An algorithm and application to efficiently analyze DNA fiber data

The duplication of genetic information (DNA replication) is central to life. Numerous control mechanisms ensure the exact course of the process during each cell division. Disturbances of DNA replication have severe consequences for the affected cell, and current models link them to cancer development. One of the most accurate methods for studying DNA replication is labeling newly synthesized DNA molecules with halogenated nucleotides, followed by immunofluorescence and microscopy detection, known as DNA fiber labeling. The method allows the registration of the activity of single replication complexes by measuring the length of the "trace" left by each of them. The major difficulty of the method is the labor-intensive analysis, which requires measuring the lengths of a large number of labeled fragments. Recently, the interest in this kind of image analysis has grown rapidly. In this manuscript, we provide a detailed description of an algorithm and a lightweight Java application to automatically analyze single DNA molecule images we call "DNA size finder". DNA size finder significantly simplified the analysis of the experimental data while increasing reliability by the standardized measurement of a greater number of DNA molecules. It is freely available and does not require any paid platforms or services to be used. We hope that the application will facilitate both the study of DNA replication control and the effects of various compounds used in human activity on the process of DNA replication.

Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii

DNA replication is fundamental to all living organisms. In yeast and animals, it is triggered by an assembly of pre-replicative complex including ORC, CDC6 and MCMs. Cyclin Dependent Kinase (CDK) regulates both assembly and firing of the pre-replicative complex. We tested temperature-sensitive mutants blocking Chlamydomonas DNA replication. The mutants were partially or completely defective in DNA replication and did not produce mitotic spindles. After a long G1, wild type Chlamydomonas cells enter a division phase when it undergoes multiple rapid synchronous divisions (‘multiple fission’). Using tagged transgenic strains, we found that MCM4 and MCM6 were localized to the nucleus throughout the entire multiple fission division cycle, except for transient cytoplasmic localization during each mitosis. Chlamydomonas CDC6 was transiently localized in nucleus in early division cycles. CDC6 protein levels were very low, probably due to proteasomal degradation. CDC6 levels were severely reduced by inactivation of CDKA1 (CDK1 ortholog) but not the plant-specific CDKB1. Proteasome inhibition did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 might upregulate CDC6 at the transcriptional level. All of the DNA replication proteins tested were essentially undetectable until late G1. They accumulated specifically during multiple fission and then were degraded as cells completed their terminal divisions. We speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. We also developed a simple assay for salt-resistant chromatin binding of MCM4, and found that tight MCM4 loading was dependent on ORC1, CDC6 and MCM6, but not on RNR1 or CDKB1. These results provide a microbial framework for approaching replication control in the plant kingdom.

Combination of HIV-1 and Diabetes Enhances Blood Brain Barrier Injury via Effects on Brain Endothelium and Pericytes

Despite combined antiretroviral therapy (ART) achieving efficient HIV replication control, HIV-associated neurocognitive disorders (HAND) continue to be highly prevalent in HIV-infected patients. Diabetes mellitus (DM) is a well-known comorbidity of HAND in HIV-infected patients. Blood brain barrier (BBB) dysfunction has been linked recently to dementia development, specifically in DM patients. BBB injury exists both in HIV and DM, likely contributing to cognitive decline. However, its extent, exact cellular targets and mechanisms are largely unknown. In this report, we found a decrease in pericyte coverage and expression of tight junction proteins in human brain tissues from HIV patients with DM and evidence of HAND when compared to HIV-infected patients without DM or seronegative DM patients. Using our in vitro BBB models, we demonstrated diminution of barrier integrity, enhanced monocyte adhesion, changes in cytoskeleton and overexpression of adhesion molecules in primary human brain endothelial cells or human brain pericytes after exposure to HIV and DM-relevant stimuli. Our study demonstrates for the first-time evidence of impaired BBB function in HIV-DM patients and shows potential mechanisms leading to it in brain endothelium and pericytes that may result in poorer cognitive performance compared to individuals without HIV and DM.

Mitochondrial Inheritance in Phytopathogenic Fungi—Everything Is Known, or Is It?

