Human Polynucleotide Phosphorylase (hPNPaseold-35 or PNPT1) is an evolutionarily conserved 3′→5′ exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPaseold-35 in cellular physiology, we knocked-down and overexpressed hPNPaseold-35 in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPaseold-35 resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPaseold-35 caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPaseold-35 knockdown and overexpression datasets allowed us to identify 77 potential “direct” and 61 potential “indirect” targets of hPNPaseold-35 which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPaseold-35 expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes.
Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specific-promoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.
Progression Elevated Gene-3; Sabutoclax; Apogossypol; BI-97C1; Gossypol; AP-1; PEA3; ETV4; E1AF; c-fos; c-jun; Cancer Terminator Virus
The present study aims at understanding the timing and nature of mitochondrial DNA (mtDNA) alterations in urothelial cell carcinoma (UCC) and their detection in urine sediments. The entire 16.5 kb mitochondrial genome was sequenced in matched normal lymphocytes, tumor and urine sediments from 31 UCC patients and compared with different clinical stages and histological grades. The mtDNA content index was examined in all the specimens. Sixty five percent (20/31) of the patients harbored at least 1 somatic mtDNA mutation. A total of 25 somatic mtDNA mutations were detected, which were more frequent in the respiratory complex coding regions (Complex-I, III, IV and V) of the mtDNA and significantly affected respiratory complex-III compared to the other complexes (P=0.021–0.039). Compared to stage Ta, mtDNA mutation was higher in stage T1 and significantly higher in stage T2 (P=0.01) patients. MtDNA mutation was also significantly higher (P=0.04) in stage T2 compared to stage T1 patients. Ninety percent (18/20) of the patients harboring mtDNA mutation in the tumor also had mutation in their urine sediments. Eighty percent (20/25) of the tumor-associated mtDNA mutations was detectable in the urine sediments. Compared to the normal lymphocytes, the mtDNA content increased significantly in the tumor (P=0.0013) and corresponding urine sediments (P=0.0025) in 19/25 (76%) patients analyzed. Our results indicate that mtDNA alterations occur frequently in progressive stages of UCC patients and are readily detectable in the urine sediments. MtDNA mutations appear to provide a promising tool for developing early detection and monitoring strategies for UCC patients.
Urothelial cell carcinoma; mitochondria; mtDNA alteration; urine detection
The purpose of this study was to identify key genetic pathways involved in non-small cell lung cancer (NSCLC) and understand their role in tumor progression. We performed a genome wide scanning using paired tumors and corresponding 16 mucosal biopsies from four follow-up lung cancer patients on Affymetrix 250K-NSpI array platform. We found that a single gene SH3GL2 located on human chromosome 9p22 was most frequently deleted in all the tumors and corresponding mucosal biopsies. We further validated the alteration pattern of SH3GL2 in a substantial number of primary NSCLC tumors at DNA and protein level. We also overexpressed wild-type SH3GL2 in three NSCLC cell lines to understand its role in NSCLC progression. Validation in 116 primary NSCLC tumors confirmed frequent loss of heterozygosity of SH3GL2 in overall 51 % (49/97) of the informative cases. We found significantly low (p=0.0015) SH3GL2 protein expression in 71 % (43/60) primary tumors. Forced over-expression of wild-type (wt) SH3GL2 in three NSCLC cell lines resulted in a marked reduction of active epidermal growth factor receptor (EGFR) expression and an increase in EGFR internalization and degradation. Significantly decreased in vitro (p=0.0015–0.030) and in vivo (p=0.016) cellular growth, invasion (p=0.029–0.049), and colony formation (p=0.023–0.039) were also evident in the wt-SH3GL2-transfected cells accompanied by markedly low expression of activated AKT(Ser473), STAT3 (Tyr705), and PI3K. Downregulation of SH3GL2 interactor USP9X and activated β-catenin was also evident in the SH3GL2-transfected cells. Our results indicate that SH3GL2 is frequently deleted in NSCLC and regulates cellular growth and invasion by modulating EGFR function.
