Molecular models for the tissue specificity of DNA mismatch repair-deficient carcinogenesis

A common feature of all the known cancer genetic syndromes is that they predispose only to selective types of malignancy. However, many of the genes mutated in these syndromes are ubiquitously expressed, and influence seemingly universal processes such as DNA repair or cell cycle control. The tissue specificity of cancers that arise from malfunction of these apparently universal traits remains a key puzzle in cancer genetics. Mutations in DNA mismatch repair (MMR) genes cause the most common known cancer genetic syndrome, hereditary non-polyposis colorectal cancer, and the fundamental biology of MMR is one of the most intensively studied processes in laboratories all around the world. This review uses MMR as a model system to understand mechanisms that may explain the selective development of tumors in particular cell types despite the universal nature of this process. We evaluate recent data giving insights into the specific tumor types that are attributable to defective MMR in humans and mice under different modes of inheritance, and propose models that may explain the spectrum of cancer types observed.     

Mutation rates of TGFBR2 and ACVR2 coding microsatellites in human cells with defective DNA mismatch repair

Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFbeta family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1-/-, hMSH6-/-, hMSH3-/-, and MMR-proficient) to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP) gene, allowing a -1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7-35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a -1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2) in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes) and M2 (bright, representing full mutants) were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91 x 10(-4)) and 15 (2.18 x 10(-4)) times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was approximately 3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The -1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background.     

UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells

DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions. Immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as the immediate repair of CPDs, indicating that a single 6-4PP lesion is severalfold more toxic than a CPD in the cells. Because UV light-induced DNA damage is not repaired at all in nucleotide excision repair-deficient cells, proliferation of these cells after UV light irradiation must be achieved by tolerance of the damage at replication. We found that RNA interference designed to suppress polymerase zeta activity made the cells more sensitive to UV light. This increase in sensitivity was prevented by photorepair of 6-4PPs but not by photorepair of CPDs, indicating that polymerase zeta is involved in the tolerance of 6-4PPs in human cells.     

Novel roles for MLH3 deficiency and TLE6-like amplification in DNA mismatch repair-deficient gastrointestinal tumorigenesis and progression

DNA mismatch repair suppresses gastrointestinal tumorgenesis. Four mammalian E. coli MutL homologues heterodimerize to form three distinct complexes: MLH1/PMS2, MLH1/MLH3, and MLH1/PMS1. To understand the mechanistic contributions of MLH3 and PMS2 in gastrointestinal tumor suppression, we generated Mlh3^−/−;Apc^1638N and Mlh3^−/−;Pms2^−/−;Apc^1638N (MPA) mice. Mlh3 nullizygosity significantly increased Apc frameshift mutations and tumor multiplicity. Combined Mlh3;Pms2 nullizygosity further increased Apc base-substitution mutations. The spectrum of MPA tumor mutations was distinct from that observed in Mlh1^−/−;Apc^1638N mice, implicating the first potential role for MLH1/PMS1 in tumor suppression. Because Mlh3;Pms2 deficiency also increased gastrointestinal tumor progression, we used array-CGH to identify a recurrent tumor amplicon. This amplicon contained a previously uncharacterized Transducin enhancer of Split (Tle) family gene, Tle6-like. Expression of Tle6-like, or the similar human TLE6D splice isoform in colon cancer cells increased cell proliferation, colony-formation, cell migration, and xenograft tumorgenicity. Tle6-like;TLE6D directly interact with the gastrointestinal tumor suppressor RUNX3 and antagonize RUNX3 target transactivation. TLE6D is recurrently overexpressed in human colorectal cancers and TLE6D expression correlates with RUNX3 expression. Collectively, these findings provide important insights into the molecular mechanisms of individual MutL homologue tumor suppression and demonstrate an association between TLE mediated antagonism of RUNX3 and accelerated human colorectal cancer progression.     

