scholarly journals UAF1 DNA Binding Activity Is Critical for RAD51-Mediated Homologous DNA Pairing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2497-2497
Author(s):  
Fengshan Liang ◽  
Adam S Miller ◽  
Carolilne Tang ◽  
Patrick Sung ◽  
Gary M. Kupfer
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Activity ◽  
Loop Formation ◽  
Dna Binding Activity ◽  
D Loop ◽  
In Cells ◽  
Dna Pairing

Background: In the Fanconi anemia (FA) DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer by the FA core complex through its inherent E3 ligase activity. The timely deubiquitination of ID2 by USP1-UAF1 deubiquitinase complex is also critically important for the FA DNA repair. UAF1 has a DNA binding activity, which is required for FANCD2 deubiquitination. UAF1 also enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1. UAF1 deficient cells are impaired for DNA repair by homologous recombination (HR).The biochemical and cellular functions of UAF1 DNA binding activity in HR remain elusive. Methods:UAF1 wild type and DNA binding mutant proteins were purified and used to define its biochemical properties in HR. In vitroD-loop formation and synaptic complex assembly assay were performed to discover the DNA binding of UAF1 in RAD51 recombinase enhancement. U2OS-DR-GFP cell lines with impaired UAF1 or RAD51AP1DNA binding were generated to examine HR efficiency and DNA damage resistance. Results:UAF1 preferentially binds an HR-intermediate-like DNA substrate (D-loop, Fig.1). The DNA binding deficient mutant of UAF1 is unable to stimulate RAD51AP1 promotion of RAD51-mediated D-loop (Fig. 2) and the ability to recruit homologous DNA to form the presynaptic complex formation in HR (Fig. 3). In cells, the UAF1 DNA-binding mutant is compromised for the ability to repair DNA damage and to implement HR (Fig. 4). Such activity correlates with the ability to confer resistance to DNA cross linking agents such as mitomycin C (Fig. 4). The DNA binding of UAF1 and RAD51AP1 have a coordinated role in HR-directed DNA damage repair (Fig. 5). Conclusions: UAF1 DNA binding activity is indispensable for its function in enhancing RAD51-mediated homologous DNA pairing within the context of the UAF1-RAD51AP1 complex. UAF1 DNA binding deficiency causes DNA damage sensitivity and impairs HR efficiency in cells. Translational Applicability:Our findings reveal a critical role of UAF1 DNA binding in DNA repair and genome maintenance. The identification of UAF1's role in repair will enable targeted efforts to improve molecular approaches for FA therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of UAF1 in the Fanconi Anemia Pathway Regulation of Homologous Recombination-Mediated Genome Maintenance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1041-1041
Author(s):  
Fengshan Liang ◽  
Simonne Longerich ◽  
Caroline Tang ◽  
Olga Buzovestsky ◽  
Yong Xiong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Binding ◽  
Homologous Recombination ◽  
Complex Formation ◽  
Binding Activity ◽  
Genome Maintenance ◽  
Synaptic Complex ◽  
Dna Binding Activity ◽  
D Loop

Abstract Background: Fanconi anemia (FA), a cancer-prone genetic disease, is caused by defects in the FA-DNA repair pathway. In response to DNA interstrand crosslink (ICL)-induced DNA damage, FANCI-FANCD2 mono-ubiquitination licenses the execution of downstream DNA damage signaling and repair steps, including repair by homologous recombination (HR) that utilizes the recombinase RAD51 and its cohort of accessory factors. Timely deubiquitination of FANCD2 by the UAF1-USP1 deubiquitinating enzyme complex is also critically important for the FA pathway. As such, UAF1 depletion results in persistent FANCD2 ubiquitination and DNA damage hypersensitivity. UAF1 deficient cells are also impaired for DNA repair by homologous recombination. UAF1 physically associates with RAD51AP1, a protein that enhances the activity of the RAD51 recombinase. It remains to be defined how UAF1 regulates homologous recombination and genome stability. Methods: Highly purified proteins were used to define the DNA binding activity and protein interaction of UAF1. In vitroD-loop formation reaction and synaptic complex assembly assay were used to discover the function of UAF1 in RAD51 recombinase enhancement. HeLa and U2OS-DR-GFP cell lines with impaired UAF1-RAD51AP1 interaction or UAF1 DNA binding were generated to examine DNA-damage agent sensitivity and HR efficiency. Results: (1) UAF1 possesses a DNA binding activity capable of engaging ssDNA, dsDNA and has a preference for the D-loop DNA substrate. We further identified that the N-terminus but not C-terminal SLD domain of UAF1 binds DNA. (2) UAF1 forms a dimeric complex with RAD51AP1. Our results also revealed a trimeric complex of RAD51-RAD51AP1-UAF1, with RAD51AP1 providing a tethering function between the other two proteins. (3) The RAD51AP1-UAF1 interaction interface was defined showing a novel SIM motif in the middle portion of RAD51AP1and the SLD1-SLD2 domain of UAF1 mediate protein complex formation. Based on the domain mapping results, point mutants of RAD51AP1 and UAF1 that are specifically compromised for the formation of the RAD51AP1-UAF1 complex were generated. (4) UAF1 synergizes with RAD51AP1 in the RAD51-mediated D-loop reaction and that this functional synergy requires the RAD51AP1-UAF1 complex and also the DNA and RAD51 binding attributes of RAD51AP1. (5) RAD51AP1-UAF1 works in conjunction with the RAD51 presynaptic filament in the capture of the duplex DNA partner and in the assembly of the synaptic complex. (6) Human cell lines impaired for RAD51AP1-UAF1 complex formation are compromised for the ability to repair DNA damage and to execute HR. (7) DNA repair function of the RAD51AP1-UAF1 complex is likely USP1-independent. Conclusions: The physical interaction between UAF1 and RAD51AP1 is indispensable for functional synergy in vitro and, accordingly, for the biological function of UAF1 in HR and DNA damage repair. Our findings provide insights into a novel USP1-independent regulatory mechanism of UAF1 on homologous recombination-mediated genome maintenance. Disclosures No relevant conflicts of interest to declare.

