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Visualization of topoisomerases in live Bacillus subtilis cells showed that Topo I, Topo IV, and DNA gyrase differentially localize on the nucleoids but are absent at cytosolic spaces surrounding the nucleoids, suggesting that these topoisomerases interact with many regions of the chromosome. While both subunits of Topo IV were uniformly distributed throughout the nucleoids, Topo I and gyrase formed discrete accumulations, or foci, on the nucleoids in a large fraction of the cells, which showed highly dynamic movements. Three-dimensional time lapse microscopy showed that gyrase foci accumulate and dissipate within a 1-min time scale, revealing dynamic assembly and disassembly of subcelluar topoisomerase centers. Gyrase centers frequently colocalized with the central DNA replication machinery, suggesting a major role for gyrase at the replication fork, while Topo I foci were frequently close to or colocalized with the structural maintenance of chromosomes (SMC) chromosome segregation complex. The findings suggest that different areas of supercoiling exist on the B. subtilis nucleoids, which are highly dynamic, with a high degree of positive supercoiling attracting gyrase to the replication machinery and areas of negative supercoiling at the bipolar SMC condensation centers recruiting Topo I.

The role of topoisomerase IV (Topo IV) and of the structural maintenance of chromosomes (SMC) complex in chromosome compaction and in global protein synthesis was investigated. Lowering of the levels of Topo IV led to chromosome decondensation, while overproduction induced chromosome hyper-compaction, showing that Topo IV has an influence on the compaction of the whole chromosome, in a manner similar to that of the SMC protein, though different in mechanism. Increased synthesis of Topo IV in smc-deleted cells partially rescued the growth and condensation defect of the deletion, but not the segregation defect, revealing that the two systems interact at a genetic level. Two-dimensional gel investigations showed that global protein synthesis is highly aberrant in smc-deleted cells, and, to a different extent, also in cells lacking ScpA or ScpB, which form the SMC complex together with SMC protein. Overproduction of Topo IV partially rescued the defect in protein synthesis in smc mutant cells, indicating that Topo IV can restore the loss of negative super-coiling caused by the absence of SMC protein, but does not fully rescue the segregation defect. The data also show that the SMC protein has a dual function, in chromosome supercoiling and in active segregation.

Abstract Background DprA is a widely conserved bacterial protein and has been shown to confer an important function during transformation in competent cells, possibly through protection of incoming DNA. B. subtilis DprA (called Smf) and has been shown to play an important role during transformation with chromosomal DNA, but its mode of action is unknown. Results We show that B. subtilis DprA/Smf is more important for transformation with plasmid DNA than with chromosomal DNA. A functional Smf-YFP fusion localized as discrete foci to the cell pole in a subset of cells grown to competence, dependent on the ComK master transcription factor. Smf-YFP foci colocalized with ComGA-CFP. However, a considerable number of cells having high ComK activity contained Smf dispersed throughout the cytosol and lacked a polar Smf assembly. The absence of polar Smf-YFP foci in these cells strongly correlated with the absence of ComGA-CFP foci, and comGA mutant cells mostly lacked polar Smf-YFP foci. Smf formed polar assemblies in the absence of RecA, and RecA formed dynamic threads after addition of DNA in a smf deletion strain. Upon addition of DNA, Smf-YFP foci relocalized from the poles to the cell centre, dependent on the presence of RecA protein. Conclusion Our data show that Smf is recruited to the polar competence machinery, and that polar Smf assembly requires a functional DNA uptake complex. High ComK levels drive expression of Smf in 20% of all cells grown to competence, but not all competent cells contain a polar DNA uptake machinery, showing that ComK activity is necessary but not sufficient to achieve assembly of the uptake machinery in all cells. Smf and RecA localize independently of each other, in agreement with our finding that Smf is much more important for plasmid transformation than RecA, but RecA influences the dynamic localization pattern of Smf. Our data show that DprA/Smf acts downstream of the DNA uptake machinery, and support the idea that Smf protects incoming ssDNA, possibly in conjunction with RecA.

