scholarly journals The Single-Strand DNA Binding Activity of Human PC4 Prevents Mutagenesis and Killing by Oxidative DNA Damage

2004 ◽  
Vol 24 (13) ◽  
pp. 6084-6093 ◽  
Author(s):  
Jen-Yeu Wang ◽  
Altaf Hossain Sarker ◽  
Priscilla K. Cooper ◽  
Michael R. Volkert
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Binding Activity ◽  
Single Strand ◽  
Physical Interaction ◽  
Ssdna Binding ◽  
Dna Binding Activity ◽  
Resistance Pathway ◽  
Domain Of Unknown Function ◽  
Human Proteins

ABSTRACT Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Saccharomyces cerevisiae mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide-induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub1Δ mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show that XPG recruits PC4 to a bubble-containing DNA substrate with a resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

scholarly journals Cu,Zn-superoxide dismutase deficiency in mice leads to organ-specific increase in oxidatively damaged DNA and NF-κB1 protein activity.

2010 ◽  
Vol 57 (4) ◽  
Author(s):  
Agnieszka Siomek ◽  
Kamil Brzoska ◽  
Barbara Sochanowicz ◽  
Daniel Gackowski ◽  
Rafal Rozalski ◽  
...  
Keyword(s):  
Dna Damage ◽  
Superoxide Dismutase ◽  
Dna Binding ◽  
Urinary Excretion ◽  
Oxidative Dna Damage ◽  
Binding Activity ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Organ Specific ◽  
Damaged Dna

Earlier experimental studies have demonstrated that: i) Cu,Zn-superoxide dismutase deficiency leads to oxidative stress and carcinogenesis; ii) dysregulation of NF-κB pathway can mediate a wide variety of diseases, including cancer. Therefore, we decided, for the first time, to examine the level of oxidative DNA damage and the DNA binding activity of NF-κB proteins in SOD1 knockout, heterozygous and wild-type mice. Two kinds of biomarkers of oxidatively damaged DNA: urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA were analysed using HPLC-GC-MS and HPLC-EC. The DNA binding activity of p50 and p65 proteins in a nuclear extracts was assessed using NF-κB p50/p65 EZ-TFA transcription factor assay. These parameters were determined in the brain, liver, kidney and urine of SOD1 knockout, heterozygous and wild-type mice. The level of 8-oxodG in DNA was higher in the liver and kidney of knockout mice than in wild type. No differences were found in urinary excretion of 8-oxoGua and 8-oxodG between wild type and the SOD1-deficient animals. The activity of the p50 protein was higher in the kidneys, but surprisingly not in the livers of SOD1-deficient mice, whereas p65 activity did not show any variability. Our results indicate that in Cu,Zn-SOD-deficient animals the level of oxidative DNA damage and NF-κB1 activity are elevated in certain organs only, which may provide some explanation for organ-specific ROS-induced carcinogenesis.

Aberrant Non-Homologous End Joining in Multiple Myeloma: A Role in Genomic Instability and As Potential Prognostic Marker.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2932-2932 ◽  
Author(s):  
Teresa Calimeri ◽  
Mariateresa Fulciniti ◽  
Jianhong Lin ◽  
Mehmet Kemal Samur ◽  
Anne S. Calkins ◽  
...  
Keyword(s):  
Dna Damage ◽  
Board Of Directors ◽  
Genomic Instability ◽  
Cell Lines ◽  
Binding Activity ◽  
End Joining ◽  
Advisory Committees ◽  
Nhej Pathway ◽  
Dna Binding Activity ◽  
Nuclear Extracts

