scholarly journals Motifs of the C-terminal Domain of MCM9 Direct Localization to Sites of Mitomycin-C Damage for RAD51 Recruitment

2020 ◽  
Author(s):  
David R. McKinzey ◽  
Shivasankari Gomathinayagam ◽  
Wezley C. Griffin ◽  
Kathleen N. Klinzing ◽  
Elizabeth P. Jeffries ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Amino Acid ◽  
Mitomycin C ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Impaired Ability ◽  
Direct Localization ◽  
Functional Components ◽  
Positively Charged

AbstractThe MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC) induced DNA damage. First, an unconventional ‘bipartite-like’ nuclear localization (NLS) motif consisting of two positively charged amino acid stretches separated by a long intervening sequence is required for the nuclear import of both MCM8 and MCM9. Second, a variant of the BRC motif (BRCv), similar to that found in other HR helicases, is necessary for localization to sites of MMC damage. The MCM9-BRCv directly interacts with and recruits RAD51 downstream to MMC-induced damage to aid in DNA repair. Patient lymphocytes devoid of functional MCM9 and discrete MCM9 knockout cells have a significantly impaired ability to form RAD51 foci after MMC treatment. Therefore, the disordered CTE in MCM9 is functionally important in promoting MCM8/9 activity and in recruiting downstream interactors; thus, requiring full length MCM9 for proper DNA repair.

scholarly journals Nuclear Import of the Retrotransposon Tf1 Is Governed by a Nuclear Localization Signal That Possesses a Unique Requirement for the FXFG Nuclear Pore Factor Nup124p

2000 ◽  
Vol 20 (20) ◽  
pp. 7798-7812 ◽  
Author(s):  
Van-Dinh Dang ◽  
Henry L. Levin
Keyword(s):  
Amino Acid ◽  
Nuclear Envelope ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Heterologous Protein ◽  
Simian Virus 40 ◽  
Nuclear Pore ◽  
Surprising Result ◽  
Gag Protein ◽  
Nuclear Localization Signals

ABSTRACT Retroviruses, such as human immunodeficiency virus, that infect nondividing cells generate integration precursors that must cross the nuclear envelope to reach the host genome. As a model for retroviruses, we investigated the nuclear entry of Tf1, a long-terminal-repeat-containing retrotransposon of the fission yeastSchizosaccharomyces pombe. Because the nuclear envelope of yeasts remains intact throughout the cell cycle, components of Tf1 must be transported through the envelope before integration can occur. The nuclear localization of the Gag protein of Tf1 is different from that of other proteins tested in that it has a specific requirement for the FXFG nuclear pore factor, Nup124p. Using extensive mutagenesis, we found that Gag contained three nuclear localization signals (NLSs) which, when included individually in a heterologous protein, were sufficient to direct nuclear import. In the context of the intact transposon, mutations in the NLS that mapped to the first 10 amino acid residues of Gag significantly impaired Tf1 retrotransposition and abolished nuclear localization of Gag. Interestingly, this NLS activity in the heterologous protein was specifically dependent upon the presence of Nup124p. Deletion analysis of heterologous proteins revealed the surprising result that the residues in Gag with the NLS activity were independent from the residues that conveyed the requirement for Nup124p. In fact, a fragment of Gag that lacked NLS activity, residues 10 to 30, when fused to a heterologous protein, was sufficient to cause the classical NLS of simian virus 40 to require Nup124p for nuclear import. Within the context of the current understanding of nuclear import, these results represent the novel case of a short amino acid sequence that specifies the need for a particular nuclear pore complex protein.

scholarly journals Functional Identification of a Nuclear Localization Signal of MYB2 Protein in Giardia lamblia

2020 ◽  
Vol 58 (6) ◽  
pp. 675-679
Author(s):  
Juri Kim ◽  
Mee Young Shin ◽  
Soon-Jung Park
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Giardia Lamblia ◽  
Nuclear Protein ◽  
Functional Identification ◽  
Localization Signal ◽  
Nuclear Location