scholarly journals The DNA-binding activity of USP1-associated factor 1 is required for efficient RAD51-mediated homologous DNA pairing and homology-directed DNA repair

2020 ◽  
Vol 295 (24) ◽  
pp. 8186-8194 ◽  
Author(s):  
Fengshan Liang ◽  
Adam S. Miller ◽  
Caroline Tang ◽  
David Maranon ◽  
Elizabeth A. Williamson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Binding ◽  
Fanconi Anemia ◽  
Complementation Group ◽  
Binding Activity ◽  
Associated Factor ◽  
Dna Binding Activity ◽  
Damage Repair ◽  
Dna Pairing

USP1-associated factor 1 (UAF1) is an integral component of the RAD51-associated protein 1 (RAD51AP1)–UAF1-ubiquitin-specific peptidase 1 (USP1) trimeric deubiquitinase complex. This complex acts on DNA-bound, monoubiquitinated Fanconi anemia complementation group D2 (FANCD2) protein in the Fanconi anemia pathway of the DNA damage response. Moreover, RAD51AP1 and UAF1 cooperate to enhance homologous DNA pairing mediated by the recombinase RAD51 in DNA repair via the homologous recombination (HR) pathway. However, whereas the DNA-binding activity of RAD51AP1 has been shown to be important for RAD51-mediated homologous DNA pairing and HR-mediated DNA repair, the role of DNA binding by UAF1 in these processes is unclear. We have isolated mutant UAF1 variants that are impaired in DNA binding and tested them together with RAD51AP1 in RAD51-mediated HR. This biochemical analysis revealed that the DNA-binding activity of UAF1 is indispensable for enhanced RAD51 recombinase activity within the context of the UAF1–RAD51AP1 complex. In cells, DNA-binding deficiency of UAF1 increased DNA damage sensitivity and impaired HR efficiency, suggesting that UAF1 and RAD51AP1 have coordinated roles in DNA binding during HR and DNA damage repair. Our findings show that even though UAF1's DNA-binding activity is redundant with that of RAD51AP1 in FANCD2 deubiquitination, it is required for efficient HR-mediated chromosome damage repair.

Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme.

1992 ◽  
Vol 11 (9) ◽  
pp. 3323-3335 ◽  
Author(s):  
S. Xanthoudakis ◽  
G. Miao ◽  
F. Wang ◽  
Y.C. Pan ◽  
T. Curran
Keyword(s):  
Dna Repair ◽  
Dna Binding ◽  
Binding Activity ◽  
Repair Enzyme ◽  
Dna Binding Activity ◽  
Redox Activation

Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein

Science ◽  
1995 ◽  
Vol 269 (5220) ◽  
pp. 81-83 ◽  
Author(s):  
C. Yu ◽  
D. Meyer ◽  
G. Campbell ◽  
A. Larner ◽  
C Carter-Su ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Related Protein ◽  
Dna Binding Activity ◽  
In Cells

Nicotinamide Increases the Megakaryocytic Maturation of Human Hematopoietic Stem Cells Primarily Due to SIRT Inhibition