Abstract Abstract 2932 Multiple Myeloma (MM) is a hematologic malignancy characterized by a complex combination of structural and numerical chromosomal abnormalities. However, the underlying molecular basis of the genomic instability remains largely unknown. The ability to repair DNA damages, especially double-strand breaks (DSBs), is essential to suppress genetic instability. Non-homologous end joining (NHEJ) is one of the most important mechanisms responsible for repair of these breaks. Since both impaired and aberrant NHEJ seem to be linked to genomic instability in solid as well as other hematologic tumors, we have investigated its altered function in MM. To confirm involvement of an aberrant NHEJ pathway in MM genomic instability, we measured the end joining (EJ) capacity of 6 different MM cell lines using a plasmid based assay containing both the test gene (Luciferase - LUC) measuring end joining as well as a reporter gene (Alkaline Phosphatase - SEAP) to control for transfection efficiency. MM and normal control cells were transfected with this plasmid and the LUC and SEAP activity was detected directly in the supernatant of the cells at 24 h. Increased EJ activity was observed in all the MM cell lines tested compared to peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs) from healthy donor. To confirm the role of the NHEJ pathway in this increased DNA EJ activity, nuclear extracts from 9 different MM cell lines were used to determine the DNA-binding-activity of Ku86, a key protein of this repair mechanism involved in the recognition of the broken DNA ends and in the initiation of the DSBs repair process. As in EJ activity, all the MM cell lines showed an increased Ku86 binding respect to normal cells confirming the aberrant activation of the NHEJ pathway in MM cells. Interestingly, we did not observe significant differences in Ku86 level in nuclear extracts between PBMCs and MM cell lines suggesting that the difference in the Ku86 DNA-binding-activity was likely a functional and not due to disparity in the protein levels. We further investigated the link between this aberrant NHEJ activity and MM genomic instability using an immune-fluorescent based assay for DSBs. We observed an increased constitutive DNA damage in the absence of treatment with DSB-inducing agents in 5 of 6 MM cell lines compared with normal PBMCs. Most importantly, we noticed a direct correlation between the basal level of DSBs and the Ku86-binding-affinity. Furthermore, all the MM cell lines showed little or no ability to repair ionizing radiation (IR)-induced DNA damage compared to normal cells as well as no change in the Ku86-binding-affinity after stimulation suggesting that the aberrant NHEJ pathway in MM might represent a response to the constitutive endogenous DNA damage in these cells. We have also observed that 2 key NHEJ genes (Ku86 and Artemis) are overexpressed in MM compared to MGUS and normal plasma cells and their overexpression correlates with a shortened overall survival in MM suggesting that an hyper-activation of this pathway could have a potential role in MM progression and prognosis. Ongoing experiments are assessing the NHEJ activity in primary MM cells to correlate with clinical outcome. In conclusion, our data suggests that an aberrant NHEJ in the context of a constitutive endogenous DNA damage might contribute to the high frequency of chromosome abnormalities in MM cells, thus potentially playing a central role in the tumor progression and as an important prognostic marker in this disease. Disclosures: Anderson: Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

scholarly journals umuDC-Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD2C Complex Involved in a DNA Damage Checkpoint Control

2001 ◽  
Vol 183 (4) ◽  
pp. 1215-1224 ◽  
Author(s):  
Mark D. Sutton ◽  
Graham C. Walker
Keyword(s):  
Dna Damage ◽  
Polymerase Activity ◽  
Binding Activity ◽  
Cell Cycle Checkpoint ◽  
Cold Sensitivity ◽  
Gene Products ◽  
Checkpoint Control ◽  
Sos Mutagenesis ◽  
Dna Binding Activity ◽  
C Complex

ABSTRACT The umuDC genes are part of the Escherichia coli SOS response, and their expression is induced as a consequence of DNA damage. After induction, they help to promote cell survival via two temporally separate pathways. First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD′2C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA. Furthermore, elevated expression of the umuDC gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis. Here, using two mutant umuC alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (umuC104; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (umuC125; A39V), we show thatumuDC-mediated cold sensitivity can be genetically separated from the role of UmuD′2C in SOS mutagenesis. Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate thatumuDC-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC. Taken together, our analyses suggest that umuDC-mediated cold sensitivity is conferred by an activity of the UmuD2C complex and not by the separate actions of the UmuD and UmuC proteins. Finally, we present evidence for structural differences between UmuD and UmuD′ in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of theumuDC gene products.

scholarly journals APE2 Zf-GRF facilitates 3′-5′ resection of DNA damage following oxidative stress

2016 ◽  
Vol 114 (2) ◽  
pp. 304-309 ◽  
Author(s):  
Bret D. Wallace ◽  
Zachary Berman ◽  
Geoffrey A. Mueller ◽  
Yunfeng Lin ◽  
Timothy Chang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Nucleic Acid ◽  
Oxidative Dna Damage ◽  
Strand Break ◽  
Binding Activity ◽  
Nucleic Acid Binding ◽  
Catalytic Core ◽  
Single Strand Break ◽  
X Ray

The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3′-5′ nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structure and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3′-5′ exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.

scholarly journals Adenovirus E1B 55-Kilodalton Oncoprotein Inhibits p53 Acetylation by PCAF

2000 ◽  
Vol 20 (15) ◽  
pp. 5540-5553 ◽  
Author(s):  
Yue Liu ◽  
April L. Colosimo ◽  
Xiang-Jiao Yang ◽  
Daiqing Liao
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Target Genes ◽  
Binding Activity ◽  
Physical Interaction ◽  
Dna Binding Activity ◽  
C Terminus ◽  
Adenovirus E1b

ABSTRACT The adenovirus E1B 55-kDa protein binds to cellular tumor suppressor p53 and inactivates its transcriptional transactivation function. p53 transactivation activity is dependent upon its ability to bind to specific DNA sequences near the promoters of its target genes. It was shown recently that p53 is acetylated by transcriptional coactivators p300, CREB bidning protein (CBP), and PCAF and that acetylation of p53 by these proteins enhances p53 sequence-specific DNA binding. Here we show that the E1B 55-kDa protein specifically inhibits p53 acetylation by PCAF in vivo and in vitro, while acetylation of histones and PCAF autoacetylation is not affected. Furthermore, the DNA-binding activity of p53 is diminished in cells expressing the E1B 55-kDa protein. PCAF binds to the E1B 55-kDa protein and to a region near the C terminus of p53 encompassing Lys-320, the specific PCAF acetylation site. We further show that the E1B 55-kDa protein interferes with the physical interaction between PCAF and p53, suggesting that the E1B 55-kDa protein inhibits PCAF acetylase function on p53 by preventing enzyme-substrate interaction. These results underscore the importance of p53 acetylation for its function and suggest that inhibition of p53 acetylation by viral oncoproteins prevent its activation, thereby contributing to viral transformation.