Single nucleotide polymorphism array; Lung cancer; SH3GL2; Deletion
Mitochondrial DNA (mtDNA) mutations were reported in different cancers. However, the nature and role of mtDNA mutation in never-smoker lung cancer patients including patients with EGFR and KRAS gene mutation are unknown. In the present study, we sequenced entire mitochondrial genome (16.5 kb) in matched normal and tumors obtained from 30 never-smoker and 30 current-smoker lung cancer patients, and determined the mtDNA content. All the patients’ samples were sequenced for KRAS (exon 2) and EGFR (exon 19 and 21) gene mutation. The impact of forced overexpression of a respiratory complex-I gene mutation was evaluated in a lung cancer cell line. We observed significantly higher (P=0.006) mtDNA mutation in the never-smokers compared to the current-smoker lung cancer patients. MtDNA mutation was significantly higher (P=0.026) in the never-smoker Asian compared to the current-smoker Caucasian patients’ population. MtDNA mutation was significantly (P=0.007) associated with EGFR gene mutation in the never-smoker patients. We also observed a significant increase (P=0.037) in mtDNA content among the never-smoker lung cancer patients. The majority of the coding mtDNA mutations targeted respiratory complex-I and forced overexpression of one of these mutations resulted in increased in vitro proliferation, invasion and superoxide production in lung cancer cells. We observed a higher prevalence and new relationship between mtDNA alterations among never-smoker lung cancer patients and EGFR gene mutation. Moreover, a representative mutation produced strong growth effects after forced overexpression in lung cancer cells. Signature mtDNA mutations provide a basis to develop novel biomarkers and therapeutic strategies for never-smoker lung cancer patients.
Lung cancer; never-smokers; MtDNA mutation; Respiratory Complex-I; EGFR mutation
Despite significant improvements in therapeutic protocols, Head and Neck Squamous Cell Carcinoma (HNSCC) remains a major health problem worldwide. The 5-year post therapeutic survival rate is among the lowest of the major cancers with loco-regional relapse being the main cause of death. Moreover, in most instances, the quality of life of the afflicted patient is severely compromised. The poor prognosis for HNSCC is primarily due to disease detection at advanced stages. Accordingly, development of early detection and preventive strategies are essential. Recent advances in our understanding of the molecular biology and etiology of HNSCC should facilitate development of improved intervention and therapeutic approaches. The present review discusses the potential role of such factors for developing preventive and early diagnostic strategies for HNSCC management.
Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Analysis of the 2.5-kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many β-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a β-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes (PHFA) through the NF-κB signaling pathway. The NF-κB binding site at −272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.
Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers.
Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments.
We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.
We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients.
To determine the analytical sensitivity, we examined the tumor and the matched serum DNA for aberrant methylation of fifteen gene promoters from 10 patients with primary lung tumors by using Quantitative methylation specific PCR. We then tested this 15 gene set to identify the more useful DNA methylation changes in the serum of a limited number of lung cancer patients and controls. In an independent set, we tested the six most promising genes (APC, CDH1, MGMT, DCC, RASSF1A and AIM) for further elucidation of the diagnostic application of this panel of markers.
Promoter hypermethylation of at least one of the genes studied was detected in all 10 lung primary tumors. In majority of cases, aberrant methylation in serum DNA was accompanied by methylation in the matched tumor samples. In the independent set, using a single gene that had 100% specificity (DCC), 35.5% (95% CI 25%, 47%) of the 76 lung cancer patients were correctly identified. For patients without methylated DCC, addition of a logistic regression score that was based on the five remaining genes improved sensitivity from 35.5% to 75% (95% CI: 64%, 84%) but decreased the specificity from 100% to 73% (95% CI:54%, 88%).
This approach needs to be evaluated in a larger test set to determine the role of this gene set in early detection and surveillance of lung cancer.
DNA methylation/epigenetics; serum; lung cancer
Mitochondria control essential cellular activities including generation of ATP via oxidative phosphorylation. Mitochondrial DNA (mtDNA) mutations in the regulatory D-loop region and somatic mtDNA mutations are common in primary human cancers. The biological impact of a given mutation may vary, depending on the nature of the mutation and the proportion of mutant mtDNAs carried by the cell. Identification of mtDNA mutations in precancerous lesions supports their early contribution to cell transformation and cancer progression. Introduction of mtDNA mutations in transformed cells has been associated with increased ROS production and tumor growth. Studies reveal that increased and altered mtDNA plays a role in the development of cancer but further work is required to establish the functional significance of specific mitochondrial mutations in cancer and disease progression. This review offers some insight into the extent of mtDNA mutations, their functional consequences in tumorigenesis, mitochondrial therapeutics, and future clinical application.
