Mph1 requires mismatch repair-independent and -dependent functions of MutSα to regulate crossover formation during homologous recombination repair

In budding yeast the DNA helicase Mph1 prevents genome rearrangements during ectopic homologous recombination (HR) by suppressing the formation of crossovers (COs). Here we show that during ectopic HR repair, the anti-CO function of Mph1 is intricately associated with the mismatch repair (MMR) factor, MutSα. In particular, during HR repair using a completely homologous substrate, we reveal an MMR-independent function of MutSα in generating COs that is specifically antagonized by Mph1, but not Sgs1. In contrast, both Mph1 and MutSα are required to efficiently suppress COs in the presence of a homeologous substrate. Mph1 acts redundantly with Sgs1 in this respect since mph1Δ sgs1Δ double mutant cells pheno-copy MutSα mutants and completely fail to discriminate homologous and homeologous sequences during HR repair. However, this defect of mph1Δ sgs1Δ cells is not due to an inability to carry out MMR but rather is accompanied by elevated levels of gene conversion (GC) and bi-directional GC tracts specifically in non-crossover products. Models describing how Mph1, MutSα and Sgs1 act in concert to suppress genome rearrangements during ectopic HR repair are discussed.     

Repression of integrin-linked kinase by antidiabetes drugs through cross-talk of PPARγ- and AMPKα-dependent signaling: Role of AP-2α and Sp1

Nasopharyngeal carcinoma (NPC) is one of the most common cancers of the head and neck, particularly in Southern China and Southeast Asia with high treatment failure due to the development of local recurrence and distant metastasis. The molecular mechanisms related to the progression of NPC have not been fully understood. In this study, we showed that antidiabetes drugs rosiglitazone and metformin inhibit NPC cell growth through reducing the expression of integrin-linked kinase (ILK). Blockade of PPARγ and AMPKα overcame the effects of rosiglitazone and metformin on ILK protein. Importantly, overexpression of ILK abrogated the effect of rosiglitazone and metformin on NPC cell growth. Furthermore, these agents reduced ILK promoter activity, which was not observed in AP-2α, but not Sp1 site mutation in ILK gene promoter. In addition, silencing of AP-2α or overexpression of Sp1 reversed the effect of these agents on ILK protein expression and cell growth. Chromatin immunoprecipitation (ChIP) assay showed that rosiglitazone induced AP-2α, while metformin reduced Sp1 protein binding to the DNA sequences in the ILK gene promoter. Intriguingly, overexpression of Sp1 abolished the effect of rosiglitazone on AP-2α protein expression. Collectively, we show that rosiglitazone and metformin inhibit ILK gene expression through PPARγ- and AMPKα-dependent signaling pathways that are involved in the regulation of AP-2α and Sp1 protein expressions. The effect of combination of rosiglitazone and metformin demonstrates greater extent than single agent alone. The cross-talk of PPARγ and AMPKα signaling enhances the synergistic effects of rosiglitazone and metformin. This study unveils novel mechanisms by which oral antidiabetes drugs inhibit the growth of human NPC cells.     

Co-localization of chicken DNA topoisomerase IIα, but not β, with sites of DNA replication and possible involvement of a C-terminal region of α through its binding to PCNA

Clones for DNA topoisomerase IIalpha and beta (topo-IIalpha and beta) were isolated from a cDNA expression library of chicken MSB-1 cells by immunoscreening. The deduced sequences of chicken topo-IIalpha and beta were about 80% identical for the N-terminal ATPase domain and the central core domain but only 37% for the C-terminal domain. Polyclonal antibodies were raised against C-terminal polypeptides specific to topo-IIalpha and beta. Indirect immunofluorescence with these antibodies to chicken embryonic fibroblasts demonstrated that topo-IIalpha was distributed in discrete intranuclear spots, which coincided with sites of DNA replication as indicated by incorporation of 5-bromo-2'-deoxyuridine, whereas topo-IIbeta was distributed rather uniformly within a nucleus. Examination of intranuclear distribution patterns of chimeric constructs between topo-IIalpha and beta suggested that a sequence region (residues 1280-1294) in the C-terminal domain of topo-IIalpha was effective in co-localization with sites of DNA replication. This region consists of a QTxhxF motif (x, any residue; h, hydrophobic residue) followed by a KR-rich sequence, which resembles those found in several proteins known to associate with proliferating cell nuclear antigen (PCNA) or targeted to the replication factory. An in vitro pull-down assay with glutathione-S-transferase-PCNA and (His)6-tagged truncated forms of topo-IIalpha demonstrated that polypeptides containing the above region (residues 1158-1553 or 1158-1294) bound to PCNA in vitro.     

