Distorted DNA Structures Research Articles

Molecular dynamics simulation study of DNA conformation changes caused by the dinuclear platinum(II) complexes with the bisphosphonate group

Bisphosphonate (BP) has been widely used as a bone-targeting group, and the BP-modified platinum(II) complexes have shown potential to as anticancer drugs against bone-related diseases, such as osteosarcoma. DNA conformation changes induced by the BP-modified dinuclear platinum(II) complexes have been investigated using molecular dynamics simulations. The results indicated that the BP-modified dinuclear platinum(II) complexes coordinated to DNA results in DNA structural distortions, including twisting, unwinding and bending. Furthermore, the rigidity of the bridging linkers in the BP-modified platinum(II) complex may induce more significant DNA structural distortions with same spans. The results provide the detail information of DNA conformational changes induced by the BP-modified platinum(II) complexes with different flexibility of bridging linkers, and are helpful for exploring novel platinum-based antitumor drugs.

Abstract 1512: Architectural protein HMGB3 interacts with cisplatin-resistance associated overexpressed protein (CROP/LUC7L3) in human cancer cells and modulates cisplatin-DNA adduct removal

Abstract Cancer treatment and management have improved substantially in recent years resulting in increased overall patient survival. However, upon recurrence, many tumors can eventually stop responding to treatment and become resistant to therapy. Therefore, sensitizing cancer cells to therapy is an important approach to increase overall patient survival and improve their quality of life. We have previously shown that targeting the chromatin associated architectural protein High Mobility Group Box 3 (HMGB3) can sensitize cisplatin resistant human ovarian cancer cells to cisplatin. HMGB proteins are non-histone chromatin proteins, which bind to distorted DNA structures with higher affinity compared to double-stranded DNA. HMGB proteins have been shown to play important roles in DNA metabolism, such as replication, transcription and repair. We found that the sensitization of cisplatin resistant human A2780/CP70 ovarian cancer cells by siRNA-mediated targeting of HMGB3 is achieved, at least in part, by significantly lowering the expression of the DNA damage response (DDR) proteins ATR and CHK1. In addition, depletion of HMGB3 repressed global transcription by ~50% in human osteosarcoma (U2OS) cells. Here we report that GFP-tagged HMGB3 protein forms distinct foci in human A2780 ovarian cancer cells upon cisplatin treatment. Further, depleting HMGB3 in cisplatin resistant A2780/CP70 cells reduces the rate of cisplatin-DNA adduct removal, suggesting a possible role in chemoresistance. In addition, mass spectrometry studies reveal that HMGB3 interacts with the cisplatin resistance-associated overexpressed protein (CROP, also known as LUC7L3 ), following treatment with the DNA interstrand crosslink-inducing agent, psoralen (plus UVA irradiation). LUC7L3 is associated with the spliceosome and is overexpressed in A2780/CP70 cells compared to A2780 ovarian cancer cells. SiRNA-mediated depletion of HMGB3 reduces the transcription levels of LUC7L3. Further, siRNA-mediated depletion of LUC7L3 reduced the clone forming capacity of U2OS cells and ovarian cancer cells. Analysis of cancer patient databases revealed that LUC7L3 mRNA is overexpressed in many cancer types including ovarian cancer. Further analysis showed that ovarian cancer, gastric cancer, liver cancer and lung cancer patients overexpressing LUC7L3 have a significantly reduced overall survival than those with lower levels. Taken together, these data indicate that in addition to the architectural roles of HMGB3, which is important for DNA metabolism, transcriptional modulation of DDR proteins through interaction with the spliceosome may be another way that HMGB3 modulates chemotherapeutic DNA damage processing in human cancer cells. Citation Format: Anirban Mukherjee, Lorraine-Rana Benhamou, Karen M. Vasquez. Architectural protein HMGB3 interacts with cisplatin-resistance associated overexpressed protein (CROP/LUC7L3) in human cancer cells and modulates cisplatin-DNA adduct removal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1512.

Extended magnesium and calcium force field parameters for accurate ion-nucleic acid interactions in biomolecular simulations.