MYB2 protein was identified as a transcription factor that showed encystation-induced expression in <i>Giardia lamblia</i>. Although nuclear import is essential for the functioning of a transcription factor, an evident nuclear localization signal (NLS) of <i>G. lamblia</i> MYB2 (GlMYB2) has not been defined. Based on putative GlMYB2 NLSs predicted by 2 programs, a series of plasmids expressing hemagglutinin (HA)-tagged GlMYB2 from the promoter of <i>G. lamblia</i> glutamate dehydrogenase were constructed and transfected into Giardia trophozoites. Immunofluorescence assays using anti-HA antibodies indicated that GlMYB2 amino acid sequence #507–#530 was required for the nuclear localization of GlMYB2, and this sequence was named as NLS<sub>GlMYB2</sub>. We further verified this finding by demonstrating the nuclear location of a protein obtained by the fusion of NLS<sub>GlMYB2</sub> and <i>G. lamblia</i> glyceraldehyde 3-phosphate dehydrogenase, a non-nuclear protein. Our data on GlMYB2 will expand our understanding on NLSs functioning in <i>G. lamblia.</i>

scholarly journals Evolution of natural transformation: testing the DNA repair hypothesis in Bacillus subtilis and Haemophilus influenzae.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 755-761 ◽  
Author(s):  
R J Redfield
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Bacillus Subtilis ◽  
Haemophilus Influenzae ◽  
Ultraviolet Radiation ◽  
Mitomycin C ◽  
Gene Fusion ◽  
Natural Transformation ◽  
Transformation Systems ◽  
Inducible Gene

Abstract The hypothesis that the primary function of bacterial transformation is DNA repair was tested in the naturally transformable bacteria Bacillus subtilis and Haemophilus influenzae by determining whether competence for transformation is regulated by DNA damage. Accordingly, DNA damage was induced by mitomycin C and by ultraviolet radiation at doses that efficiently induced a known damage-inducible gene fusion, and the ability of the damaged cultures to transform was monitored. Experiments were carried out both under conditions where cells do not normally become competent and under competence-inducing conditions. No induction or enhancement of competence by damage was seen in either organism. These experiments strongly suggest that the regulation of competence does not involve a response to DNA damage, and thus that explanations other than DNA repair must be sought for the evolutionary functions of natural transformation systems.

scholarly journals How Mg2+ stimulates DNA repair in prokaryotic (6-4) photolyases

2018 ◽  
Author(s):  
Hongju Ma ◽  
Daniel Holub ◽  
Natacha Gillet ◽  
Gero Kaeser ◽  
Katharina Thoulass ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Dna Repair ◽  
Amino Acid ◽  
Dna Binding ◽  
Freshwater Environment ◽  
Dna Lesion ◽  
Dna Binding Site ◽  
Positively Charged ◽  
Overlapping Region ◽  
Charge Stabilization

AbstractProkaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. In the Agrobacterium (6-4) photolyase PhrB, the repair of DNA with UV-induced (6-4) pyrimidin dimers is stimulated by Mg2+. We show that Mg2+ is required for efficient lesion binding and for charge stabilization after electron transfer from the FADH- chromophore to the DNA lesion. Two highly conserved Asp residues close to the DNA binding site are essential for the Mg2+ effect. Simulations showed that two Mg2+ bind to the region around these residues. DNA repair by eukaryotic (6-4) photolyases is not increased by Mg2+. Here, the structurally overlapping region contains no Asp but positively charged Lys or Arg. During evolution, charge stabilization and DNA binding by Mg2+ was therefore replaced by a positive amino acid. We argue that this transition has evolved in a freshwater environment. Prokaryotic (6-4) photolyases usually contain an FeS cluster. DNA repair of a cyanobacterial member of this group which is missing the FeS cluster was also found to be stimulated by Mg2+.

scholarly journals RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity

2021 ◽  
Author(s):  
Alex L. Payne-Dwyer ◽  
Aisha H. Syeda ◽  
Jack W. Shepherd ◽  
Lewis Frame ◽  
Mark. C. Leake
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Dna Repair ◽  
Mitomycin C ◽  
Single Molecule ◽  
Reca Protein ◽  
Live Cells ◽  
Dna Breaks ◽  
Fluorescent Reporters ◽  
Double Stranded Dna

