34 research outputs found
Biochemical characterization of the Minichromosome maintenance (MCM) helicase from Methanothermobacter thermautotrophicus
DNA replication requires coordination of numerous proteins to duplicate genetic information in a precise and timely manner. One of the key players in replication is the replicative helicase that unwinds the duplex DNA to provide the single-stranded template for the DNA polymerases. Minichromosome maintenance (MCM) protein is the replicative helicase in archaea. This dissertation focuses on the MCM helicase from the euryarchaeon Methanothermobacter thermautotrophicus (Mth).
Archaeal MCM proteins can be divided into two major parts, the N terminal and C terminal domains. The N terminal domain is essential for DNA binding and multimerization, while the C-terminus contains the catalytic domains. The objective of this dissertation is to elucidate the mechanism by which the N terminal domain communicates with the catalytic domain to facilitate helicase activity. To address this question, two approaches were taken. One approach identified conserved residues found in the N terminus and investigated their properties using various biochemical and biophysical methods. By analyzing several proteins with mutations in the conserved residues, a loop that is essential for MCM helicase activity was identified. The study suggests that the loop is involved in coupling the N-terminal DNA binding function and the catalytic activity of the AAA+ domain. Some other conserved residues, however, did not directly affect the MCM helicase activity but showed differences in biochemical properties suggesting that they may play a role in maintaining the structural integrity of the MCM helicase. Another approach determined the differences in thermal stability of the MCM protein in the presence of various cofactors and DNA substrates. The study shows that the protein has two unfolding transitions when ATP and the DNA are present, while non-hydrolyzable ATP results in one transition. This study suggests possible conformational changes arising from decoupling of two domains that occur during the ATP hydrolysis in the presence of DNA. Furthermore, both DNA binding function by the N terminal domain and ATP binding by the catalytic domain are essential for the change
Different Residues on the Surface of the Methanothermobacter thermautotrophicus MCM Helicase Interact with Single- and Double-Stranded DNA
The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea, separating the two strands of chromosomal DNA during replication. The catalytic activity resides within the C-terminal region of the MCM protein, while the N-terminal portion plays an important role in DNA binding and protein multimerization. An alignment of MCM homologues from several archaeal species revealed a number of conserved amino acids. Here several of the conserved residues located on the surface of the helicase have been mutated and their roles in MCM functions determined. It was found that some mutations result in increased affinity for ssDNA while the affinity for dsDNA is decreased. Other mutants exhibit the opposite effect. Thus, the data suggest that these conserved surface residues may participate in MCM-DNA interactions
Cloning, Purification, and Partial Characterization of the Halobacterium sp. NRC-1 Minichromosome Maintenance (MCM) Helicase
The MCM gene from the archaeon Halobacterium, with and without its intein, was cloned into an Escherichia coli expression vector, overexpressed and the protein was purified and antibodies were generated. The antibodies were used to demonstrate that in vivo only the processed enzyme, without the intein, could be detected
Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein
Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme
Thermoplasma acidophilum Cdc6 protein stimulates MCM helicase activity by regulating its ATPase activity
The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. In most archaeal species studied, the interaction between MCM and the initiator protein, Cdc6, inhibits helicase activity. To date, the only exception is the helicase and Cdc6 proteins from the archaeon Thermoplasma acidophilum. It was previously shown that when the Cdc6 protein interacts with MCM it substantially stimulates helicase activity. It is shown here that the mechanism by which the Cdc6 protein stimulates helicase activity is by stimulating the ATPase activity of MCM. Also, through the use of site-specific substitutions, and truncated and chimeric proteins, it was shown that an intact Cdc6 protein is required for this stimulation. ATP binding and hydrolysis by the Cdc6 protein is not needed for the stimulation. The data suggest that binding of Cdc6 protein to MCM protein changes the structure of the helicase, enhancing the catalytic hydrolysis of ATP and helicase activity