Magnesium and calcium play an essential role in the folding and function of nucleic acids. To correctly describe their interactions with DNA and RNA in biomolecular simulations, an accurate parameterization is crucial. In most cases, the ion parameters are optimized based on a set of experimental solution properties such as solvation free energies, radial distribution functions, water exchange rates, and activity coefficient derivatives. However, the transferability of such bulk-optimized ion parameters to quantitatively describe biomolecular systems is limited. Here, we extend the applicability of our previous bulk-optimized parameters by including experimental binding affinities toward the phosphate oxygen on nucleic acids. In particular, we systematically adjust the combination rules that are an integral part of the pairwise interaction potentials of classical force fields. This allows us to quantitatively describe specific ion binding to nucleic acids without changing the solution properties in the most simple and efficient way. We show the advancement of the optimized Lorentz combination rule for two representative nucleic acid systems. For double-stranded DNA, the optimized combination rule for Ca2+ significantly improves the agreement with experiments, while the standard combination rule leads to unrealistically distorted DNA structures. For the add A-riboswitch, the optimized combination rule for Mg2+ improves the structure of two specifically bound Mg2+ ions as judged by the experimental distance to the binding site. Including experimental binding affinities toward specific ion binding sites on biomolecules, therefore, provides a promising perspective to develop a more accurate description of metal cations for biomolecular simulations.

The DNA damage-sensing NER repair factor XPC-RAD23B does not recognize bulky DNA lesions with a missing nucleotide opposite the lesion

The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were −2.5–3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 β−hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes.

CDH1 binds MAD2L2 in a Rev1-like pattern

MAD2L2 (i.e. Rev7) is a central regulatory protein important in several processes, such as translesion synthesis (TLS), DNA damage response and mitosis. In TLS, MAD2L2 binds Rev3 to form Pol zeta (ζ) and promotes formation of the Pol ζ- REV1 complex allowing extension beyond distorted DNA structures. MAD2L2 is also part of the heterotetrameric shieldin complex that regulates DNA repair at sites of damage, where similarly to TLS, it bridges the interaction between SHLD2 and SHLD3. Lastly, during mitosis, MAD2L2 prevents premature activation of the anaphase promoting complex/cyclosome (APC/C), by sequestering its activator, CDH1. MAD2L2 exits in a ‘closed’ active conformation binding Rev3 and Rev1, or SHLD2 and SHLD3, and an ‘open’ inactive conformation, with no binding partners. Moreover, Pol ζ- REV1 forms a homodimer using a protein-protein interaction (PPI) domain comprised of a central αC helix, promoting Rev3-MAD2L2 interaction and C-terminus β-sheets, enabling Rev1-MAD2L2 interaction. While the role of MAD2L2 in TLS is well established, molecular details regarding the CDH1-MAD2L2 interaction and MAD2L2 homodimerization are still missing. Here we demonstrate, in a human cell line, using a series of MAD2L2 mutants, that MAD2L2’s C-terminus interface is essential for the CDH1-MAD2L2 binding as well as for homodimerization. In addition, we show that CDH1 interacts with MAD2L2 in a Rev1-like pattern, using the same C-terminus residues on MAD2L2 which Rev1 binds. Thus, identification of CDH1 as an additional Rev1-like binding protein strengthens the versatility of MAD2L2 as a regulatory protein and emphasizes the complexity involved in MAD2L2’s preferential complex formation.

Distortion of double-stranded DNA structure by the binding of the restriction DNA glycosylase R.PabI

R.PabI is a restriction DNA glycosylase that recognizes the sequence 5′-GTAC-3′ and hydrolyses the N-glycosidic bond of adenine in the recognition sequence. R.PabI drastically bends and unwinds the recognition sequence of double-stranded DNA (dsDNA) and flips the adenine and guanine bases in the recognition sequence into the catalytic and recognition sites on the protein surface. In this study, we determined the crystal structure of the R.PabI-dsDNA complex in which the dsDNA is drastically bent by the binding of R.PabI but the base pairs are not unwound. This structure is predicted to be important for the indirect readout of the recognition sequence by R.PabI. In the complex structure, wedge loops of the R.PabI dimer are inserted into the minor groove of dsDNA to stabilize the deformed dsDNA structure. A base stacking is distorted between the two wedge-inserted regions. R.PabI is predicted to utilize the distorted base stacking for the detection of the recognition sequence.

DNA Structural Distortions Induced by a Monofunctional Trinuclear Platinum Complex with Various Cross-Links Using Molecular Dynamics Simulation.