AbstractThe RecA protein and RecBCD complex are key bacterial components for the maintenance and repair of DNA, RecBCD a helicase-nuclease that uses homologous recombination to resolve double-stranded DNA breaks and also facilitating decoration of single-stranded DNA with RecA to form RecA filaments, a vital step in the double-stranded break DNA repair pathway. However, questions remain about the mechanistic roles of RecA and RecBCD in live cells. Here, we use millisecond super-resolved fluorescence microscopy to pinpoint the spatial localization of fluorescent reporters of RecA and the RecB at physiological levels of expression in individual live Escherichia coli cells. By introducing the DNA crosslinker mitomycin C, we induce DNA damage and quantify the resulting changes in stoichiometry, copy number and molecular mobilities of RecA and RecB. We find that both proteins accumulate in molecular hotspots to effect repair, resulting in RecA filamental stoichiometries equivalent to several hundred molecules that act largely in RecA tetramers before DNA damage, but switch to approximately hexameric subunits when mature filaments are formed. Unexpectedly, we find that the physiologically predominant form of RecB is a dimer.

scholarly journals Antiviral activity of a purine synthesis enzyme reveals a key role of deamidation in regulating protein nuclear import

2019 ◽  
Vol 5 (10) ◽  
pp. eaaw7373 ◽  
Author(s):  
Junhua Li ◽  
Jun Zhao ◽  
Simin Xu ◽  
Shu Zhang ◽  
Junjie Zhang ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Lytic Replication ◽  
Metabolic Enzyme ◽  
Purine Synthesis ◽  
Localization Signal ◽  
Positively Charged

Protein nuclear translocation is highly regulated and crucial for diverse biological processes. However, our understanding concerning protein nuclear import is incomplete. Here we report that a cellular purine synthesis enzyme inhibits protein nuclear import via deamidation. Employing human Kaposi’s sarcoma-associated herpesvirus (KSHV) to probe the role of protein deamidation, we identified a purine synthesis enzyme, phosphoribosylformylglycinamidine synthetase (PFAS) that inhibits KSHV transcriptional activation. PFAS deamidates the replication transactivator (RTA), a transcription factor crucial for KSHV lytic replication. Mechanistically, deamidation of two asparagines flanking a positively charged nuclear localization signal impaired the binding of RTA to an importin β subunit, thus diminishing RTA nuclear localization and transcriptional activation. Finally, RTA proteins of all gamma herpesviruses appear to be regulated by PFAS-mediated deamidation. These findings uncover an unexpected function of a metabolic enzyme in restricting viral replication and a key role of deamidation in regulating protein nuclear import.

scholarly journals Karyopherin α-2 Mediates MDC1 Nuclear Import through a Functional Nuclear Localization Signal in the tBRCT Domain of MDC1

2020 ◽  
Vol 21 (7) ◽  
pp. 2650
Author(s):  
Kamalakannan Radhakrishnan ◽  
Seon-Joo Park ◽  
Seok Won Kim ◽  
Gurusamy Hariharasudhan ◽  
Seo-Yeon Jeong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Ring Finger ◽  
Nucleocytoplasmic Transport ◽  
Ring Finger Protein ◽  
Finger Protein ◽  
Localization Signal ◽  
Functional Nuclear Localization Signal

Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in DNA damage response (DDR) by coordinating the repair of double strand breaks (DSBs). Here, we identified a novel interaction between MDC1 and karyopherin α-2 (KPNA2), a nucleocytoplasmic transport adaptor, and showed that KPNA2 is necessary for MDC1 nuclear import. Thereafter, we identified a functional nuclear localization signal (NLS) between amino acid residues 1989–1994 of the two Breast Cancer 1 (BRCA1) carboxyl-terminal (tBRCT) domain of MDC1 and demonstrated disruption of this NLS impaired interaction between MDC1 and KPNA2 and reduced nuclear localization of MDC1. In KPNA2-depleted cells, the recruitment of MDC1, along with the downstream signaling p roteins Ring Finger Protein 8 (RNF8), 53BP1-binding protein 1 (53BP1), BRCA1, and Ring Finger Protein 168 (RNF168), to DNA damage sites was abolished. Additionally, KPNA2-depleted cells had a decreased rate of homologous recombination (HR) repair. Our data suggest that KPNA2-mediated MDC1 nuclear import is important for DDR signaling and DSB repair.