Oncogenic Ras and ΔNp63α cooperate to recruit immunosuppressive polymorphonuclear myeloid-derived suppressor cells in a mouse model of squamous cancer pathogenesis
IntroductionAmplification of human chromosome 3q26-29, which encodes oncoprotein ΔNp63 among other isoforms of the p63 family, is a feature common to squamous cell carcinomas (SCCs) of multiple tissue origins. Along with overexpression of ΔNp63, activation of the protooncogene, RAS, whether by overexpression or oncogenic mutation, is frequently observed in many cancers. In this study, analysis of transcriptome data from The Cancer Genome Atlas (TCGA) demonstrated that expression of TP63 mRNA, particularly ΔNp63 isoforms, and HRAS are significantly elevated in advanced squamous cell carcinomas of the head and neck (HNSCCs), suggesting pathological significance. However, how co-overexpressed ΔNp63 and HRAS affect the immunosuppressive tumor microenvironment (TME) is incompletely understood.MethodsHere, we established and characterized an immune competent mouse model using primary keratinocytes with retroviral-mediated overexpression of ΔNp63α and constitutively activated HRAS (v-rasHa G12R) to evaluate the role of these oncogenes in the immune TME.ResultsIn this model, orthotopic grafting of wildtype syngeneic keratinocytes expressing both v-rasHa and elevated levels of ΔNp63α consistently yield carcinomas in syngeneic hosts, while cells expressing v-rasHa alone yield predominantly papillomas. We found that polymorphonuclear (PMN) myeloid cells, experimentally validated to be immunosuppressive and thus representing myeloid-derived suppressor cells (PMN-MDSCs), were significantly recruited into the TME of carcinomas arising early following orthotopic grafting of ΔNp63α/v-rasHa-expressing keratinocytes. ΔNp63α/v-rasHa-driven carcinomas expressed higher levels of chemokines implicated in recruitment of MDSCs compared to v-rasHa-initiated tumors, providing a heretofore undescribed link between ΔNp63α/HRAS-driven carcinomas and the development of an immunosuppressive TME.ConclusionThese results support the utilization of a genetic carcinogenesis model harboring specific genomic drivers of malignancy to study mechanisms underlying the development of local immunosuppression
Papillomaviruses use recombination-dependent replication to vegetatively amplify their genomes in differentiated cells.
CIN612-9E cells were derived from a cervical lesion and contain hundreds of copies of extrachromosomally replicating HPV31 genomes [<b>52</b>]<b>.</b>
<p>These cells can be induced to differentiate with high calcium–containing medium, which switches on vegetative viral DNA replication <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003321#ppat.1003321-Moody1" target="_blank">[3]</a>. Many of these cells contain multiple small replication foci <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003321#ppat.1003321-Moody1" target="_blank">[3]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003321#ppat.1003321-Gillespie1" target="_blank">[4]</a>; but numerous cells contain one large foci, as shown here, perhaps indicative of a temporal evolution. The nucleus shown has been stained with DAPI (grey) and antibodies to γH2AX to identify the viral replication foci (shown in cyan), and RAD51 to identify centers of homologous recombination (shown in red). 3D reconstruction of Z-stacks of confocal images was performed using Bitplane Imaris.</p
The papillomavirus life cycle is closely coupled with differentiation of the host epithelium.
<p>The virus infects the dividing basal cells through a microabrasion. The viral DNA is maintained at a low copy number in these cells. When basal cells divide, some daughter cells move up in the epithelium and begin the process of terminal differentiation. Papillomaviruses are finely tuned to this process and turn on late transcription, translation, and late DNA replication in specific stages of the differentiation process. Vegetative viral DNA replication takes place in cells that are in either the G2 phase of the cell cycle or have exited the cell cycle. By inducing the DNA damage response and homologous recombination repair pathways, the virus can efficiently replicate progeny genomes in differentiated cells without competition from host DNA synthesis.</p