scholarly journals Effects of Conserved Wedge Domain Residues on DNA Binding Activity of Deinococcus radiodurans RecG Helicase

2021 ◽  
Vol 12 ◽  
Author(s):  
Sun-Wook Jeong ◽  
Min-Kyu Kim ◽  
Lei Zhao ◽  
Seul-Ki Yang ◽  
Jong-Hyun Jung ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Repair ◽  
Dna Binding ◽  
Deinococcus Radiodurans ◽  
Bacterial Species ◽  
Binding Activity ◽  
Holliday Junctions ◽  
Dna Binding Activity ◽  
Domain Structures ◽  
Repair Efficiency

Deinococcus radiodurans is extremely resistant to ionizing radiation and has an exceptional ability to repair DNA damage caused by various DNA-damaging agents. D. radiodurans uses the same DNA-repair strategies as other prokaryotes, but certain proteins involved in the classical DNA repair machinery have characteristics different from their counterparts. RecG helicase, which unwinds a variety of branched DNA molecules, such as Holliday junctions (HJ) and D-loops, plays important roles in DNA repair, recombination, and replication. Primary sequence analysis of RecG from a number of bacterial species revealed that three amino acids (QPW) in the DNA-binding wedge domain (WD) are well-conserved across the Deinococcus RecG proteins. Interactions involving these conserved residues and DNA substrates were predicted in modeled domain structures of D. radiodurans RecG (DrRecG). Compared to the WD of Escherichia coli RecG protein (EcRecG) containing FSA amino acids corresponding to QPW in DrRecG, the HJ binding activity of DrRecG-WD was higher than that of EcRecG-WD. Reciprocal substitution of FSA and QPW increased and decreased the HJ binding activity of the mutant WDs, EcRecG-WDQPW, and DrRecG-WDFSA, respectively. Following γ-irradiation treatment, the reduced survival rate of DrRecG mutants (ΔrecG) was fully restored by the expression of DrRecG, but not by that of EcRecG. EcRecGQPW also enhanced γ-radioresistance of ΔrecG, whereas DrRecGFSA did not. ΔrecG cells complemented in trans by DrRecG and EcRecGQPW reconstituted an intact genome within 3 h post-irradiation, as did the wild-type strain, but ΔrecG with EcRecG and DrRecGFSA exhibited a delay in assembly of chromosomal fragments induced by γ-irradiation. These results suggested that the QPW residues facilitate the association of DrRecG with DNA junctions, thereby enhancing the DNA repair efficiency of DrRecG.

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

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 ATPases of cohesin interface with regulators to modulate cohesin-mediated DNA tethering

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Gamze Çamdere ◽  
Vincent Guacci ◽  
Jeremiah Stricklin ◽  
Douglas Koshland
Keyword(s):  
Dna Repair ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Sister Chromatid ◽  
Active Sites ◽  
Binding Activity ◽  
Second Step ◽  
Dna Binding Activity ◽  
Chromatid Cohesion ◽  
Biochemical Analyses

Cohesin tethers together regions of DNA, thereby mediating higher order chromatin organization that is critical for sister chromatid cohesion, DNA repair and transcriptional regulation. Cohesin contains a heterodimeric ATP-binding Cassette (ABC) ATPase comprised of Smc1 and Smc3 ATPase active sites. These ATPases are required for cohesin to bind DNA. Cohesin’s DNA binding activity is also promoted by the Eco1 acetyltransferase and inhibited by Wpl1. Recently we showed that after cohesin stably binds DNA, a second step is required for DNA tethering. This second step is also controlled by Eco1 acetylation. Here, we use genetic and biochemical analyses to show that this second DNA tethering step is regulated by cohesin ATPase. Furthermore, our results also suggest that Eco1 promotes cohesion by modulating the ATPase cycle of DNA-bound cohesin in a state that is permissive for DNA tethering and refractory to Wpl1 inhibition.

scholarly journals Clp-dependent proteolysis of the LexA N-terminal domain in Staphylococcus aureus

Microbiology ◽  
2011 ◽  
Vol 157 (3) ◽  
pp. 677-684 ◽  
Author(s):  
Marianne T. Cohn ◽  
Peter Kjelgaard ◽  
Dorte Frees ◽  
José R. Penadés ◽  
Hanne Ingmer
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Bacterial Species ◽  
Transcriptional Regulator ◽  
Sos Response ◽  
Binding Activity ◽  
Dimerization Domain ◽  
Dna Binding Activity ◽  
Lexa Repressor ◽  
Induced Mutagenesis

The SOS response is governed by the transcriptional regulator LexA and is elicited in many bacterial species in response to DNA damaging conditions. Induction of the SOS response is mediated by autocleavage of the LexA repressor resulting in a C-terminal dimerization domain (CTD) and an N-terminal DNA-binding domain (NTD) known to retain some DNA-binding activity. The proteases responsible for degrading the LexA domains have been identified in Escherichia coli as ClpXP and Lon. Here, we show that in the human and animal pathogen Staphylococcus aureus, the ClpXP and ClpCP proteases contribute to degradation of the NTD and to a lesser degree the CTD. In the absence of the proteolytic subunit, ClpP, or one or both of the Clp ATPases, ClpX and ClpC, the LexA domains were stabilized after autocleavage. Production of a stabilized variant of the NTD interfered with mitomycin-mediated induction of sosA expression while leaving lexA unaffected, and also significantly reduced SOS-induced mutagenesis. Our results show that sequential proteolysis of LexA is conserved in S. aureus and that the NTD may differentially regulate a subset of genes in the SOS regulon.

scholarly journals LEU3 of Saccharomyces cerevisiae encodes a factor for control of RNA levels of a group of leucine-specific genes.

