scholarly journals Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

2015 ◽  
Vol 112 (51) ◽  
pp. E7055-E7064 ◽  
Author(s):  
Longhua Hu ◽  
Anthony G. Vecchiarelli ◽  
Kiyoshi Mizuuchi ◽  
Keir C. Neuman ◽  
Jian Liu
Keyword(s):  
Copy Number ◽  
Detailed Mechanism ◽  
Ratchet Mechanism ◽  
Partition System ◽  
Plasmid Segregation ◽  
Emergent Phenomenon ◽  
Mechanochemical Coupling ◽  
Low Copy Number

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA–nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos—an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.

scholarly journals Mapping of the interactions between partition proteins Delta and Omega of plasmid pSM19035 from Streptococcus pyogenes

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1009-1020 ◽  
Author(s):  
Michal Dmowski ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Streptococcus Pyogenes ◽  
Dna Sequences ◽  
Copy Number ◽  
Terminal Part ◽  
Partition System ◽  
Plasmid Partition ◽  
Nucleoprotein Complex ◽  
Interaction Domains

Formation of the segrosome, a nucleoprotein complex crucial for proper functioning of plasmid partition systems, involves interactions between specific partition proteins (ParA-like and ParB-like), ATP and specific DNA sequences (the centromeric sites). Although partition systems have been studied for many years, details of the segrosome formation are not yet clear. Organization of the pSM19035-encoded partition system is unique; in contrast with other known par systems, here, the δ and ω genes do not constitute an operon. Moreover, Omega [a ParB-like protein which has a Ribbon-Helix-Helix (RHH) structure] recognizes multiple centromeric sequences located in the promoters of δ, ω and copS (copy-number control gene). The ParA-like protein Delta is a Walker-type ATPase. In this work, we identify the interaction domains and requirements for dimerization and hetero-interactions of the Delta and Omega proteins of pSM19035 plasmid. The RHH structures are involved in Omega dimerization in vivo and its N-terminal unstructured part is indispensable for association with Delta, both in vivo and in vitro. Omega does not need to form dimers to interact with Delta. ATP binding is not required for Delta dimerization but is important for interaction with Omega in vivo. The in vitro interaction between Delta and Omega depends on ATP but does not require the presence of specific DNA segments (the centromere) recognized by Omega. The C-terminal part of the Delta protein (aa 198–284) is indispensable for interaction with Omega. Delta most probably interacts with Omega as a dimer since two amino acid substitutions in a conserved region between the A′ and B motifs abolish both the dimerization of Delta and its interaction with Omega.

scholarly journals pTAR-Encoded Proteins in Plasmid Partitioning

2000 ◽  
Vol 182 (7) ◽  
pp. 1889-1894 ◽  
Author(s):  
Kirill Kalnin ◽  
Svetlana Stegalkina ◽  
Michael Yarmolinsky
Keyword(s):  
Copy Number ◽  
Transcription Start ◽  
Par Proteins ◽  
E Coli ◽  
Partition System ◽  
Segregational Stability ◽  
Low Copy Number ◽  
Single Protein ◽  
Operator Functions

ABSTRACT Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent cis-acting centromere analog to which one or perhaps both proteins bind. The diminutive partition region of pTAR of Agrobacterium spp. was reported to be exceptional, encoding only a single protein, ParA (D. R. Gallie and C. I. Kado, J. Mol. Biol. 193:465–478, 1987). However, resequencing of the region revealed two small downstream genes,parB and orf-84, of which only parBwas found to be essential for partitioning in A. tumefaciens. Purified ParA exhibited a weak ATPase activity that was modestly increased by nonspecific DNA. ParB bound in vitro to repeated sequences present in a region, parS, that possesses centromere and operator functions and within which we identified the primary transcription start site by primer extension. In certain respects the Par proteins behave normally in the foreign hostEscherichia coli. In E. coli, as in A. tumefaciens, ParB repressed the partition operon; ParA, inactive alone, augmented this repression. Functional similarities between the partition system of pTAR and those of other plasmids and bacteria are prominent, despite differences in size, organization, and amino acid sequence.