Base excision repair, which is initiated by the DNA N -glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. Several base excision repair genes are deregulated in cancer and affect cellular outcomes to chemotherapy and carcinogenesis. Endonuclease VIII-like 3 (NEIL3) is a DNA glycosylase protein that is involved in oxidative and interstrand crosslink DNA damage repair. Our previous work has showed that NEIL3 is required to maintain replication fork integrity. It is unknown whether NEIL3 overexpression could contribute to cancer phenotypes, and its prognostic value and use as potential drug target remain unexplored. Our analysis of cancer genomics data sets reveals that NEIL3 frequently undergoes overexpression in several cancers. Furthermore, patients who exhibited NEIL3 overexpression with pancreatic adenocarcinoma, lung adenocarcinoma, lower grade glioma, kidney renal clear cell carcinoma, and kidney papillary cell carcinoma had worse overall survival. Importantly, NEIL3 overexpressed tumors accumulate mutation and chromosomal variations. Furthermore, NEIL3 overexpressed tumors exhibit simultaneous overexpression of homologous recombination genes (BRCA1/2) and mismatch repair genes (MSH2/MSH6). However, NEIL3 overexpression is negatively correlated with tumor overexpressing nucleotide excision repair genes (XPA, XPC, ERCC1 / 2). Our results suggest that NEIL3 might be a potential prognosis marker for high-risk patients, and/or an attractive therapeutic target for selected cancers. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is an idiopathic multisystem disease affecting 5–7% of pregnant women. Placental oxidative stress is a characteristic feature of PE and occurs when the production of reactive oxygen species (ROS) within the placenta overwhelms the intrinsic anti-oxidant defenses. We hypothesize that excessive oxidative DNA damage at the fetal-maternal interface coupled with a defective DNA damage/repair response is causally related to PE. Here we demonstrate that γH2AX (a sensitive marker of DNA damage) is expressed in the maternal decidua but not trophoblast of normal placentas, and that expression is significantly higher in PE placental tissues in vivo. Using primary in vitro cultures of maternal decidual stromal cells (DSCs) and fetal cytotrophoblast cells (CTs), we show an increase in γH2AX foci in DSCs cultured with vs without H2O2 (70.6% vs 11.6%; P<0.0001) or under hypoxia-reperfusion vs normoxia (20- vs 3-fold; P = 0.01); no foci were seen in CTs. We further demonstrate that Base Excision Repair (BER) intermediates are significantly increased in DSCs (not CTs) under these same conditions. Our data show that DNA damage is significantly more common in PE placentas, and that this DNA damage is localized to the maternal and not fetal side of the placenta. CTs may be selectively resistant to DNA damage in an effort to protect the fetus. [ABSTRACT FROM AUTHOR]

Objective: Progesterone supplementation prevents preterm birth (PTB) in some high-risk women, but its mechanism of action is not known. One third of PTB is associated with preterm premature rupture of membranes (PPROM).We hypothesize that progesterone may block proinflammatory cytokine-induced apoptosis of fetal membrane, thereby preventing PPROM and PTB. Methods: Fetal membranes were collected at elective repeat cesarean at term (no labor, no infection [n = 12]), washed, and cultured with/ without progesterone (125-500 ng/mL), 17a-hydroxyprogesterone caproate (125-500 ng/mL [17P]), or medroxyprogesterone acetate (10-7-10-6 mol/L [MPA]) for 24 hours. Membranes were then treated with/without lipopolysaccharide ([LPS] 100 ng/mL) or tumor necrosis factor alpha ([TNF-α] 50 ng/ mL) for 24 to 72 hours, harvested, and homogenized. Apoptosis was determined by evaluating caspase-3, -8, and -9 activities. Caspase activity in relative light units was measured on a luminometer and corrected for total protein. Results: Both TNF-α and LPS significantly increased caspase-3, -8, and -9 activity in term fetal membranes in a time-dependent fashion. Progesterone, 17P, and MPA significantly reduced TNF-α, but not LPS, induced caspase-3 activity. Interestingly, progesterone and MPA, but not 17P, also inhibited basal caspase-3 activity. Conclusion: Progesterone inhibits basal and TNF-α-induced apoptosis in term fetal membranes. This novel observation may explain in part the mechanism by which progesterone supplementation prevents PPROM and PTB in some high-risk women. The effect of progesterone on the basal levels of apoptosis suggests that this mechanism may also be important for normal labor at term. [ABSTRACT FROM AUTHOR]