A role for DNA polymerase in the specificity of nucleotide incorporation opposite N-acetyl-2-aminofluorene adducts

Escherichia coli DNA polymerase I (Klenow fragment), DNA polymerase α from both calf thymus and human lymphoma cells and DNA polymerase β from calf thymus and Novikoff hepatoma cells can incorporate nucleotides opposite N-guanin-8-yl-acetyl-2-aminofluorene in DNA. The polymerases incorporate dCTP opposite some AAF-dG⁴ lesions when Mg²⁺ is the divalent cation. Substitution of Mn²⁺ for Mg²⁺ broadens the specificity of insertion: E. coli DNA polymerase I (Klenow fragment) also inserts A, and at specific sites G or T; DNA polymerase α inserts any of the four dNTPs with A and C incorporated preferentially to G and T. Polymerase β is specific, inserting mainly C even in the presence of Mn²⁺. The K_m for addition of dATP opposite a lesion by E. coli polymerase I (Klenow fragment) in the presence of Mn²⁺ is about 0.5 mm. dNMPs increase the insertion of nucleotides opposite AAF-dG in the presence of Mg²⁺ and increase both the rate and number of sites at which incorporation occurs in the presence of Mn²⁺. dNTPαS and recA protein increase only the insertion of C.We suppose that the incorporation of dCTP reflects normal base-pairing with the AAF-deoxyguanine in the anti conformation, whereas insertion of the other nucleotides (including some of the C) reflects insertion opposite the AAF adduct in its preferred syn conformation. The fact that the DNA polymerase plays a role in determining the specificity of insertion opposite a lesion terminating DNA synthesis suggests that the spectrum of base substitution mutagenesis seen in vivo may reflect the properties of the protein components, including the polymerase, involved in bypass synthesis.     

UV-induced histone H2AX phosphorylation and DNA damage related proteins accumulate and persist in nucleotide excision repair-deficient XP-B cells

DNA double strand breaks (DSB) may be caused by ionizing radiation. In contrast, UV exposure forms dipyrimidine photoproducts and is not considered an inducer of DSB. We found that uniform or localized UV treatment induced phosphorylation of the DNA damage related (DDR) proteins H2AX, ATM and NBS1 and co-localization of γ-H2AX with the DDR proteins p-ATM, p-NBS1, Rad51 and FANCD2 that persisted for about 6h in normal human fibroblasts. This post-UV phosphorylation was observed in the absence of nucleotide excision repair (NER), since NER deficient XP-B cells (lacking functional XPB DNA repair helicase) and global genome repair-deficient rodent cells also showed phosphorylation and localization of these DDR proteins. Resolution of the DDR proteins was dependent on NER, since they persisted for 24h in the XP-B cells. In the normal and XP-B cells p53 and p21 was detected at 6h and 24h but Mdm2 was not induced in the XP-B cells. Post-UV induction of Wip1 phosphatase was detected in the normal cells but not in the XP-B cells. DNA DSB were detected with a neutral comet assay at 6h and 24h post-UV in the normal and XP-B cells. These results indicate that UV damage can activate the DDR pathway in the absence of NER. However, a later step in DNA damage processing involving induction of Wip1 and resolution of DDR proteins was not observed in the absence of NER.     

Positive selection within the Schizophrenia-associated GABA(A) receptor beta(2) gene

The gamma-aminobutyric acid type-A (GABA(A)) receptor plays a major role in inhibitory neurotransmissions. Intronic SNPs and haplotypes in GABRB2, the gene for GABA(A) receptor beta(2) subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced beta(2) isoforms. In the present study, using chimpanzee as an ancestral reference, high frequencies were observed for the derived (D) alleles of the four SNPs rs6556547, rs187269, rs1816071 and rs1816072 in GABRB2, suggesting the occurrence of positive selection for these derived alleles. Coalescence-based simulation showed that the population frequency spectra and the frequencies of H56, the haplotype having all four D alleles, significantly deviated from neutral-evolution expectation in various demographic models. Haplotypes containing the derived allele of rs1816072 displayed significantly less diversity compared to haplotypes containing its ancestral allele, further supporting positive selection. The variations in DD-genotype frequencies in five human populations provided a snapshot of the evolutionary history, which suggested that the positive selections of the D alleles are recent and likely ongoing. The divergence between the DD-genotype profiles of schizophrenic and control samples pointed to the schizophrenia-relevance of positive selections, with the schizophrenic samples showing weakened selections compared to the controls. These DD-genotypes were previously found to increase the expression of beta(2), especially its long isoform. Electrophysiological analysis showed that this long beta(2) isoform favored by the positive selections is more sensitive than the short isoform to the inhibition of GABA(A) receptor function by energy depletion. These findings represent the first demonstration of positive selection in a schizophrenia-associated gene.     