scholarly journals LiquidCNA: tracking subclonal evolution from longitudinal liquid biopsies using somatic copy number alterations

2021 ◽  
Author(s):  
Eszter Lakatos ◽  
Helen Hockings ◽  
Maximilian Mossner ◽  
Michelle Lockley ◽  
Trevor A. Graham
Keyword(s):  
Copy Number ◽  
Evolutionary Dynamics ◽  
Cost Effective ◽  
Copy Number Alterations ◽  
Liquid Biopsies ◽  
Metastatic Lung ◽  
Low Pass ◽  
Somatic Copy Number Alterations

AbstractCell-free DNA (cfDNA) measured via liquid biopsies provides a way for minimally-invasive monitoring of tumour evolutionary dynamics during therapy. Here we present liquidCNA, a method to track subclonal evolution from longitudinally collected cfDNA samples based on somatic copy number alterations (SCNAs). LiquidCNA utilises SCNA profiles derived through cost-effective low-pass whole genome sequencing to automatically and simultaneously genotype and quantify the size of the dominant subclone without requiring prior knowledge of the genetic identity of the emerging clone. We demonstrate the accuracy of liquidCNA in synthetically generated sample sets and in vitro and in silico mixtures of cancer cell lines. Application in vivo in patients with metastatic lung cancer reveals the progressive emergence of a novel tumour sub-population. LiquidCNA is straightforward to use, computationally inexpensive and enables continuous monitoring of subclonal evolution to understand and control therapy-induced resistance.

scholarly journals Clonal expansion and epigenetic reprogramming following deletion or amplification of mutant IDH1

2017 ◽  
Vol 114 (40) ◽  
pp. 10743-10748 ◽  
Author(s):  
Tali Mazor ◽  
Charles Chesnelong ◽  
Aleksandr Pankov ◽  
Llewellyn E. Jalbert ◽  
Chibo Hong ◽  
...  
Keyword(s):  
Copy Number ◽  
Cpg Island ◽  
Clonal Expansion ◽  
Low Grade ◽  
Copy Number Alterations ◽  
Wild Type ◽  
Mutant Idh1

IDH1 mutation is the earliest genetic alteration in low-grade gliomas (LGGs), but its role in tumor recurrence is unclear. Mutant IDH1 drives overproduction of the oncometabolite d-2-hydroxyglutarate (2HG) and a CpG island (CGI) hypermethylation phenotype (G-CIMP). To investigate the role of mutant IDH1 at recurrence, we performed a longitudinal analysis of 50 IDH1 mutant LGGs. We discovered six cases with copy number alterations (CNAs) at the IDH1 locus at recurrence. Deletion or amplification of IDH1 was followed by clonal expansion and recurrence at a higher grade. Successful cultures derived from IDH1 mutant, but not IDH1 wild type, gliomas systematically deleted IDH1 in vitro and in vivo, further suggestive of selection against the heterozygous mutant state as tumors progress. Tumors and cultures with IDH1 CNA had decreased 2HG, maintenance of G-CIMP, and DNA methylation reprogramming outside CGI. Thus, while IDH1 mutation initiates gliomagenesis, in some patients mutant IDH1 and 2HG are not required for later clonal expansions.

scholarly journals P1 Plasmid Segregation: Accurate Redistribution by Dynamic Plasmid Pairing and Separation

2009 ◽  
Vol 192 (5) ◽  
pp. 1175-1183 ◽  
Author(s):  
Manjistha Sengupta ◽  
Henrik Jorck Nielsen ◽  
Brenda Youngren ◽  
Stuart Austin
Keyword(s):  
Large Cell ◽  
Growth Conditions ◽  
Fine Tuning ◽  
Cell Axis ◽  
Partition System ◽  
Plasmid Segregation ◽  
Daughter Cells ◽  
Low Copy Number ◽  
Type Ia ◽  
Analytical Tools