Prognosis for patients with non-small cell lung cancer (NSCLC) is poor. The potential value of modulating EGFR for treatment is reflected by the recent approval of specific drugs that inhibit its activity. Mutations in EGFR were reported in lung cancer and generated interest, once they enable the identification of lung cancers likely to respond to various targeted small molecules.
We tested 3 key genetic and epigenetic alterations (EGFR, RASSF1A, and BRAF) of this pathway on a series of primary NSCLC [Total 111; adenocarcinoma 49, squamous cell carcinoma (SCC) 48 and others 14]. The mutational status of KRAS (and p53) was known for these samples. The purpose of this study was to define the pattern of erbB pathway alterations in NSCLC and to test for associations with clinical parameters.
Five EGFR mutations were identified: 3 in adenocarcinoma (6 %), 1 in SCC (2%) and 1 in adenocarcinoma with bronchoalveolar component tumor (7%). EGFR mutations included 3 in-frame deletions in exon 19 and 2 point mutations in exon 21. Promoter methylation of RASSF1A was detected in 25 of 45 adenocarcinomas and 18 of 46 SCC. Mutations of EGFR, BRAF and KRAS in adenocarcinoma were mutually exclusive and inversely correlated with RASSF1A methylation (p = −0.394; p=0.007). Overall, genetic and/or epigenetic alterations of erbB pathway genes were detected in 80% (39/49) of adenocarcinomas.
Nearly half of primary adenocarcinoma harbor molecular alterations of the erbB pathway. Careful characterization of these alterations and response to anti-EGFR therapies is warranted to determine better and accurate determinants of clinical response.
EGFR mutation; DNA methylation/epigenetics; RASSF1A
Mitochondrial DNA (mtDNA) mutations were reported in primary head and neck squamous cell carcinoma (HNSCC) patients. However, very little information is available on the mtDNA mutation pattern in the histologically negative surgical margins and tumors of HNSCC patients who experienced tumor recurrence. The present study aimed at understanding the nature and timing of mtDNA mutation in histologically negative margins, and tumors in HNSCC patients who developed local recurrence during the follow ups. The entire 16.5-kb mitochondrial genome was sequenced in matched normal lymphocytes, histologically normal margins, and tumors of 50 recurrent HNSCC patients. The mtDNA mutations were then compared with clinical parameters. Forty-eight percent (24 of 50) patients harbored at least one somatic mtDNA mutation in the tumor, and a total of 37 somatic mtDNA mutations were detected. The mtDNA mutations were mostly heteroplasmic in nature and nucleotide transitions (A↔G; T↔C). Forty-six percent of the mutations (17 of 37) were detected in the tumors and were also detectable in the corresponding histologically normal margin of the patients. The mtDNA mutations involved both coding and noncoding regions of the mtDNA. Majority (9 of 17, 53%) of the noncoding mutations involved tRNAs. Seventy-five percent (15 of 20) of the coding mtDNA mutations were nonsynonymous in nature and mainly affected cytochrome c oxidase (Complex IV), frequently altered in different human mitochondrial diseases including cancer. Analysis of mtDNA mutation could be an invaluable tool for molecular assessment of histologically negative margins and as well for monitoring HNSCC patients with locoregional recurrences.
The glycosylphosphatidylinositol transamidase complex (GPIT) consists of five subunits: PIG-U, PIG-T, GPAA1, PIG-S and GPI8, and is important in attaching GPI anchors to target proteins. On the basis of our previous reports incriminating PIG-U as an oncogene in bladder cancer and PIG-T and GPAA1 as oncogenes in breast cancer, we evaluated the expression pattern of the GPIT subunits in 19 different human cancers at both mRNA and protein levels. In general, our results demonstrate a more frequent expression of GPIT subunits in cancers than in normal. Among the 19 anatomic sites compared; breast, ovary and uterus showed consistent evidence of overexpression of specific GPIT subunits. There was also overexpression of PIG-U and GPI8 in lymphoma. In addition, non-small cell lung carcinoma showed significant overexpression of the GPIT subunits as compared to small cell lung carcinoma and normal lung tissue. Also, deregulation of specific GPIT subunits was seen in various other cancers. Forced overexpression of two GPIT subunits; PIG-S and GPI8 alone or in combination induced increased proliferation and invasion of breast cancer cells. Collectively, our study defines a trend involving the deregulated expression and the functional contribution of the GPIT subunits in various cancers with potential implications in diagnosis, prognosis and therapeutic intervention.