Activin-type II receptor B (ACVR2B) and follistatin haplotype associations with muscle mass and strength in humans.

Genetic variation in myostatin, a negative regulator of skeletal muscle, in cattle has shown remarkable influence on skeletal muscle, resulting in a double-muscled phenotype in certain breeds; however, DNA sequence variation within this gene in humans has not been consistently associated with skeletal muscle mass or strength. Follistatin and activin-type II receptor B (ACVR2B) are two myostatin-related genes involved in the regulation and signaling of myostatin. We sought to identify associations between genetic variation and haplotype structure in both follistatin and ACVR2B with skeletal muscle-related phenotypes. Three hundred fifteen men and 278 women aged 19-90 yr from the Baltimore Longitudinal Study of Aging were genotyped to determine respective haplotype groupings (Hap Groups) based on HapMap data. Whole body soft tissue composition was measured by dual-energy X-ray absorptiometry. Quadriceps peak torque (strength) was measured using an isokinetic dynamometer. Women carriers of ACVR2B Hap Group 1 exhibited significantly less quadriceps muscle strength (shortening phase) than women homozygous for Hap Group 2 (109.2+/-1.9 vs. 118.6+/-4.1 N.m, 30 degrees/s, respectively, P=0.036). No significant association was observed in men. Male carriers of follistatin Hap Group 3 exhibited significantly less total leg fat-free mass than noncarriers (16.6+/-0.3 vs. 17.5+/-0.2 kg, respectively, P=0.012). No significant associations between these haplotype groups were observed in women. These results indicate that haplotype structure at the ACVR2B and follistatin loci may contribute to interindividual variation in skeletal muscle mass and strength, although these data indicate sex-specific relationships.     

2'-anthraquinone-conjugated oligonucleotide as an electrochemical probe for DNA mismatch

We previously prepared the oligonucleotides (ODNs) conjugated to an anthraquinone (AQ) group via one carbon linker at the 2'-sugar position. When these modified ODNs bind to cDNA sequences, the AQ moiety can be intercalated into the predetermined base-pair pocket of a duplex DNA. In this paper, 2'-AQ-modified ODNs are shown to be an excellent electrochemical probe to clarify the effect of a mismatch base on the charge transfer (CT) though DNA. Two types of DNA-modified electrodes were constructed by assembly of disulfide-terminated 2'-AQ-ODN duplexes onto gold electrodes. One type of electrodes (system I) contains fully matched base pairs or a single-base mismatch in duplex DNA between the redox center and the electrode. The other (system II) consists of the mismatch but at the outside of the redox center. The modified electrodes were analyzed by cyclic voltammetry to estimate the CT rate through duplex DNA. In system I, the CT rate was found to be approximately 50 s (-1) for the fully matched AQ-ODN duplexes, while the CT rates of the mismatched DNA were considerably slower than that of the fully matched DNA. In system II, the AQ-ODN duplexes showed almost similar CT rates ( approximately 50 s (-1)) for the fully matched DNA and for the mismatched DNAs. The detection of a single-base mismatch was then performed by chronocoulometry (CC). All the DNA duplexes containing a mismatch base in system I gave the reduced electrochemical responses when compared to the fully matched DNA. In particular, the mismatched DNAs including G--A mismatch can be differentiated from fully matched DNA without using any electrochemical catalyst. We further tested the usefulness of single-stranded (ss) AQ-ODN immobilized on a gold electrode in the electrochemical detection of a single-base mismatch through hybridization assay. The ss-AQ-ODN electrodes were immersed in target-containing buffer at room temperature, and the CC measurements were carried out to see the changes in the integrated charge. Within 60 min, the mismatched DNA was clearly distinguishable by the CC differences from the fully matched target. Thus, the electrochemical hybridization assay provides an easy and convenient detection for DNA mutation that does not require any extra reagents, catalyst, target labeling, and washing steps.     