Background: DprA is a widely conserved bacterial protein and has been shown to confer an important function during transformation in competent cells, possibly through protection of incoming DNA. B. subtilis DprA (called Smf) and has been shown to play an important role during transformation with chromosomal DNA, but its mode of action is unknown. Results: We show that B. subtilis DprA/Smf is more important for transformation with plasmid DNA than with chromosomal DNA. A functional Smf-YFP fusion localized as discrete foci to the cell pole in a subset of cells grown to competence, dependent on the ComK master transcription factor. Smf-YFP foci colocalized with ComGA-CFP. However, a considerable number of cells having high ComK activity contained Smf dispersed throughout the cytosol and lacked a polar Smf assembly. The absence of polar Smf-YFP foci in these cells strongly correlated with the absence of ComGA-CFP foci, and comGA mutant cells mostly lacked polar Smf-YFP foci. Smf formed polar assemblies in the absence of RecA, and RecA formed dynamic threads after addition of DNA in a smf deletion strain. Upon addition of DNA, Smf-YFP foci relocalized from the poles to the cell centre, dependent on the presence of RecA protein. Conclusion: Our data show that Smf is recruited to the polar competence machinery, and that polar Smf assembly requires a functional DNA uptake complex. High ComK levels drive expression of Smf in 20% of all cells grown to competence, but not all competent cells contain a polar DNA uptake machinery, showing that ComK activity is necessary but not sufficient to achieve assembly of the uptake machinery in all cells. Smf and RecA localize independently of each other, in agreement with our finding that Smf is much more important for plasmid transformation than RecA, but RecA influences the dynamic localization pattern of Smf. Our data show that DprA/Smf acts downstream of the DNA uptake machinery, and support the idea that Smf protects incoming ssDNA, possibly in conjunction with RecA. [ABSTRACT FROM AUTHOR]

Base excision repair, which is initiated by the DNA N -glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. Several base excision repair genes are deregulated in cancer and affect cellular outcomes to chemotherapy and carcinogenesis. Endonuclease VIII-like 3 (NEIL3) is a DNA glycosylase protein that is involved in oxidative and interstrand crosslink DNA damage repair. Our previous work has showed that NEIL3 is required to maintain replication fork integrity. It is unknown whether NEIL3 overexpression could contribute to cancer phenotypes, and its prognostic value and use as potential drug target remain unexplored. Our analysis of cancer genomics data sets reveals that NEIL3 frequently undergoes overexpression in several cancers. Furthermore, patients who exhibited NEIL3 overexpression with pancreatic adenocarcinoma, lung adenocarcinoma, lower grade glioma, kidney renal clear cell carcinoma, and kidney papillary cell carcinoma had worse overall survival. Importantly, NEIL3 overexpressed tumors accumulate mutation and chromosomal variations. Furthermore, NEIL3 overexpressed tumors exhibit simultaneous overexpression of homologous recombination genes (BRCA1/2) and mismatch repair genes ( MSH2/MSH6 ). However, NEIL3 overexpression is negatively correlated with tumor overexpressing nucleotide excision repair genes ( XPA, XPC, ERCC1 / 2 ). Our results suggest that NEIL3 might be a potential prognosis marker for high-risk patients, and/or an attractive therapeutic target for selected cancers.

The transcription factor RelB-NFκB2, activated by the noncanonical NFκB pathway, positively regulates corticotropin-releasing hormone (CRH) and prostaglandin production in the term human placenta and may play an important role in the timing of human parturition. Here we explored whether RelB-NFκB2 signaling plays a role in parturition in nonhuman anthropoid primates. We performed immunohistochemical staining to assess the correlation between CRH and nuclear activity of RelB-NFκB2 heterodimers in term placentas from humans, 3 catarrhine primate species, and a single platyrrhine primate species. Consistent with our previous studies, the human placenta showed cytoplasmic staining for CRH and nuclear staining for RelB-NFκB2. Similar staining patterns were noted in the 3 catarrhine primates (chimpanzee, baboon, and rhesus macaque). The platyrrhine (marmoset) placentas stained positively for CRH and RelB but not for NFκB2. Catarrhine (but not platyrrhine) nonhuman primate term placentas demonstrate the same CRH staining and nuclear localization patterns of RelB and NFκB2 as does human placenta. These results suggest that catarrhine primates, particularly rhesus macaques, may serve as useful animal models to study the biologic significance of the noncanonical NFκB pathway in human pregnancy.