GPIT complex; gene expression; cancer
Mitochondria encoded Cytochrome B (CYTB) gene mutations were reported in tumors of different anatomic origin but the functional significance of these mutations are not well studied. Earlier, we found a 7-amino acid deletion mutation in the CYTB gene in a primary bladder cancer patient. In the present study, we overexpressed this 7-amino acid deletion mutation of CYTB gene in SV-40 transformed human uroepithelial HUC-1 cells. The nuclear transcribed mitochondrial CYTB (mtCYTB) was targeted into the mitochondria and generated increased copies of mitochondria and mitochondrial COX-I protein in the transfected HUC-1 cells. The pro-apoptotic protein Bax largely remained confined to the cytoplasm of the mtCYTB transfected HUC-1 cells without release of Cytochrome C. The downstream apoptotic proteins PARP also remained uncleaved along with increased Lamin B1 in the mtCYTB transfected cells. Our results demonstrate that forced overexpression of mtCYTB in transformed human uroepithelial HUC-1 cells triggered mitochondrial proliferation and induction of an anti-apoptotic signaling cascade favoring sustained cellular growth. Coding mitochondrial DNA mutations appear to have significant functional contribution in tumor progression.
Mitochondria; Cytochrome B mutation; apoptosis; mtDNA content
Bacteria have the ability to adapt to different growth conditions and to survive in various environments. They have also the capacity to enter into dormant states and some bacteria form spores when exposed to stresses such as starvation and oxygen deprivation. Sporulation has been demonstrated in a number of different bacteria but Mycobacterium spp. have been considered to be non-sporulating bacteria. We recently provided evidence that Mycobacterium marinum and likely also Mycobacterium bovis bacillus Calmette–Guérin can form spores. Mycobacterial spores were detected in old cultures and our findings suggest that sporulation might be an adaptation of lifestyle for mycobacteria under stress. Here we will discuss our current understanding of growth, cell division, and sporulation in mycobacteria.
Mycobacteria; Bacterial cell division; Septum formation; Sporulation
The aims of our study were to elucidate the role of methylation of a large panel of genes during multistage pathogenesis of bladder cancer and to correlate our findings with patient age and other clinicopathologic features.
We studied the methylation status of 21 genes by quantitative methylation-specific PCR in an evaluation set of 25 tumor and 5 normal samples. Based on methylation frequency in tumors and normals in gene evaluation set, we selected 7 candidate genes and tested an independent set of 93 tumors and 26 normals. The presence or absence of methylation was evaluated for an association with cancer using cross-tabulations and χ2 or Fisher’s exact tests as appropriate. All statistical tests were two-sided.
Most primary tumors (89 of 93, 96%) had methylation of one or more genes of independent set; 53 (57%) CCNA1, 29 (31%) MINT1, 36 (39%) CRBP, 53 (57%) CCND2, 66 (71%) PGP9.5, 60 (65%) CALCA, and 78 (84%) AIM1. Normal uroepithelium samples from 26 controls revealed no methylation of the CCNA1 and MINT1 genes, whereas methylation of CRBP, CCND2, PGP9.5, and CALCA was detected at low levels. All the 7 genes in independent set were tightly correlated with each other and 3 of these genes showed increased methylation frequencies in bladder cancer with increasing age. PGP9.5 and AIM1 methylation correlated with primary tumor invasion.
Our results indicate that the methylation profile of novel genes in bladder cancers correlates with clinicopathologic features of poor prognosis and is an age-related phenomenon.
Mitochondrial DNA (mtDNA) mutations are reported in different tumors. However, there is no information on the temporal development of the mtDNA mutations/content alteration and their extent in normal and abnormal mucosa continuously exposed to tobacco smoke in lung cancer patients.
We examined the pattern of mtDNA alteration (mtDNA mutation and content index) in 25 airway mucosal biopsies, corresponding tumors and normal lymph nodes obtained from three patients with primary lung cancers. In addition, we examined the pattern of mtDNA mutation in corresponding tumors and normal lymph nodes obtained from eight other patients with primary lung cancers. The entire 16.5 kb mitochondrial genome was sequenced on Affymetrix Mitochip v2.0 sequencing platform in every sample. To examine mtDNA content index, we performed real-time PCR analysis.