Simulations of DNA topoisomerase 1B bound to supercoiled DNA reveal changes in the flexibility pattern of the enzyme and a secondary protein–DNA binding site

Human topoisomerase 1B has been simulated covalently bound to a negatively supercoiled DNA minicircle, and its behavior compared to the enzyme bound to a simple linear DNA duplex. The presence of the more realistic supercoiled substrate facilitates the formation of larger number of protein–DNA interactions when compared to a simple linear duplex fragment. The number of protein–DNA hydrogen bonds doubles in proximity to the active site, affecting all of the residues in the catalytic pentad. The clamp over the DNA, characterized by the salt bridge between Lys369 and Glu497, undergoes reduced fluctuations when bound to the supercoiled minicircle. The linker domain of the enzyme, which is implicated in the controlled relaxation of superhelical stress, also displays an increased number of contacts with the minicircle compared to linear DNA. Finally, the more complex topology of the supercoiled DNA minicircle gives rise to a secondary DNA binding site involving four residues located on subdomain III. The simulation trajectories reveal significant changes in the interactions between the enzyme and the DNA for the more complex DNA topology, which are consistent with the experimental observation that the protein has a preference for binding to supercoiled DNA.     

Estrogen improved metabolic syndrome through down-regulation of VEGF and HIF-1α to inhibit hypoxia of periaortic and intra-abdominal fat in ovariectomized female rats

Metabolic syndrome (MBS), a cluster of metabolic abnormalities and visceral fat accumulation, increases cardiovascular risks in postmenopausal women. In addition to visceral fat, perivascular adipose tissue has been recently found to play an important role in vascular pathophysiology. Hence, the present study investigates the effects of estrogen on both intra-abdominal fat (visceral fat) and periaortic fat (perivascular fat) accumulation as well as hypoxia in ovariectomized female rats. Female rats were divided into sham operation, ovariectomy and ovariectomy with 17β-estradiol supplementation groups. Twelve weeks later, we found that estrogen improved MBS via reducing body weight gain, the weight of periaortic and intra-abdominal fat, hepatic triglyceride, and total serum cholesterol levels. Estrogen also increased insulin sensitivity through restoring glucose and serum leptin levels. For periaortic fat, western blot showed estrogen inhibited hypoxia by reducing the levels of VEGF and HIF-1α, which is consistent with the results from immunohistochemical staining. The correlation analysis indicated that perivascular fat had a positive correlation with body weight, intra-abdominal fat or serum total cholesterol, but a negative correlation with insulin sensitivity index. For intra-abdominal fat, real-time fluorescent RT-PCR showed estrogen improved fat dysfunction via reducing the levels of relative leptin, MCP-1 but increasing adiponectin mRNA. Estrogen reduced the levels of VEGF and HIF-1α to inhibit hypoxia but restored the levels of PPARγ and Srebp-1c, which are important for lipid capacity function of intra-abdominal fat. These results demonstrated estrogen improved MBS through down-regulating VEGF and HIF-1α to inhibit hypoxia of periaortic and intra-abdominal fat in ovariectomized female rats.     

Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK,and MEK1/2-dependent upregulation of TNF-α and IL-6

Despite their differential cell tropisms, HIV-1 and HCV dramatically influence disease progression in coinfected patients. Macrophages are important target cells of HIV-1. We hypothesized that secreted HCV core protein might modulate HIV-1 replication. We demonstrate that HCV core significantly enhances HIV-1 replication in human macrophages by upregulating TNF-α and IL-6 via TLR2-, JNK-, and MEK1/2-dependent pathways. Furthermore, we show that TNF-α and IL-6 secreted from HCV core-treated macrophages reactivates monocytic U1 cells latently infected with HIV-1. Our studies reveal a previously unrecognized role of HCV core by enhancing HIV-1 infection in macrophages.     

AP endonuclease 1 prevents the extension of a T/G mismatch by DNA polymerase β to prevent mutations in CpGs during base excision repair

Dynamics of DNA methylation and demethylation at CpG clusters are involved in gene regulation. CpG clusters have been identified as hot spots of mutagenesis because of their susceptibility to oxidative DNA damage. Damaged Cs and Gs at CpGs can disrupt a normal DNA methylation pattern through modulation of DNA methylation and demethylation, leading to mutations and deregulation of gene expression. DNA base excision repair (BER) plays a dual role of repairing oxidative DNA damage and mediating an active DNA demethylation pathway on CpG clusters through removal of a T/G mismatch resulting from deamination of a 5mC adjacent to a guanine that can be simultaneously damaged by oxidative stress. However, it remains unknown how BER processes clustered lesions in CpGs and what are the consequences from the repair of these lesions. In this study, we examined BER of an abasic lesion next to a DNA demethylation intermediate, the T/G mismatch in a CpG dinucleotide, and its effect on the integrity of CpGs. Surprisingly, we found that the abasic lesion completely abolished the activity of thymine DNA glycosylase (TDG) for removing the mismatched T. However, we found that APE1 could still efficiently incise the abasic lesion leaving a 3-terminus mismatched T, which was subsequently extended by pol β. This in turn resulted in a C to T transition mutation. Interestingly, we also found that APE1 3′–5′ exonuclease activity efficiently removed the mismatched T, thereby preventing pol β extension of the mismatched nucleotide and the resulting mutation. Our results demonstrate a crucial role of APE1 3′–5′ exonuclease activity in combating mutations in CpG clusters caused by an intermediate of DNA demethylation during BER.     