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2445-2445 ◽  
Author(s):  
Swapna Panuganti ◽  
Lisa M. Giammona ◽  
Jan M. Kemper ◽  
Pani Apostolidis ◽  
Stephan Lindsey ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dna Binding ◽  
De Novo ◽  
Binding Activity ◽  
Salvage Pathway ◽  
Hematopoietic Stem ◽  
Dna Binding Activity ◽  
Cd34 Cells ◽  
High Ploidy ◽  
In Cells

Abstract Introduction: Megakaryocytic cells (Mks), the precursors to platelets, are among the least understood blood cell types. A primary aspect of Mk differentiation is endomitosis, whereby Mks duplicate their DNA content without undergoing cytokinesis and form cells with 4N, 8N, 16N, etc. Mk ploidy strongly correlates with platelet production. Thrombocytopenia accompanies several hematologic malignancies including myelodysplastic syndromes and is often associated with low in vivo Mk ploidy. Elucidation of the factors that regulate Mk endomitosis will aid in developing treatments for Mk-related disorders. We have previously shown that the B3 vitamin nicotinamide (NIC) causes a dose-dependent increase in Mk size and the fraction of high-ploidy (≥ 8N) Mks and leads to more complex proplatelet formation without affecting Mk commitment, ultrastructure, apoptosis, or viability in cultures of CD34+ cells (Giammona LM, et al. Br J Haem 135 (2006): 554). We examined whether NIC’s roles as an inhibitor of the sirtuin family of histone/protein deacetylases (SIRTs) and as a precursor for NAD+ were responsible for its effects on Mk ploidy. Methods: CD34+ cells, isolated from healthy G-CSF-mobilized peripheral blood donors, were maintained in serum-free X-VIVO 20 media supplemented with 100 ng/mL thrombopoietin (Tpo). On day 5, cells were treated with 6.25 mM NIC, 10 μM cambinol (SIRT1/2 inhibitor), or 10 μM AGK2 (SIRT2 inhibitor) or maintained with Tpo alone. Flow cytometry was used to determine Mk commitment (CD41+), viability, apoptosis, ploidy, and intracellular levels of total and acetylated p53. The intracellular concentration of NAD(H) (NAD+ plus NADH) was determined using an enzymatic assay. Immunoblots were used to detect acetylated and total nucleosomes, as well as the NAD processing enzyme Nmnat1. p53 DNA-binding activity was determined using EMSA analysis. Results: Adding NIC to CD34+ cell cultures increased the percentage of high-ploidy Mks by 3-fold. The SIRT1/2 inhibitor cambinol increased Mk ploidy to a similar extent as NIC, while the SIRT2 inhibitor AGK2 was only 30% as effective. NIC and cambinol more than tripled the fractions of 16N and 32N Mks (Figure). None of the additives affected Mk commitment, viability, or apoptosis. Functional inhibition of SIRT1/2 by NIC was confirmed by increased acetylation of several SIRT1/2 target proteins. Both SIRTs deacetylate histones and we observed up to 3-fold greater nucleosome acetylation in cells treated with NIC. Flow cytometry showed that the ratio of AcK382p53 to total p53 was 3-fold higher in cells treated with NIC as compared to Tpo alone. Consistent with reports that acetylation increases p53 DNA-binding activity, EMSA analysis showed that p53 binding to the p53 consensus sequence was 50% greater in NIC-treated Mks. We have previously shown that p53 knockdown increases Mk ploidy in culture (Fuhrken PG, et al. J Biol Chem 283 (2008): 15589). These results suggest that increased p53 acetylation differentially affects different p53 target genes. NIC increased intracellular levels of NAD(H) by 5-fold. In contrast, an NAD+de novo pathway precursor had minimal impact on ploidy. NIC is incorporated into NAD+ via the salvage pathway, which is localized to the nucleus in yeast, whereas the de novo pathway is distributed throughout the cell. This suggests that NAD+ production in the nucleus may also play a role in NIC-mediated increases in Mk ploidy, and is consistent with higher nuclear levels of the NAD+ salvage pathway enzyme Nmnat1 detected in cells treated with NIC. Conclusions: Inhibition of SIRT1 and SIRT2 appears to be the primary mechanism for NIC-mediated increases in Mk ploidy, and increased p53 acetylation is likely to play an important role in this process. Further study of SIRT targets associated with DNA repair, apoptosis, and cell cycle regulation may provide additional insight into Mk polyploidization. Figure Figure

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.

scholarly journals Evidence that complex formation by Bas1p and Bas2p (Pho2p) unmasks the activation function of Bas1p in an adenine-repressible step of ADE gene transcription.