The monofunctional trinuclear platinum complex (MTPC), as a promising antitumor agent, can form MTPC-DNA adducts via bifunctional and trifunctional cross-links. Molecular dynamics simulations were used to investigate DNA structural distortions of the MTPC-DNA adducts. MTPC coordinating to DNA results in the decrease of base-pair thermal stability and DNA structural distortions. It is found that there are more significant DNA structural distortions in the trifunctional cross-link than in the bifunctional cross-link, in the 1,4-GG than in the 1,3-GG cross-link, and in the intrastrand than in the interstrand cross-link with the same spans. The results provide a better understanding of DNA structural distortions induced by MTPC with various cross-links at the nucleotide level and are helpful for exploring novel Pt-based anticancer drugs.

Modulating DNA by polyamides to regulate transcription factor PU.1-DNA binding interactions

Hairpin polyamides are synthetic small molecules that bind DNA minor groove sequence-selectively and, in many sequences, induce widening of the minor groove and compression of the major groove. The structural distortion of DNA caused by polyamides has enhanced our understanding of the regulation of DNA-binding proteins via polyamides. Polyamides have DNA binding affinities that are comparable to those proteins, therefore, can potentially be used as therapeutic agents to treat diseases caused by aberrant gene expression. In fact, many diseases are characterized by over- or under-expressed genes. PU.1 is a transcription factor that regulates many immune system genes. Aberrant expression of PU.1 has been associated with the development of acute myeloid leukemia (AML). We have, therefore, designed and synthesized ten hairpin polyamides to investigate their capacity in controlling the PU.1-DNA interaction. Our results showed that nine of the polyamides disrupt PU.1-DNA binding and the inhibition capacity strongly correlates with binding affinity. One molecule, FH1024, was observed forming a FH1024-PU.1-DNA ternary complex instead of inhibiting PU.1-DNA binding. This is the first report of a small molecule that is potentially a weak agonist that recruits PU.1 to DNA. This finding sheds light on the design of polyamides that exhibit novel regulatory mechanisms on protein-DNA binding.

Visual detection of cyclobutane pyrimidine dimer DNA damage lesions by Hg2+ and carbon dots

Cyclobutane pyrimidine dimmers (CPDs) and 6–4-[pyrimidine-2′-one] pyrimidine (6-4 PP) are major UV induced DNA damage lesions formed from solar radiation and other sources. CPD lesions are presumably mutagenic and carcinogenic that inhibit polymerases and interfere in DNA replication. An easy and cost effective way for visual detection of these lesions by using fluorescence based method is shown here. Artificial UVA and UVB lights were used for the generation of CPD and 6-4 PPs in selected DNA samples. Binding of Hg2+ ions with DNA before and after induction of CPD and 6-4 PP lesions was evaluated in the presence of highly fluorescent blue emitting carbon dots (CDs). Induction of CPD and 6-4 PPs in DNA causes distortion of DNA structure which hinders the binding of Hg2+ ions to DNA nucleobases. Quenching of fluorescence intensity of CDs by unbound Hg2+ ions was found to be proportional to the amount of CPD and 6-4 PP lesions induced by UV irradiation of DNA samples that offer a biosensing platform for the sensitive detection of CPD lesions in DNA. The fluorescent quenching was visually detectable using hand held UV light without the intervention of any equipment.

Dual function of Ixr1 in transcriptional regulation and recognition of cisplatin-DNA adducts is caused by differential binding through its two HMG-boxes

Ixr1 is a transcriptional factor involved in the response to hypoxia, which is also related to DNA repair. It binds to DNA through its two in-tandem high mobility group box (HMG-box) domains. Each function depends on recognition of different DNA structures, B-form DNA at specific consensus sequences for transcriptional regulation, or distorted DNA, like cisplatin-DNA adducts, for DNA repair. However, the contribution of the HMG-box domains in the Ixr1 protein to the formation of different protein-DNA complexes is poorly understood. We have biophysically and biochemically characterized these interactions with specific DNA sequences from the promoters regulated by Ixr1, or with cisplatin-DNA adducts. Both HMG-boxes are necessary for transcriptional regulation, and they are not functionally interchangeable. The in-tandem arrangement of their HMG-boxes is necessary for functional folding and causes sequential cooperative binding to specific DNA sequences, with HMG-box A showing a higher contribution to DNA binding and bending than the HMG-box B. Binding of Ixr1 HMG boxes to specific DNA sequences is entropy driven, whereas binding to platinated DNA is enthalpy driven for HMG-box A and entropy driven for HMG-box B. This is the first proof that HMG-box binding to different DNA structures is associated with predictable thermodynamic differences. Based on our study, we present a model to explain the dual function of Ixr1 in the regulation of gene expression and recognition of distorted DNA structures caused by cisplatin treatment.