Structure and nucleotide specificity of Staphylococcus aureus dihydrodipicolinate reductase (DapB)

Lysine biosynthesis proceeds by the nucleotide-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) by dihydrodipicolinate reductase (DHDPR). The S. aureus DHDPR structure reveals different conformational states of this enzyme even in the absence of a substrate or nucleotide-cofactor. Despite lacking a conserved basic residue essential for NADPH interaction, S. aureus DHDPR differs from other homologues as NADPH is a more preferred co-factor than NADH. The structure provides a rationale-Lys35 compensates for the co-factor site mutation. These observations are significant for bi-ligand inhibitor design that relies on ligand-induced conformational changes as well as co-factor specificity for this important drug target.     

Histone peptide AKRHRK enhances H(2)O(2)-induced DNA damage and alters its site specificity

Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H(2)O(2), leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH(3)CO-AKRHRK-CONH(2), which has a metal-binding site. This histone peptide enhanced DNA damage induced by H(2)O(2) and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5'-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and ESR spin-trapping signal from H(2)O(2) and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H(2)O(2), may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition.     

DDB2 (Damaged-DNA binding protein 2) in nucleotide excision repair and DNA damage response

DDB2 was identified as a protein involved in the Nucleotide Excision Repair (NER), a major DNA repair mechanism that repairs UV damage to prevent accumulation of mutations and tumorigenesis. However, recent studies indicated additional functions of DDB2 in the DNA damage response pathway. Herein, we discuss the proposed mechanisms by which DDB2 activates NER and programmed cell death upon DNA damage through its E3 ligase activity.     

Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived nucleotide substrates

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with KS values of approximately 8 and 4mm, respectively, in 0.1 m KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.     

Benzimidazoles as benzamide replacements within cyclohexane-based CC chemokine receptor 2 (CCR2) antagonists

We describe the design, synthesis, and evaluation of benzimidazoles as benzamide replacements within a series of trisubstituted cyclohexane CCR2 antagonists. 7-Trifluoromethylbenzimidazoles displayed potent binding and functional antagonism of CCR2 while being selective over CCR3. These benzimidazoles were also incorporated into lactam-containing antagonists, thus completely eliminating the customary bis-amide.     

Immunocytochemical study of activin type IB receptor (XALK4) in Xenopus oocytes

Studies have shown that the activin type IB receptor is specific for activin/nodal signaling. Activin is produced by follicle cells in the ovary, and is incorporated into the oocytes. Antisera against three peptides were prepared, encompassing the extracellular, intracellular and serine/threonine kinase domains of the Xenopus type IB activin receptor (XALK4). Immunocytochemistry was done using these antisera to investigate the distribution of XALK4 in the Xenopus ovary. All three antisera stained the mitochondrial cloud of Xenopus previtellogenic oocytes. Purified antibody against the intracellular domain also recognized the mitochondrial cloud. Immunoelectron microscopy localized XALK4 on the endoplasmic reticulum of the mitochondrial cloud, although not on mitochondria.     

Modeling the interactions of the nucleotide excision repair UvrA(2) dimer with DNA