Mitochondria are important organelles in eukaryotes that provide energy for cellular processes. Their function is highly conserved and depends on the expression of nuclear encoded genes and genes encoded in the organellar genome. Mitochondrial DNA replication is independent of the replication control of nuclear DNA and as such, mitochondria may behave as selfish elements, so they need to be controlled, maintained and reliably inherited to progeny. Phytopathogenic fungi meet with special environmental challenges within the plant host that might depend on and influence mitochondrial functions and services. We find that this topic is basically unexplored in the literature, so this review largely depends on work published in other systems. In trying to answer elemental questions on mitochondrial functioning, we aim to introduce the aspect of mitochondrial functions and services to the study of plant-microbe-interactions and stimulate phytopathologists to consider research on this important organelle in their future projects.

Competition between PAF1 and MLL1/COMPASS confers the opposing function of LEDGF/p75 in HIV latency and proviral reactivation

Transcriptional status determines the HIV replicative state in infected patients. However, the transcriptional mechanisms for proviral replication control remain unclear. In this study, we show that, apart from its function in HIV integration, LEDGF/p75 differentially regulates HIV transcription in latency and proviral reactivation. During latency, LEDGF/p75 suppresses proviral transcription via promoter-proximal pausing of RNA polymerase II (Pol II) by recruiting PAF1 complex to the provirus. Following latency reversal, MLL1 complex competitively displaces PAF1 from the provirus through casein kinase II (CKII)–dependent association with LEDGF/p75. Depleting or pharmacologically inhibiting CKII prevents PAF1 dissociation and abrogates the recruitment of both MLL1 and Super Elongation Complex (SEC) to the provirus, thereby impairing transcriptional reactivation for latency reversal. These findings, therefore, provide a mechanistic understanding of how LEDGF/p75 coordinates its distinct regulatory functions at different stages of the post-integrated HIV life cycles. Targeting these mechanisms may have a therapeutic potential to eradicate HIV infection.

Conservation and divergence of DNA replication control in Chlamydomonas reinhardtii

ABSTRACTWe recently isolated temperature-sensitive cell cycle mutants in Chlamydomonas reinhardtii for which the causative mutations were located in genes annotated for potential involvement in DNA replication. Chlamydomonas has a very long G1 period during which cells grow up to ~10-fold without division, followed by rapid cycles of DNA replication and mitosis (‘multiple fission’). All of the candidate DNA replication mutants tested were defective in completion of the first round of DNA replication, and also failed to produce mitotic spindles. For a subset of the mutants, we rescued temperature-sensitive lethality with tagged transgenes and used the resulting strains to analyze abundance and localization control of the tagged protein. All of the DNA replication proteins tested were essentially undetectable until late G1, accumulated during the period of multiple fission and then were degraded as cells completed their terminal divisions. MCM4 and MCM6 were localized to the nucleus during the division cycle except for transient cytoplasmic localization during mitosis. CDC45 showed strict protein location to the nucleus and co-localized to spindles during mitosis. In contrast, CDC6 was detected in the nucleus only transiently during early divisions within the overall multiple fission cycle. Cdc6 protein levels were very low, but increased upon treatment with MG132, a proteasome inhibitor. We also tested if these DNA replication proteins are regulated by cyclin dependent kinase (CDK). There are two main CDKs in Chlamydomonas, CDKA1 and CDKB1. We found that CDC6 protein level was severely reduced in a cdka1 mutant, but not in a cdkb1 mutant. MG132 did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 upregulates CDC6 at the transcription level. Since MCM4, MCM6 and CDC6 were all essentially undetectable during the long G1 before DNA replication cycles began, we speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. These results provide a microbial framework for approaching replication control in the plant kingdom.