DNA with Damage in Both Strands as Affinity Probes and Nucleotide Excision Repair Substrates.

Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dC(FAB) and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dC(FAB)/dG (probe I), dC(FAB)/nFlu+4 (probe II), and dC(FAB)/nFlu-3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC-HR23B (Kdum > KdI > KdII ≈ KdIII) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dC(FAB) results in (i) decreased melting temperature (ΔTm=-3°C) and (ii) 12° DNA bending. The extended dC(FAB)/dG-DNA (137bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC-HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dC(FAB)/nFlu+4, dC(FAB)/nFlu-3) or irreparable (nFlu/nFlu+4, nFlu/nFlu-3, nAnt/nFlu+4, nAnt/nFlu-3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis.

Guanine nucleobase adducts formed by a monofunctional complex: [Pt(N-(6-methyl-2-picolyl)-N-(2-picolyl)amine)Cl]Cl

Monofunctional Pt(II) complexes bind to G residues in DNA and, if the carrier ligands are bulky, cause DNA structural distortions that lead to anticancer activity. We assessed the steric effects of the tridentate carrier ligand, N(H)6-Medpa (N-(6-methyl-2-picolyl)-N-(2-picolyl)amine), bearing a 6-methyl group and a 6′-proton projecting toward the nucleobase in Pt(N(H)6-Medpa)G adducts (G=9-ethylguanine, 3′-GMP, 5′-GMP, 5′-GTP). Pt(N(H)6-Medpa)G adducts form syn and anti rotamers with the guanine O6 and the central N–H of N(H)6-Medpa on the same or opposite side of the coordination plane, respectively. Pt(N(H)6-Medpa)G adducts have some properties (ease of rotamer interchange and extent of conversion to bis adducts, Pt(N(H)6-Medpa)G2) intermediate to properties reported for analogs having a tridentate ligand with zero or two methyl groups. However, in comparison, the syn rotamer of Pt(N(H)6-Medpa)G adducts has an unexpectedly high abundance. This result is attributable to guanine base canting, such that the 6-membered guanine ring is positioned away from the bulky 6-Me group. This canting both relieves electrostatic repulsion between the partially positive H6′ and the guanine H8 protons and creates a favorable electrostatic attraction between the H6′ proton and the partially negative guanine O6. This combined information provides insight useful for designing monofunctional anticancer agents.

A nuclear factor of high mobility group box protein in Toxoplasma gondii.

High mobility group box 1 (HMGB1) is a nuclear factor that usually binds DNA and modulates gene expression in multicellular organisms. Three HMGB1 orthologs were predicted in the genome of Toxoplasma gondii, an obligate intracellular protozoan pathogen, termed TgHMGB1a, b and c. Phylogenetic and bioinformatic analyses indicated that these proteins all contain a single HMG box and which shared in three genotypes. We cloned TgHMGB1a, a 33.9 kDa protein that can stimulates macrophages to release TNF-α, and, we demonstrated that the TgHMGB1a binds distorted DNA structures such as cruciform DNA in electrophoretic mobility shift assays (EMSA). Immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites but translocated into the cytoplasm while the parasites release to extracellular. There were no significant phenotypic changes when the TgHMGB1a B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR analyses showed that the expression levels of many important genes, including virulence factors, increased when TgHMGB1a was overexpressed, but no significant changes were observed in TgHMGB1a B box-deficient parasites. Our findings demonstrated that TgHMGB1a is indeed a nuclear protein that maintains HMG box architectural functions and is a potential proinflammatory factor during the T.gondii infection. Further studies that clarify the functions of TgHMGB1s will increase our knowledge of transcriptional regulation and parasite virulence, and might provide new insight into host–parasite interactions for T. gondii infection.

The combination of a nuclear HMGB1-positive and HMGB2-negative expression is potentially associated with a shortened survival in patients with pancreatic ductal adenocarcinoma.

