O6MeG in the presence of active-site Mg(2+) or Mn(2+). The Mg(2+)-bound structures show that both the O6MeG·dCTP/dTTP-Mg(2+) complexes adopt an open protein conformation, staggered base pair, and one active-site metal ion. The Mn(2+)-bound structures reveal that, whereas the O6Me·dCTP-Mn(2+) complex assumes the similar altered conformation, the O6MeG·dTTP-Mn(2+) complex adopts a catalytically competent state with a closed protein conformation and pseudo-Watson-Crick base pair. On the basis of these observations, we conclude that polß slows nucleotide incorporation opposite O6MeG by inducing an altered conformation suboptimal for catalysis and promotes mutagenic replication by allowing Watson-Crick-mode for O6MeG·T but not for O6MeG·C in the enzyme active site. The O6MeG·dTTP-Mn(2+) ternary structure, which represents the first structure of mismatched polß ternary complex with a closed protein conformation and coplanar base pair, the first structure of pseudo-Watson-Crick O6MeG·T formed in the active site of a DNA polymerase, and a rare, if not the first, example of metal-dependent conformational activation of a DNA polymerase, indicate that catalytic metal-ion coordination is utilized as a kinetic checkpoint by polß and is crucial for the conformational activation of polß. Overall, our structural studies not only explain the promutagenic polß catalysis across O6MeG but also provide new insights into the replication fidelity of polß.

Structural basis for promutagenicity of 8-halogenated guanine.

8-Halogenated guanine (haloG), a major DNA adduct formed by reactive halogen species during inflammation, is a promutagenic lesion that promotes misincorporation of G opposite the lesion by various DNA polymerases. Currently, the structural basis for such misincorporation is unknown. To gain insights into the mechanism of misincorporation across haloG by polymerase, we determined seven x-ray structures of human DNA polymerase ß (polß) bound to DNA bearing 8-bromoguanine (BrG). We determined two pre-catalytic ternary complex structures of polß with an incoming nonhydrolyzable dGTP or dCTP analog paired with templating BrG. We also determined five binary complex structures of polß in complex with DNA containing BrG·C/T at post-insertion and post-extension sites. In the BrG·dGTP ternary structure, BrG adopts syn conformation and forms Hoogsteen base pairing with the incoming dGTP analog. In the BrG·dCTP ternary structure, BrG adopts anti conformation and forms Watson-Crick base pairing with the incoming dCTP analog. In addition, our polß binary post-extension structures show Hoogsteen BrG·G base pair and Watson-Crick BrG·C base pair. Taken together, the first structures of haloG-containing DNA bound to a protein indicate that both BrG·G and BrG·C base pairs are accommodated in the active site of polß. Our structures suggest that Hoogsteen-type base pairing between G and C8-modified G could be accommodated in the active site of a DNA polymerase, promoting G to C mutation.

Synthesis and structure of 16,22-diketocholesterol bound to oxysterol-binding protein Osh4.

We have synthesized 16,22-diketocholesterol, a novel ligand for oxysterol-binding protein Osh4, and determined X-ray structure of the diketocholesterol in complex with Osh4. The X-ray structure shows that α7 helix of Osh4 assumes open conformation while the rest of Osh4, closed conformation, implying this diketocholesterol-bound Osh4 structure may represent a structural intermediate between the two conformations.

Synthesis of C17-OH-north unit of ritterazine G via "Red-Ox" modifications of hecogenin acetate.

The C17-OH-north unit of ritterazine G was prepared in 13 steps from hecogenin acetate. This synthesis features a highly efficient and stereoselective introduction of the C17-OH via E-ring cleavage/F-ring formation, D-ring oxidation, and F-ring cleavage/E-ring formation.