Essential Role of mGBP7 for Survival of Toxoplasma gondii Infection

ABSTRACT Members of the murine guanylate-binding protein family (mGBP) are induced by interferon gamma (IFN-γ) and have been shown to be important factors in cell-autonomous immunity toward the intracellular pathogen Toxoplasma gondii. Previously, we identified that mGBP2 mediates disruption of the parasitophorous vacuole membrane (PVM) and directly assaults the plasma membrane of the parasite. Here, we show that mGBP7-deficient mice are highly susceptible to T. gondii infection. This is demonstrated by the loss of parasite replication control, pronounced development of ascites, and death of the animals in the acute infection phase. Interestingly, live-cell microscopy revealed that mGBP7 recruitment to the PVM occurs after mGBP2 recruitment, followed by disruption of the PVM and T. gondii integrity and accumulation of mGBP7 inside the parasite. This study defines mGBP7 as a crucial effector protein in resistance to intracellular T. gondii. IMPORTANCE Guanylate-binding proteins (GBPs) are induced by the inflammatory cytokine interferon gamma (IFN-γ) and have been shown to be important factors in the defense of the intracellular pathogen Toxoplasma gondii. In previous studies, we showed that members of the mouse GBP family, such as mGBP2 and mGBP7, accumulate at the parasitophorous vacuole of T. gondii, which is the replicatory niche of the parasite. In this study, we show that mice deficient in mGBP7 succumb early after infection with T. gondii, showing a complete failure of resistance to the pathogen. On a molecular level, mGBP7 is found directly at the parasite, likely mediating its destruction.

Engineering and Characterization of Oncolytic Vaccinia Virus Expressing Truncated Herpes Simplex Virus Thymidine Kinase

Oncolytic viruses are a promising class of anti-tumor agents; however, concerns regarding uncontrolled viral replication have led to the development of a replication-controllable oncolytic vaccinia virus (OVV). The engineering involves replacing the native thymidine kinase (VV-tk) gene, in a Wyeth strain vaccinia backbone, with the herpes simplex virus thymidine kinase (HSV-tk) gene, which allows for viral replication control via ganciclovir (GCV, an antiviral/cytotoxic pro-drug). Adding the wild-type HSV-tk gene might disrupt the tumor selectivity of VV-tk deleted OVVs; therefore, only engineered viruses that lacked tk activity were selected as candidates. Ultimately, OTS-412, which is an OVV containing a mutant HSV-tk, was chosen for characterization regarding tumor selectivity, sensitivity to GCV, and the influence of GCV on OTS-412 anti-tumor effects. OTS-412 demonstrated comparable replication and cytotoxicity to VVtk- (control, a VV-tk deleted OVV) in multiple cancer cell lines. In HCT 116 mouse models, OTS-412 replication in tumors was reduced by >50% by GCV (p = 0.004); additionally, combination use of GCV did not compromise the anti-tumor effects of OTS-412. This is the first report of OTS-412, a VV-tk deleted OVV containing a mutant HSV-tk transgene, which demonstrates tumor selectivity and sensitivity to GCV. The HSV-tk/GCV combination provides a safety mechanism for future clinical applications of OTS-412.

Polyploidy in halophilic archaea: regulation, evolutionary advantages, and gene conversion

All analyzed haloarachea are polyploid. In addition, haloarchaea contain more than one type of chromosome, and thus the gene dosage can be regulated independently on different replicons. Haloarchaea and several additional archaea have more than one replication origin on their major chromosome, in stark contrast with bacteria, which have a single replication origin. Two of these replication origins of Haloferax volcanii have been studied in detail and turned out to have very different properties. The chromosome copy number appears to be regulated in response to growth phases and environmental factors. Archaea typically contain about two Origin Recognition Complex (ORC) proteins, which are homologous to eukaryotic ORC proteins. However, haloarchaea are the only archaeal group that contains a multitude of ORC proteins. All 16 ORC protein paralogs from H. volcanii are involved in chromosome copy number regulation. Polyploidy has many evolutionary advantages for haloarchaea, e.g. a high resistance to desiccation, survival over geological times, and the relaxation of cell cycle-specific replication control. A further advantage is the ability to grow in the absence of external phosphate while using the many genome copies as internal phosphate storage polymers. Very efficient gene conversion operates in haloarchaea and results in the unification of genome copies. Taken together, haloarchaea are excellent models to study many aspects of genome biology in prokaryotes, exhibiting properties that have not been found in bacteria.