High-mobility group box (HMGB) proteins are ubiquitous, abundant nuclear non-histone chromosomal proteins that play a critical role in binding to distorted DNA structures and subsequently regulating DNA transcription, replication, repair, and recombination. Both HMGB1 and HMGB2 exhibit a high expression in several human cancers and are closely associated with tumor progression and a poor prognosis. However, the expression patterns of these molecules in pancreatic ductal adenocarcinoma (PDAC) remain to be elucidated. As most cases of postoperative relapse of PDAC occur within the first 2 years, the clinical significance of accurate biomarkers is needed. Therefore, we investigated the correlation between the immunohistochemical HMGB1 and HMGB2 expression and the clinicopathological characteristics and prognosis using 62 paraffin-embedded tumor samples obtained from patients with surgically resected PDAC. The HMGB1/2 expression was considered to be positive when 10 % or more of the cancer cells showed positive nuclear, not merely cytoplasmic, staining. Consequently, the expression of HMGB1/2 was observed in 54 (87.1 %) and 31 (50.0 %) patients, respectively. Unexpectedly, a positive HMGB1 expression was found to have a significantly close relationship with a negative HMGB2 expression. The univariate and multivariate analyses demonstrated that the patients with a HMGB1+ and HMGB2- status had markedly lower disease-specific survival rates, especially within the first 2 years postoperatively, whereas those with a HMGB1+ status alone did not. Therefore, the combination of a HMGB1+ and HMGB2- expression potentially predicts a poor prognosis in patients with PDAC, and these new biomarkers may be useful parameters for clinical management in the early postoperative phase.

Cytosolic HMGB1 expression in human renal clear cell cancer indicates higher pathological T classifications and tumor grades.

High mobility group box (HMGB) proteins are nuclear nonhistone chromosomal proteins that bend DNA, bind preferentially to distorted DNA structures, and promote the assembly of site-specific DNA binding proteins. Recent reports indicate that HMGB1 functions as a proinflammatory cytokine. Here, we studied expressions of HMGB1 and HMGB2 in human renal cancer. Immunohistological expressions of HMGB1 and HMGB2 were assessed in 39 surgically resected human renal cancer specimens. HMGB1 was expressed in the nucleus in 37 out of 39 (94.9%) renal clear cell cancers,while its expression in the cytosol was noted in 19 cases (48.7%). Cytosolic HMGB1 is expressed more frequently in cancers beyond the pT1b classification than in those at the pT1a classification. Higher tumor grades (≥ G2) were also significantly linked with the cytosolic expression of HMGB1. HMGB2 was expressed in the nucleus in 35 of 39 (89.7%) renal clear cell cancers, while its expression in the cytosol was observed in only 7 case (17.9%). Linkage between cytosolic expression of HMGB2 and T classifications was weakly observed, while that between nuclear expression and T classifications was not. HMGB1 expressed in the cytoplasm may be an effective marker indicating higher T classifications and tumor grades.

Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties.

The chaperone like function of the nonhistone protein HMGB1

Almost all essential nuclear processes as replication, repair, transcription and recombination require the chromatin template to be correctly unwound and than repackaged. The major strategy that the cell uses to overcome the nucleosome barrier is the proper removal of the histone octamer and subsequent deposition onto DNA. Important factors in this multi step phenomenon are the histone chaperones that can assemble nucleosome arrays in vitro in the absence of ATP. The nonhistone protein HMGB1 is a good candidate for a chaperone as its molecule consists of two DNA binding motives, Box’s A and B, and a long nonstructured C tail highly negatively charged. HMGB1 protein is known as a nuclear “architectural” factor for its property to bind preferentially to distorted DNA structures and was reported to kink the double helix. Our experiments show that in the classical stepwise dialysis method for nucleosome assembly the addition of HMGB1 protein stimulates more than two times the formation of middle-positioned nucleosomes. The stimulation effect persists in dialysis free experiment when the reconstitution is possible only in the presence of a chaperone. The addition of HMGB1 protein strongly enhanced the formation of a nucleosome in a dose dependant manner. Our results show that the target of HMGB1 action as a chaperone is the DNA fragment not the histone octamer. One possible explanation for the stimulating effect of HMGB1 is the “architectural” property of the protein to associate with the middle of the DNA fragment and to kink it. The acquired V shaped DNA structure is probably conformationals more favorable to wrap around the prefolded histone octamer.We tested also the role of the post-synthetic acetylation for the chaperone function of HMGB1 protein. The presence of an acetyl groups at Lys 2 decreases strongly the stimulating effect of the protein in the stepwise salt dialysis experiment and the same tendency persisted in the dialysis free experiment.