ABSTRACT Low-copy-number plasmids, such as P1 and F, encode a type Ia partition system (P1par or Fsop) for active segregation of copies to daughter cells. Typical descriptions show a single central plasmid focus dividing and the products moving to the cell quarter regions, ensuring segregation. However, using improved optical and analytical tools and large cell populations, we show that P1 plasmid foci are very broadly distributed. Moreover, under most growth conditions, more than two foci are frequently present. Each focus contains either one or two plasmid copies. Replication and focus splitting occur at almost any position in the cell. The products then move rapidly apart for approximately 40% of the cell length. They then tend to maintain their relative positions. The segregating foci often pass close to or come to rest close to other foci in the cell. Foci frequently appear to fuse during these encounters. Such events occur several times in each cell and cell generation on average. We argue that foci pair with their neighbors and then actively separate again. The net result is an approximately even distribution of foci along the long cell axis on average. We show mathematically that trans-pairing and active separation could greatly increase the accuracy of segregation and would produce the distributions of foci that we observe. Plasmid pairing and separation may constitute a novel fine-tuning mechanism that takes the basic pattern created when plasmids separate after replication and converts it to a roughly even pattern that greatly improves the fidelity of plasmid segregation.

scholarly journals Promotion of Tumor Growth by ADAMTS4 in Colorectal Cancer: Focused on Macrophages

2018 ◽  
Vol 46 (4) ◽  
pp. 1693-1703 ◽  
Author(s):  
Jianjun Chen ◽  
Yang Luo ◽  
Yong Zhou ◽  
Shaolan Qin ◽  
Yier Qiu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Expression Profile ◽  
Copy Number ◽  
Copy Number Alterations ◽  
Extracellular Proteases ◽  
Proliferation And Invasion

Background/Aims: ADAMTSs (A disintegrin and metalloprotease domains with thrombospondins motifs) are a family of extracellular proteases that have been related to both oncogenic and tumor-suppressive functions. The aim of the present study was to investigate: 1) the mutation, copy-number alterations, and expression profile of ADAMTSs in colorectal cancer and 2) whether ADAMTSs participate in colorectal cancer (CRC) progression and invasion. Methods: The mutation, copy-number alterations, and expression profile of ADAMTSs in CRC were analyzed in the TCGA cohort using cBioportal. ADAMTS4 expression in tumor tissues and cell lines were determined by immunostaining and real-time quantitative PCR. The role of ADAMTS-4 in CRC progression and the underlying mechanisms were studied by using short hairpin RNA-mediated knockdown of ADAMTS4. The effects of ADAMTS4 in cell proliferation and invasion were determined by clone formation assay and transwell migration assay, respectively. Macrophages were depleted by liposomal clodronate in immune-competent BALB/c mice and tumor growth was analyzed. Results: ADAMTS4 was differentially expressed in CRC and predicted a poor prognosis. Elevated ADAMTS4 expression was closely associated with larger tumor size, enhanced TNM stage, and a poor clinical outcome in patients with CRC. ADAMTS4 knockdown had no inhibitory implications on cell proliferation and invasion in vitro, but significantly attenuated tumor growth in vivo. Mechanistically, we revealed that ADAMTS4 was associated macrophages infiltration and polarization in the tumor microenvironment of CRC. Macrophage depletion largely abolished the promotive effect of ADAMTS4 on tumor growth in the immune competent BALB/c mice. Conclusion: ADAMTS4 seemed to be a promising prognostic indicator in CRC. The novel link between ADAMTS4 and macrophages mirrors the potential regulatory roles of ADAMTSs in the inflammatory microenvironment of cancers.

scholarly journals Pmp22 super-enhancer deletion causes tomacula formation and conduction block in peripheral nerves