Mimosine arrests the cell cycle prior to the onset of DNA replication by preventing the binding of human Ctf4/And-1 to chromatin via Hif-1α activation in HeLa cells

Though the G₁ checkpoint in mammalian cells has been known for decades, the molecular targets that prevent S-phase entry remain unknown. Mimosine is a rare plant amino acid that arrests the cell cycle in the G₁ phase before entry into S phase. Here, we show that mimosine interrupts the binding of Ctf4 to chromatin, which is essential for the initiation of DNA replication in HeLa cells, and this effect is mediated by the Hif-1α-dependent increase in the level of p27. Depletion of Hif-1α results in an increased binding of Ctf4 to chromatin and the entry of cells into S phase even in the presence of mimosine. These results suggest that the binding of Ctf4 to chromatin is the target of the Hif-1α-dependent checkpoint pathway for cell cycle arrest in G₁ phase. Although we observed Hif-1α-dependent arrest in mimosine-treated cells, it is possible that Ctf4 may act as a common target for G₁ arrest in various other checkpoint pathways.     

CCAAT/enhancer binding protein β expression is increased in the brain during HIV-1-infection and contributes to regulation of astrocyte tissue inhibitor of metalloproteinase-1

ABSTRACT: Selective inhibition of GluN2B-containing NMDA receptor (GluN2BR) in spinal dorsal horn effectively alleviates inflammatory pain, suggesting the up-regulation of GluN2BR function involved in central sensitization. Previous studies have demonstrated that the increase in GluN2BR synaptic expression serves as a key step to enhance GluN2BR function after intradermal injection of Complete Freund's Adjuvant (CFA). Here, we showed that cAMP-dependent protein kinase (PKA) played an important role in redistributing GluN2BR at synapses, because inhibition of PKA activity impaired GluN2BR accumulation at post-synaptic density (PSD)-enriched fraction in CFA-injected mice, and direct stimulation of PKA in na?ve mice mimicked the effect of CFA by recruiting GluN2BR at PSD fraction to evoke pain sensitization. Analysis of PKA-initiated signalings unraveled that PKA was able to activate Src-family protein tyrosine kinases member Fyn, possibly by disrupting Fyn association with its inhibitory partner striatal-enriched protein tyrosine phosphatase 61. The active Fyn then promoted GluN2B phosphorylation at Tyr1472, a molecular event known to prevent GluN2BR endocytosis. As a result, pharmacological or genetic manipulation of Fyn activity greatly depressed GluN2BR accumulation at PSD-enriched fraction and ameliorated mechanical allodynia induced by PKA. Our data thus elucidated a critical role of PKA/Fyn/GluN2B signaling in triggering GluN2BR hyperfunction and pain hypersensitivity.     

Sequence of a cDNA coding for a rat class II Aα chain: Extensive DNA and protein sequence identity to H-2 Aα and HLA-DC1 α chains

The rat major histocompatibility complex (RT1-B region) codes for two sets of class II molecules (la antigens) referred to as A and E. Each class II molecule is composed of two glycoprotein chains called the A and A or E and E . Two cDNA clones encoding rat A chains were identified from cDNA derived from rat spleen mRNA using a combination of mRNA selection and colony hybridization techniques. The complete nucleotide sequence of the cDNA insert of one of these cDNA clones, pRIa.2, was determined. This sequence codes for the carboxy-terminal 129 amino acids of the rat A chain and 293 nucleotides of 3 untranslated sequence. The rat A chain was shown to be highly homologous in terms of both protein and DNA sequence identities to HLA-DC and H-2 A chains. Comparison between the coding regions of the cDNA insert of pRla.2 and the corresponding region of a cDNA insert encoding an HLA-DC1 chain showed sequence identities of 85% and 81% at the protein and DNA levels, respectively. Comparison between pRIa.2 and cDNA encoding an H-2 A chain sequence showed identities of 91% for both protein and DNA. Results are discussed which strongly suggest that the class II A and E primordial genes arose by gene duplication prior to the evolutionary divergence of the mammals.