HMGB1 and HMGB2 Expression Patterns in Human Papillary Renal Cancers

High mobility group box (HMGB) proteins are nuclear nonhistone chromosomal proteins that bend DNA, bind preferentially to distorted DNA structures, and promote the assembly of site-specific DNA binding proteins. Recent reports indicate that HMGB1 has a dual function, a cytokine in addition to a nuclear protein. The increased expression of HMGB1 has been reported for several different tumors. Here, we assessed HMGB1 and HMGB2 expressions in two cases of papillary renal cell carcinoma. One case with pT1a, Grade 2 showed HMGB1 expression in the nucleus and cytosol and HMGB2 expression in the nucleus, but not in the cytosol. In the other case, there were three renal tumors, one of which was clear cell renal cell carcinoma with pT1a, Grade 3 and two were papillary renal cell carcinomas, Grade 2 (5 mmand2 mmin the diameter). Both HMGB1 and HMGB2 were expressed in the nucleus and cytosol of papillary carcinoma. In the clear cell carcinoma of this case, HMGB1 expression was stained both in the nucleus and cytosol, while HMGB2 was observed in the nucleus, but not in the cytosol. More samples need to be further investigated in order to draw conclusions concerning HMGB expressions in papillary renal cell carcinomas.

HMGB1 and HMGB2 Expression Patterns in Human Papillary Renal Cancers

High mobility group box (HMGB) proteins are nuclear nonhistone chromosomal proteins that bend DNA, bind preferentially to distorted DNA structures, and promote the assembly of site-specific DNA binding proteins. Recent reports indicate that HMGB1 has a dual function, a cytokine in addition to a nuclear protein. The increased expression of HMGB1 has been reported for several different tumors. Here, we assessed HMGB1 and HMGB2 expressions in two cases of papillary renal cell carcinoma. One case with pT1a, Grade 2 showed HMGB1 expression in the nucleus and cytosol and HMGB2 expression in the nucleus, but not in the cytosol. In the other case, there were three renal tumors, one of which was clear cell renal cell carcinoma with pT1a, Grade 3 and two were papillary renal cell carcinomas, Grade 2 (5 mmand2 mmin the diameter). Both HMGB1 and HMGB2 were expressed in the nucleus and cytosol of papillary carcinoma. In the clear cell carcinoma of this case, HMGB1 expression was stained both in the nucleus and cytosol, while HMGB2 was observed in the nucleus, but not in the cytosol. More samples need to be further investigated in order to draw conclusions concerning HMGB expressions in papillary renal cell carcinomas.

Guanine Nucleobase Adducts Formed by [Pt(di-(2-picolyl)amine)Cl]Cl: Evidence That a Tridentate Ligand with Only in-Plane Bulk Can Slow Guanine Base Rotation

Pt(II) complexes bind preferentially at N7 of G residues of DNA, causing DNA structural distortions associated with anticancer activity. Some distortions induced by difunctional cisplatin are also found for monofunctional Pt(II) complexes with carrier ligands having bulk projecting toward the guanine base. This ligand bulk can be correlated with impeded rotation about the Pt-N7(guanine) bond. Pt(N(H)dpa)(G) adducts (N(H)dpa = di-(2-picolyl)amine, G = 5'-GMP, 5'-GDP, 5'-GTP, guanosine, 9-EtG, and 5'-IMP) were used to assess whether a tridentate carrier ligand having bulk concentrated in the coordination plane can impede guanine nucleobase rotation. Because the Pt(N(H)dpa) moiety contains a mirror plane but is unsymmetrical with respect to the coordination plane, Pt(N(H)dpa)(G) adducts can form anti or syn rotamers with the guanine O6 and the central N-H of N(H)dpa on the opposite or the same side of the coordination plane, respectively. The observation of two sharp, comparably intense guanine H8 NMR signals provided evidence that these Pt(N(H)dpa)(G) adducts exist as mixtures of syn and anti rotamers, that rotational interchange is impeded by N(H)dpa, and that the key interactions involves steric repulsions between the pyridyl and guanine rings. The relative proximity of the guanine H8 to the anisotropic pyridyl rings allowed us to conclude that the syn rotamer was usually more abundant. However, the anti rotamer was more abundant for the Pt(N(H)dpa)(5'-GTP) adduct, in which a hydrogen bond between the 5'-GTP γ-phosphate group and the N(H)dpa central N-H is geometrically possible. In all previous examples of the influence of hydrogen bond formation on rotamer abundance in Pt(II) guanine adducts, these hydrogen bonding interactions occurred between ligand groups in cis positions. Thus, the role of a trans ligand group in influencing rotamer abundance, as found here, is unusual.