The airway mucosal biopsies obtained from three lung cancer patients were histopathologically negative but exhibited multiple clonal mtDNA mutations detectable in the corresponding tumors. One of the patients was operated twice for the removal of tumor from the right upper and left lower lobe respectively within a span of two years. Both of these tumors exhibited twenty identical mtDNA mutations. MtDNA content increased significantly (P<0.001) in the lung cancer and all the histologically negative mucosal biopsies except one compared to the control lymph node.
Our results document the extent of massive clonal patches that develop in lifetime smokers and ultimately give rise to clinically significant cancers. These observations shed light on the extent of disease in the airway of smokers traceable through mtDNA mutation. MtDNA mutation could be a reliable tool for molecular assessment of respiratory epithelium exposed to continuous smoke as well as disease detection and monitoring. Functional analysis of the pathogenic mtDNA mutations may be useful to understand their role in lung tumorigenesis.
With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3′-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)6-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)6-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.
Bacterial chromosomes are organised as two replichores of opposite polarity that coincide with the replication arms from the ori to the ter region. Here, we investigated the effects of asymmetry in replichore organisation in Escherichia coli. We show that large chromosome inversions from the terminal junction of the replichores disturb the ongoing post-replicative events, resulting in inhibition of both cell division and cell elongation. This is accompanied by alterations of the segregation pattern of loci located at the inversion endpoints, particularly of the new replichore junction. None of these defects is suppressed by restoration of termination of replication opposite oriC, indicating that they are more likely due to the asymmetry of replichore polarity than to asymmetric replication. Strikingly, DNA translocation by FtsK, which processes the terminal junction of the replichores during cell division, becomes essential in inversion-carrying strains. Inactivation of the FtsK translocation activity leads to aberrant cell morphology, strongly suggesting that it controls membrane synthesis at the division septum. Our results reveal that FtsK mediates a reciprocal control between processing of the replichore polarity junction and cell division.
In most bacteria, chromosomes consist of a single replication unit. Replication initiates at a single ori and terminates in the diametrically opposite zone, ter, defining two replication arms. These are also called replichores to account for base composition and sequence motif polarity biases from ori to ter. This organisation as symmetrical replichores is conserved in bacteria. Here, we have investigated the effect of replichore asymmetry in E. coli. We show that it blocks the cell cycle at a step posterior to chromosome replication. This phenotype is not corrected by restoration of the termination of replication opposite ori, strongly suggesting that it is due to the asymmetry of replichore polarity. FtsK, a DNA-translocase associated with the division septum that processes the terminal junction of replichore polarity, is essential for growth and for the controlled blockage of cell growth in cells with asymmetric replichores. These results reveal a reciprocal control between the processing of the terminal junction of the replichores by FtsK and the progression of the cell cycle.
The recombinational rescue of chromosome replication was investigated in Escherichia coli strains with the unidirectional origin oriR1, from the plasmid R1, integrated within oriC in clockwise (intR1CW) or counterclockwise (intR1CC) orientations. Only the intR1CC strain, with replication forks arrested at the terminus, required RecA for survival. Unlike the strains with RecA-dependent replication known so far, the intR1CC strain did not require RecBCD, RecF, RecG, RecJ, RuvAB, or SOS activation for viability. The overall levels of degradation of replicating chromosomes caused by inactivation of RecA were similar in oriC and intR1CC strains. In the intR1CC strain, RecA was also needed to maintain the integrity of the chromosome when the unidirectional replication forks were blocked at the terminus. This was consistent with suppression of the RecA dependence of the intR1CC strain by inactivating Tus, the protein needed to block replication forks at Ter sites. Thus, RecA is essential during asymmetric chromosome replication for the stable maintenance of the forks arrested at the terminus and for their eventual passage across the termination barrier(s) independently of the SOS and some of the major recombination pathways.
Upon treatment of HeLa nucleoids with EcoR1 restriction enzyme, a minute fraction of the DNA remains attached to the nuclear matrix. This matrix-DNA complex appears to be composed of a unique set of proteins. Electron microscopic analysis of the DNA fragments present shows an enrichment for HeLa DNA replicating forks.