scholarly journals Cre-loxP Recombination System for Large Genome Rearrangements in Lactococcus lactis

2002 ◽  
Vol 68 (5) ◽  
pp. 2359-2367 ◽  
Author(s):  
Nathalie Campo ◽  
Marie-Line Daveran-Mingot ◽  
Kees Leenhouts ◽  
Paul Ritzenthaler ◽  
Pascal Le Bourgeois
Keyword(s):  
Homologous Recombination ◽  
Fixed Points ◽  
Lactococcus Lactis ◽  
Genome Organization ◽  
Chromosomal Rearrangements ◽  
Cre Recombinase ◽  
Genome Rearrangements ◽  
Large Genome ◽  
Gram Positive Bacteria ◽  
Recombination System

ABSTRACT We have used a new genetic strategy based on the Cre-loxP recombination system to generate large chromosomal rearrangements in Lactococcus lactis. Two loxP sites were sequentially integrated in inverse order into the chromosome either at random locations by transposition or at fixed points by homologous recombination. The recombination between the two chromosomal loxP sites was highly efficient (approximately 1 × 10−1/cell) when the Cre recombinase was provided in trans, and parental- or inverted-type chromosomal structures were isolated after removal of the Cre recombinase. The usefulness of this approach was demonstrated by creating three large inversions of 500, 1,115, and 1,160 kb in size that modified the lactococcal genome organization to different extents. The Cre-loxP recombination system described can potentially be used for other gram-positive bacteria without further modification.

scholarly journals RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

2014 ◽  
Vol 35 (2) ◽  
pp. 406-416 ◽  
Author(s):  
Su Chen ◽  
Chen Wang ◽  
Luxi Sun ◽  
Da-Liang Wang ◽  
Lu Chen ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Open Chromatin ◽  
Dsb Repair ◽  
Heterochromatin Protein ◽  
Dna Double Strand Break ◽  
Recombination Repair ◽  
Ubiquitin Conjugating Enzyme

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients.

scholarly journals Mitochondrial Genome Rearrangements in Glomus Species Triggered by Homologous Recombination between Distinct mtDNA Haplotypes

2013 ◽  
Vol 5 (9) ◽  
pp. 1628-1643 ◽  
Author(s):  
Denis Beaudet ◽  
Yves Terrat ◽  
Sébastien Halary ◽  
Ivan Enrique de la Providencia ◽  
Mohamed Hijri
Keyword(s):  
Homologous Recombination ◽  
Mitochondrial Genome ◽  
Genome Rearrangements ◽  
Mtdna Haplotypes ◽  
Glomus Species

scholarly journals The M26 Hotspot of Schizosaccharomyces pombe Stimulates Meiotic Ectopic Recombination and Chromosomal Rearrangements

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1191-1204 ◽  
Author(s):  
Jeffrey B Virgin ◽  
Jeffrey P Bailey
Keyword(s):  
Homologous Recombination ◽  
Schizosaccharomyces Pombe ◽  
Dna Sequences ◽  
Chromosomal Rearrangements ◽  
Low Frequency ◽  
Repeated Sequences ◽  
Crossing Over ◽  
Ectopic Recombination ◽  
Recombination Hotspots ◽  
Integration Sites

Abstract Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10–1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35–60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.

Benzene metabolite hydroquinone promotes DNA homologous recombination repair via the NF-κB pathway

2019 ◽  
Vol 40 (8) ◽  
pp. 1021-1030 ◽  
Author(s):  
Xuejing Yang ◽  
Yedan Lu ◽  
Fuhong He ◽  
Fenxia Hou ◽  
Caihong Xing ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Molecular Mechanisms ◽  
Chromosomal Rearrangements ◽  
Critical Role ◽  
Environmental Pollutants ◽  
Environmental Pollutant ◽  
Osteosarcoma Cell ◽  
Dna Double Strand Breaks ◽  
Hematopoietic Stem

Abstract Benzene, a widespread environmental pollutant, induces DNA double-strand breaks (DSBs) and DNA repair, which may further lead to oncogenic mutations, chromosomal rearrangements and leukemogenesis. However, the molecular mechanisms underlying benzene-induced DNA repair and carcinogenesis remain unclear. The human osteosarcoma cell line (U2OS/DR-GFP), which carries a GFP-based homologous recombination (HR) repair reporter, was treated with hydroquinone, one of the major benzene metabolites, to identify the potential effects of benzene on DSB HR repair. RNA-sequencing was further employed to identify the potential key pathway that contributed to benzene-initiated HR repair. We found that treatment with hydroquinone induced a significant increase in HR. NF-κB pathway, which plays a critical role in carcinogenesis in multiple tumors, was significantly activated in cells recovered from hydroquinone treatment. Furthermore, the upregulation of NF-κB by hydroquinone was also found in human hematopoietic stem and progenitor cells. Notably, the inhibition of NF-κB activity by small molecule inhibitors (QNZ and JSH-23) significantly reduced the frequency of hydroquinone-initiated HR (−1.36- and −1.77-fold, respectively, P < 0.01). Our results demonstrate an important role of NF-κB activity in promoting HR repair induced by hydroquinone. This finding sheds light on the underlying mechanisms involved in benzene-induced genomic instability and leukemogenesis and may contribute to the larger exploration of the influence of other environmental pollutants on carcinogenesis.

scholarly journals Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Kalpana Dulal ◽  
Benjamin Silver ◽  
Hua Zhu
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Homologous Recombination ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Bac Clone ◽  
Dna Viruses ◽  
E Coli ◽  
Recombination System ◽  
Capture Method

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained insideE. coliallows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method inE. colifor molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects forin vitrohomologous recombination for genetic cloning.

scholarly journals The Saccharomyces cerevisiae Rad6 Postreplication Repair and Siz1/Srs2 Homologous Recombination-Inhibiting Pathways Process DNA Damage That Arises in asf1 Mutants

2009 ◽  
Vol 29 (19) ◽  
pp. 5226-5237 ◽  
Author(s):  
Ellen S. Kats ◽  
Jorrit M. Enserink ◽  
Sandra Martinez ◽  
Richard D. Kolodner
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Chromosomal Rearrangements ◽  
Nuclear Antigen ◽  
Recombination Suppression ◽  
Postreplication Repair ◽  
Gross Chromosomal Rearrangements ◽  
Translesion Bypass ◽  
The Relationship ◽  
Proliferating Cell

ABSTRACT The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.

scholarly journals Regulatory Functions of Serine-46-Phosphorylated HPr in Lactococcus lactis

2001 ◽  
Vol 183 (11) ◽  
pp. 3391-3398 ◽  
Author(s):  
Vicente Monedero ◽  
Oscar P. Kuipers ◽  
Emmanuel Jamet ◽  
Josef Deutscher
Keyword(s):  
Lactococcus Lactis ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Inducer Exclusion ◽  
Gram Positive Bacteria ◽  
In Vivo Experiments ◽  
Regulatory Functions

ABSTRACT In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a β-glucoside-specific EII and a 6-P-β-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methylβ-d-thiogalactoside (TMG) and 2-deoxy-d-glucose (2-DG). In vivo experiments with theptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

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