1997 ◽  
Vol 17 (6) ◽  
pp. 3272-3283 ◽  
Author(s):  
F Zhang ◽  
M Kirouac ◽  
N Zhu ◽  
A G Hinnebusch ◽  
R J Rolfes
Keyword(s):  
Dna Binding ◽  
Complex Formation ◽  
Activation Function ◽  
Binding Activity ◽  
Activation Functions ◽  
Lacz Reporter ◽  
Activator Proteins ◽  
Dna Binding Activity ◽  
Negative Effect ◽  
High Level

Bas1p and Bas2p (Pho2p) are Myb-related and homeodomain DNA binding proteins, respectively, required for transcription of adenine biosynthetic genes in Saccharomyces cerevisiae. The repression of ADE genes in adenine-replete cells involves down-regulation of the functions of one or both of these activator proteins. A LexA-Bas2p fusion protein was found to activate transcription from a lexAop-lacZ reporter independently of both BAS1 function and the adenine levels in the medium. In contrast, a LexA-Bas1p fusion activated the lexAop reporter in a BAS2-dependent and adenine-regulated fashion. The DNA binding activity of Bas2p was not needed for its ability to support activation of the lexAop reporter by LexA-Bas1p, indicating that LexA-Bas1p recruits Bas2p to this promoter. The activation functions of both authentic Bas1p and LexA-Bas1p were stimulated under adenine-repressing conditions by overexpression of Bas2p, suggesting that complex formation by these proteins is inhibited in adenine-replete cells. Replacement of Asp-617 with Asn in Bas1p or LexA-Bas1p allowed either protein to activate transcription under repressing conditions in a manner fully dependent on Bas2p, suggesting that this mutation reduces the negative effect of adenine on complex formation by Bas1p and Bas2p. Deletions of N-terminal and C-terminal segments from the Bas1p moiety of LexA-Bas1p allowed high-level activation by the truncated proteins independently of Bas2p and adenine levels in the medium. From these results we propose that complex formation between Bas1p and Bas2p unmasks a latent activation function in Bas1p as a critical adenine-regulated step in transcription of the ADE genes.

scholarly journals Accumulation of manganese superoxide dismutase under metal-depleted conditions: proposed role for zinc ions in cellular redox balance

2004 ◽  
Vol 377 (1) ◽  
pp. 241-248 ◽  
Author(s):  
Kaoru OTSU ◽  
Yoshitaka IKEDA ◽  
Junichi FUJII
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Dna Binding ◽  
Manganese Superoxide Dismutase ◽  
Binding Activity ◽  
Rat Liver Mitochondria ◽  
Zinc Ions ◽  
Dna Binding Activity ◽  
Manganese Superoxide ◽  
In Cells

A diet low in copper results in increased levels of MnSOD (manganese superoxide dismutase), a critical antioxidative enzyme conferring protection against oxidative stress, in rat liver mitochondria. The mechanism for this was investigated using cultured HepG2 cells, a human hepatocellular carcinoma-derived line. MnSOD activity increased 5–7-fold during incubation in a medium supplemented with metal-depleted fetal bovine serum, with a corresponding elevation of its mRNA levels. Metal depletion also decreased CuZnSOD and glutathione peroxidase levels to approx. 70–80% of baseline. When zinc ions were added to the medium at micromolar levels, MnSOD accumulation was suppressed; however, copper ions had essentially no effect on MnSOD expression. Since the intracellular redox status was shifted to a more oxidized state by metal depletion, we examined the DNA-binding activity of NF-κB (nuclear factor-κB), an oxidative stress-sensitive transactivating factor that plays a primary role in MnSOD induction. A gel shift assay indicated that the DNA-binding activity of NF-κB was increased in cells maintained in metal-depleted culture, suggesting the involvement of the transactivating function of NF-κB in this induction. This was further supported by the observation that curcumin suppressed both the DNA-binding activity of NF-κB and the induction of MnSOD mRNA in cells cultivated under metal-depleted conditions. These results suggest that the level of zinc, rather than copper, is a critical regulatory factor in MnSOD expression. It is possible that a deficiency of zinc in the low-copper diet may be primarily involved in MnSOD induction.

scholarly journals Structure analysis of FAAP24 reveals single-stranded DNA-binding activity and domain functions in DNA damage response

Cell Research ◽  
2013 ◽  
Vol 23 (10) ◽  
pp. 1215-1228 ◽  
Author(s):  
Yucai Wang ◽  
Xiao Han ◽  
Fangming Wu ◽  
Justin W Leung ◽  
Megan G Lowery ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Structure Analysis ◽  
Dna Damage Response ◽  
Binding Activity ◽  
Single Stranded Dna ◽  
Dna Binding Activity ◽  
Damage Response
Sign in / Sign up

Export Citation Format

Share Document