scholarly journals DNA Damage Induces MDMX Nuclear Translocation by p53-Dependent and -Independent Mechanisms

2002 ◽  
Vol 22 (21) ◽  
pp. 7562-7571 ◽  
Author(s):  
Changgong Li ◽  
Lihong Chen ◽  
Jiandong Chen
Keyword(s):  
Dna Damage ◽  
Stress Response ◽  
Embryonic Development ◽  
Nuclear Translocation ◽  
Binding Activity ◽  
Stable Transfection ◽  
Reading Frame ◽  
Dna Binding Activity ◽  
Mdm2 Expression ◽  
Important Regulator

ABSTRACT The MDM2 homolog MDMX is an important regulator of p53 activity during embryonic development. MDMX inactivation in mice results in embryonic lethality in a p53-dependent fashion. The expression level of MDMX is not induced by DNA damage, and its role in stress response is unclear. We show here that ectopically expressed MDMX is mainly localized in the cytoplasm. DNA damage promotes nuclear translocation of MDMX in cells with or without p53. Coexpression of MDM2 or p53 is sufficient to induce MDMX nuclear translocation, suggesting that activation of p53 and induction of MDM2 expression can contribute to this process. Stable transfection of MDMX into U2OS cells does not alter p53 level but results in reduced p53 DNA-binding activity and reduced MDM2 expression. The ability of ARF (alternate reading frame of INK4a) to activate p53 is also significantly inhibited by expression of MDMX. These results suggest that MDMX function may be regulated by DNA damage. Furthermore, MDMX may complement MDM2 in regulating p53 during embryonic development due to its ability to inhibit p53 in the presence of ARF.

scholarly journals The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex.

1994 ◽  
Vol 14 (2) ◽  
pp. 1333-1346 ◽  
Author(s):  
S Obert ◽  
R J O'Connor ◽  
S Schmid ◽  
P Hearing
Keyword(s):  
Binding Activity ◽  
Mutant Protein ◽  
Stable Complex ◽  
Physical Interaction ◽  
Dimerization Domain ◽  
Fab Fragment ◽  
Dna Binding Activity ◽  
Transcription Factor E2f

Binding of the mammalian transcription factor E2F to the adenovirus E2a early promoter is modulated through interaction with the viral E4-6/7 protein. E4-6/7 induces the cooperative and stable binding of E2F in vitro to two correctly spaced and inverted E2F binding sites in the E2a promoter (E2F induction) by physical interaction in the protein-DNA complex. The E2a promoter is transactivated in vivo by the E4-6/7 product. The C-terminal 70 amino acids of E4-6/7 are necessary and sufficient for induction of E2F binding and for transactivation. To assess the mechanism(s) of E2a transactivation and the induction of cooperative E2F binding by the E4-6/7 protein, we have analyzed a series of point mutants in the functional C-terminal domain of E4-6/7. Two distinct segments of E4-6/7 are required for interaction with E2F. Additionally, and E4-6/7 mutant with a phenylalanine-to-proline substitution at amino acid 125 (F-125-P) efficiently interacts with E2F but does not induce E2F binding to the E2a promoter and is defective for transactivation. Induction of E2F stable complex formation at the E2a promoter by the F-125-P mutant protein is restored by divalent E4-6/7-specific monoclonal antibodies, but not a monovalent Fab fragment, or by appending a heterologous dimerization domain to the N terminus of the mutant protein. These and other data support the involvement of E4-6/7 dimerization in the induction of cooperative and stable E2F binding and transactivation of the E2a promoter. We present evidence that at least two cellular components are involved in E2F DNA binding activity and that both are required for E2F induction by the E4-6/7 product. The recently cloned E2F-related activities E2F-1 and DP-1 individually bind to an E2F binding site weakly, but when combined generate an activity that is indistinguishable from endogenous cellular E2F. Recombinant E2F-1, DP-1, and E4-6/7 are sufficient to form the induced E2F complex at the E2a promoter.

scholarly journals Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Thomas J Etheridge ◽  
Desiree Villahermosa ◽  
Eduard Campillo-Funollet ◽  
Alex David Herbert ◽  
Anja Irmisch ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Chromosomal Rearrangements ◽  
Binding Activity ◽  
Single Molecule Tracking ◽  
Ssdna Binding ◽  
Dna Binding Activity ◽  
Gross Chromosomal Rearrangements ◽  
Genes Encoding

The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits, we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

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