The UvrA protein initiates the DNA damage recognition process by the bacterial nucleotide excision repair (NER) system. Recently, crystallographic structures of holo-UvrA(2) dimers from two different microorganisms have been released (Protein Data Bank entries 2r6f , 2vf7 , and 2vf8 ). However, the details of the DNA binding by UvrA(2) and other peculiarities involved in the damage recognition process remain unknown. We have undertaken a molecular modeling approach to appraise the possible modes of DNA-UvrA(2) interaction using molecular docking and short-scale guided molecular dynamics [continuum field, constrained, and/or unrestricted simulated annealing (SA)], taking into account the three-dimensional location of a series of mutation-identified UvrA residues implicated in DNA binding. The molecular docking was based on the assumptions that the UvrA(2) dimer is preformed prior to DNA binding and that no major protein conformational rearrangements, except moderate domain reorientations, are required for binding of undamaged DNA. As a first approximation, DNA was treated as a rigid ligand. From the electrostatic relief of the ventral surface of UvrA(2), we initially identified three, noncollinear DNA binding paths. Each of the three resulting nucleoprotein complexes (C1, C2, and C3) was analyzed separately, including calculation of binding energies, the number and type of interaction residues (including mutated ones), and the predominant mode of translational and rotational motion of specific protein domains after SA to ensure improved DNA binding. The UvrA(2) dimer can accommodate DNA in all three orientations, albeit with different binding strengths. One of the UvrA(2)-DNA complexes (C1) fulfilled most of the requirements (high interaction energy, proximity of DNA to mutated residues, etc.) expected for a natural, high-affinity DNA binding site. This nucleoprotein presents a structural organization that is designed to clamp and bend double-stranded DNA. We examined the binding site in more detail by docking DNAs of significantly different (AT- vs CG-enriched) sequences and by submitting the complexes to DNA-unrestricted SA. It was found that in a manner independent of the DNA sequence and applied MD protocols, UvrA(2) favors binding of a bent and unwound undamaged DNA, with a kink positioned in the proximity of the Zn3 hairpins, anticollinearly aligned at the bottom of the ventral protein surface. It is further hypothesized that the Zn3 modules play an essential role in the damage recognition process and that the apparent existence of a family of DNA binding sites might be biologically relevant. Our data should prove to be useful in rational (structure-based) mutation studies.     

Flanking bases influence the nature of DNA distortion by platinum 1,2-intrastrand (GG) cross-links

The differences in efficacy and molecular mechanisms of platinum anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) are thought to be partially due to the differences in the DNA conformations of the CP and OX adducts that form on adjacent guanines on DNA, which in turn influence the binding of damage-recognition proteins that control downstream effects of the adducts. Here we report a comprehensive comparison of the structural distortion of DNA caused by CP and OX adducts in the TGGT sequence context using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations. When compared to our previous studies in other sequence contexts, these structural studies help us understand the effect of the sequence context on the conformation of Pt-GG DNA adducts. We find that both the sequence context and the type of Pt-GG DNA adduct (CP vs. OX) play an important role in the conformation and the conformational dynamics of Pt-DNA adducts, possibly explaining their influence on the ability of many damage-recognition proteins to bind to Pt-DNA adducts.     

DNA repair and antioxidant defence in the repair-deficient human cells (Marfan's syndrome) after gamma-irradiation

DNA repair synthesis (RS) was investigated in lymphocytes of healty donors and repair-deficient cells (Marfan's syndrome), treated with inhibitor of superoxidedismutase (SOD)--TRIEN--after gamma-irradiation. Significant difference was revealed in cells of healthy donors and Marfan's syndrome: in cells of healthy donors TRIEN stimulated DNA RS whereas this effect didn't observed in Marfan's syndrome cells. So it is possible to suppose that SOD activity is different in normal and gamma-repair-deficient cells.     

Reversible regulation of P2Y(2) nucleotide receptor expression in the duct-ligated rat submandibular gland

Ligation of the main excretory duct of the rat submandibular gland(SMG) produces a pronounced atrophy that is reversed upon ligatureremoval. Based on previous studies by our group and others suggestingthat P2Y₂ nucleotide receptors are upregulated in response to tissue damage, we hypothesized that P2Y₂ receptoractivity and mRNA levels would increase after duct ligation and return to control levels after ligature removal. Our results support thishypothesis. Intracellular Ca²⁺ mobilization in response tothe P2Y₂ receptor agonist UTP in SMG cells was increasedsignificantly after ligation periods of 1.5 to 7 days, whereas nosignificant response was observed in the contralateral, nonligatedgland. P2Y₂ receptor mRNA, as measured bysemiquantitative RT-PCR, increased about 15-fold after 3 days ofligation. These increases reverted to control levels by 14 days afterligature removal. In situ hybridization revealed that the changes inP2Y₂ receptor mRNA abundance occurred mostly in acinarcells, which also were more adversely affected by ligation, includingan increase in the appearance of apoptotic bodies. These findingssupport the idea that P2Y₂ receptor upregulation may be animportant component of the response to injury in SMG and that recoveryof normal physiological function may signal a decreased requirement forP2Y₂ receptors.