2020 ◽  
Vol 29 (10) ◽  
pp. 1689-1699
Author(s):  
Harrison Pantera ◽  
Bo Hu ◽  
Daniel Moiseev ◽  
Chris Dunham ◽  
Jibraan Rashid ◽  
...  
Keyword(s):  
Peripheral Neuropathy ◽  
Schwann Cell ◽  
Copy Number ◽  
Regulatory Elements ◽  
Copy Number Variations ◽  
Hereditary Neuropathy ◽  
Peripheral Myelin Protein 22 ◽  
Super Enhancer

Abstract Copy number variation of the peripheral nerve myelin gene Peripheral Myelin Protein 22 (PMP22) causes multiple forms of inherited peripheral neuropathy. The duplication of a 1.4 Mb segment surrounding this gene in chromosome 17p12 (c17p12) causes the most common form of Charcot-Marie-Tooth disease type 1A, whereas the reciprocal deletion of this gene causes a separate neuropathy termed hereditary neuropathy with liability to pressure palsies (HNPP). PMP22 is robustly induced in Schwann cells in early postnatal development, and several transcription factors and their cognate regulatory elements have been implicated in coordinating the gene’s proper expression. We previously found that a distal super-enhancer domain was important for Pmp22 expression in vitro, with particular impact on a Schwann cell-specific alternative promoter. Here, we investigate the consequences of deleting this super-enhancer in vivo. We find that loss of the super-enhancer in mice reduces Pmp22 expression throughout development and into adulthood, with greater impact on the Schwann cell-specific promoter. Additionally, these mice display tomacula formed by excessive myelin folding, a pathological hallmark of HNPP, as have been previously observed in heterozygous Pmp22 mice as well as sural biopsies from patients with HNPP. Our findings demonstrate a mechanism by which smaller copy number variations, not including the Pmp22 gene, are sufficient to reduce gene expression and phenocopy a peripheral neuropathy caused by the HNPP-associated deletion encompassing PMP22.

scholarly journals Conditional high copy number ColE1 mutants: resistance to RNA1 inhibition in vivo and in vitro.

1988 ◽  
Vol 7 (10) ◽  
pp. 3289-3297 ◽  
Author(s):  
T. Fitzwater ◽  
X. Y. Zhang ◽  
R. Elble ◽  
B. Polisky
Keyword(s):  
Copy Number ◽  
High Copy Number

scholarly journals Interplay between Plasmid Partition and Postsegregational Killing Systems

2004 ◽  
Vol 186 (8) ◽  
pp. 2504-2507 ◽  
Author(s):  
Therese Brendler ◽  
Lucretia Reaves ◽  
Stuart Austin
Keyword(s):  
Copy Number ◽  
Partition System ◽  
Plasmid Partition ◽  
Naturally Occurring ◽  
Low Copy Number ◽  
Postsegregational Killing

ABSTRACT Active partition systems and postsegregational killing (PSK) systems are present together in naturally occurring low-copy-number plasmids. Theory suggests that PSK may act as the ultimate determinant of plasmid retention, whereas the partition system may minimize the growth penalty to the host, resulting in a near-ideal symbiosis when the systems combine. Here, we prove the validity of this principle for a specific case involving the P1par system and the mvp PSK system.

scholarly journals Targeted Isolation of a Designated Region of the Bacillus subtilis Genome by Recombinational Transfer

2004 ◽  
Vol 70 (4) ◽  
pp. 2508-2513 ◽  
Author(s):  
Satoshi Tomita ◽  
Kenji Tsuge ◽  
Yo Kikuchi ◽  
Mitsuhiro Itaya
Keyword(s):  
Bacillus Subtilis ◽  
Copy Number ◽  
Positional Cloning ◽  
Deletion Mutant ◽  
Essential Gene ◽  
Transfer System ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Subtilis Genome

ABSTRACT A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

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