1987 ◽  
Vol 7 (8) ◽  
pp. 2708-2717 ◽  
Author(s):  
P Friden ◽  
P Schimmel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Coding Region ◽  
General Amino Acid Control ◽  
Dna Binding Activity ◽  
Cysteine Motif ◽  
Cell Extracts

Although the majority of genes for amino acid biosynthesis which have been examined are under general amino acid control, LEU1 and LEU2 of Saccharomyces cerevisiae respond specifically to leucine. We report here an analysis of LEU3, a putative leucine-specific regulatory locus. We show that LEU3 is necessary for expression of wild-type levels of LEU1- and LEU2-specific RNAs and, further, that the levels of LEU4-specific transcripts are also affected by LEU3. We cloned LEU3 and showed by DNA sequence analysis that it contained an open reading frame of 886 amino acids. A striking feature of the predicted LEU3 protein was a cluster of acidic amino acids (19 of 20) located in the C-terminal half of the coding region. The protein also had a repeated cysteine motif which was conserved in a number of other yeast proteins implicated in gene regulation. We show that whole-cell extracts contained a LEU3-dependent DNA-binding activity that interacted with the 5' region of LEU2. Subdivision of the LEU2 5' region established that the LEU3-dependent DNA-binding activity interacted with the segment which had the previously reported homology with LEU1.

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 Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding.

1993 ◽  
Vol 13 (9) ◽  
pp. 5370-5376 ◽  
Author(s):  
L J Walker ◽  
C N Robson ◽  
E Black ◽  
D Gillespie ◽  
I D Hickson
Keyword(s):  
Dna Repair ◽  
Dna Binding ◽  
Active Site ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Repair Enzyme ◽  
Exonuclease Iii ◽  
Terminal Domain ◽  
Dna Binding Activity ◽  
Redox Active

The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.

scholarly journals Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype.

1996 ◽  
Vol 16 (10) ◽  
pp. 5691-5700 ◽  
Author(s):  
R Moriggl ◽  
V Gouilleux-Gruart ◽  
R Jähne ◽  
S Berchtold ◽  
C Gartmann ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Mammary Epithelial Cells ◽  
Binding Activity ◽  
Dominant Negative ◽  
Terminal Deletion ◽  
Gene Promoters ◽  
Transactivation Domain ◽  
Dna Binding Activity ◽  
Carboxyl Terminal

The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; Stat5a delta 750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a delta 750 mutant. The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that Stat5a delta 750 and Stat5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters.

LEU3 of Saccharomyces cerevisiae encodes a factor for control of RNA levels of a group of leucine-specific genes

1987 ◽  
Vol 7 (8) ◽  
pp. 2708-2717
Author(s):  
P Friden ◽  
P Schimmel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Coding Region ◽  
General Amino Acid Control ◽  
Dna Binding Activity ◽  
Cysteine Motif ◽  
Cell Extracts

Although the majority of genes for amino acid biosynthesis which have been examined are under general amino acid control, LEU1 and LEU2 of Saccharomyces cerevisiae respond specifically to leucine. We report here an analysis of LEU3, a putative leucine-specific regulatory locus. We show that LEU3 is necessary for expression of wild-type levels of LEU1- and LEU2-specific RNAs and, further, that the levels of LEU4-specific transcripts are also affected by LEU3. We cloned LEU3 and showed by DNA sequence analysis that it contained an open reading frame of 886 amino acids. A striking feature of the predicted LEU3 protein was a cluster of acidic amino acids (19 of 20) located in the C-terminal half of the coding region. The protein also had a repeated cysteine motif which was conserved in a number of other yeast proteins implicated in gene regulation. We show that whole-cell extracts contained a LEU3-dependent DNA-binding activity that interacted with the 5' region of LEU2. Subdivision of the LEU2 5' region established that the LEU3-dependent DNA-binding activity interacted with the segment which had the previously reported homology with LEU1.

Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding

1993 ◽  
Vol 13 (9) ◽  
pp. 5370-5376
Author(s):  
L J Walker ◽  
C N Robson ◽  
E Black ◽  
D Gillespie ◽  
I D Hickson
Keyword(s):  
Dna Repair ◽  
Dna Binding ◽  
Active Site ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Repair Enzyme ◽  
Exonuclease Iii ◽  
Terminal Domain ◽  
Dna Binding Activity ◽  
Redox Active

The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.

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