scholarly journals Actin binding to WH2 domains regulates nuclear import of the multifunctional actin regulator JMY

2012 ◽  
Vol 23 (5) ◽  
pp. 853-863 ◽  
Author(s):  
J. Bradley Zuchero ◽  
Brittany Belin ◽  
R. Dyche Mullins
Keyword(s):  
Dna Damage ◽  
Actin Filament ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Actin Polymerization ◽  
Regulatory Protein ◽  
Actin Binding ◽  
Nuclear Localization Sequence ◽  
Filament Assembly ◽  
Localization Sequence

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. In response to DNA damage, JMY accumulates in the nucleus and promotes p53-dependent apoptosis. JMY's actin-regulatory activity relies on a cluster of three actin-binding Wiskott–Aldrich syndrome protein homology 2 (WH2) domains that nucleate filaments directly and also promote nucleation activity of the Arp2/3 complex. In addition to these activities, we find that the WH2 cluster overlaps an atypical, bipartite nuclear localization sequence (NLS) and controls JMY's subcellular localization. Actin monomers bound to the WH2 domains block binding of importins to the NLS and prevent nuclear import of JMY. Mutations that impair actin binding, or cellular perturbations that induce actin filament assembly and decrease the concentration of monomeric actin in the cytoplasm, cause JMY to accumulate in the nucleus. DNA damage induces both cytoplasmic actin polymerization and nuclear import of JMY, and we find that damage-induced nuclear localization of JMY requires both the WH2/NLS region and importin β. On the basis of our results, we propose that actin assembly regulates nuclear import of JMY in response to DNA damage.

Long-term efficacy of a new medical device containing Fernblock® and DNA repair enzyme complex in the treatment and prevention of cancerization field in patients with actinic keratosis.

2019 ◽  
Vol 2 (02) ◽  
pp. 80-89
Author(s):  
Blanca De Unamuno Bustos ◽  
Natalia Chaparr´´o Aguilera ◽  
Inmaculada Azorín García ◽  
Anaid Calle Andrino ◽  
Margarita Llavador Ros ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Medical Device ◽  
Actinic Keratosis ◽  
Statistical Significance ◽  
Dna Lesions ◽  
Enzyme Complex ◽  
Repair Enzyme ◽  
P53 Expression ◽  
Cancerization Field

Actinic keratosis (AKs) are part of the cancerization field, a region adjacent to AKs containing subclinical and histologically abnormal epidermal tissue due to Ultraviolet (UV)-induced DNA damage. The photoproducts as consequence of DNA damage induced by UV are mainly cyclobutane pyrimidine dimers (CPDs). Fernblock® demonstrated in previous studies significant reduction of the number of CPDs induced by UV radiation. Photolyases are a specific group of enzymes that remove the major UV-induced DNA lesions by a mechanism called photo-reactivation. A monocentric, prospective, controlled, and double blind interventional study was performed to evaluate the effect of a new medical device (NMD) containing a DNA-repair enzyme complex (photolyases, endonucleases and glycosilases), a combination of UV-filters, and Fernblock® in the treatment of the cancerization field in 30 AK patients after photodynamic therapy. Patients were randomized into two groups: patients receiving a standard sunscreen (SS) andpatients receiving the NMD. Clinical, dermoscopic, reflectance confocal microscopy (RCM) and histological evaluations were performed. An increase of AKs was noted in all groups after three months of PDT without significant differences between them (p=0.476). A significant increase in the number of AKs was observed in SS group after six (p=0.026) and twelve months of PDT (p=0.038); however, this increase did not reach statistical significance in the NMD group. Regarding RCM evaluation, honeycomb pattern assessment after twelve months of PDT showed significant differences in the extension and grade of the atypia in the NMD group compared to SS group (p=0.030 and p=0.026, respectively). Concerning histopathological evaluation, keratinocyte atypia grade improved from baseline to six months after PDT in all the groups, with no statistically significant differences between the groups. Twelve months after PDT, p53 expression was significantly lower in the NMD group compared to SS group (p=0.028). The product was well-tolerated, with no serious adverse events reported. Our results provide evidence of the utility of this NMD in the improvement of the cancerization field and in the prevention of the development of new AKs.  

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