Characterization of DRA0282 from Deinococcus radiodurans for its role in bacterial resistance to DNA damage

Das, Anubrata; Misra, Hari S.

doi:10.1099/mic.0.040436-0

Cited by 16 publications

(9 citation statements)

References 44 publications

(59 reference statements)

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“…Also, in vitro binding studies with circular single-stranded DNA and DNA containing hairpin loops at both ends demonstrated that eukaryotic Ku could bind to a 'closed' substrate lacking free ends with no significant difference in binding affinity compared with DNA with free ends [27,29]. Recently, a homologue of eukaryotic Ku from Deinococcus radiodurans was shown to bind supercoiled DNA with 67-fold higher affinity as compared with linear DNA, also supporting the notion that DNA binding by Ku is probably more complex than originally thought, and consistent with the possibility that Ku may associate with local secondary structures [30]. Furthermore, we recently demonstrated that the lysine-rich C-terminal repeats of M. smegmatis Ku confer on the protein the ability to promote DNA end-joining, consistent with its role in NHEJ [10].…”

Section: Introductionsupporting

confidence: 62%

“…The crystal structure of human Ku shows that it has a tripartite organization consisting of an N-terminal α/β von Willebrand factor A domain, a central βbarrel domain and a subunit-specific C-terminal SAP domain that together form a pseudosymmetrical ring-like channel that can accommodate a DNA duplex [9]. In contrast, the prokaryotic Ku proteins are homodimers [1,4,5] and much smaller (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40), consisting of the conserved central 'ring-shaped' core domain of eukaryotic Ku and lacking the N-and C-terminal domains present in Ku70/80, except for homologues from a few bacteria such as Streptomyces coelicolor that contain a SAP-like domain [1,4,5]. Also, Ku proteins from free-living mycobacterial species found in soil and natural reservoirs contain at their C-terminus low complexity repeats containing the amino acids lysine, alanine, and proline that promote DNA end-joining [10].…”

Section: Introductionmentioning

confidence: 99%

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Mycobacterium smegmatis Ku binds DNA without free ends

Kushwaha

Grove

2013

Biochemical Journal

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Ku is central to the non-homologous end-joining pathway of double-strand-break repair in all three major domains of life, with eukaryotic homologues being associated with more diversified roles compared with prokaryotic and archaeal homologues. Ku has a conserved central 'ring-shaped' core domain. While prokaryotic homologues lack the N- and C-terminal domains that impart functional diversity to eukaryotic Ku, analyses of Ku from certain prokaryotes such as Pseudomonas aeruginosa and Mycobacterium smegmatis have revealed the presence of distinct C-terminal extensions that modulate DNA-binding properties. We report in the present paper that the lysine-rich C-terminal extension of M. smegmatis Ku contacts the core protein domain as evidenced by an increase in DNA-binding affinity and a decrease in thermal stability and intrinsic tryptophan fluorescence upon its deletion. Ku deleted for this C-terminus requires free DNA ends for binding, but translocates to internal DNA sites. In contrast, full-length Ku can directly bind DNA without free ends, suggesting that this property is conferred by its C-terminus. Such binding to internal DNA sites may facilitate recruitment to sites of DNA damage. The results of the present study also suggest that extensions beyond the shared core domain may have independently evolved to expand Ku function.

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Section: Introductionsupporting

confidence: 62%

Section: Introductionmentioning

confidence: 99%

Mycobacterium smegmatis Ku binds DNA without free ends

Kushwaha

Grove

2013

Biochemical Journal

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“…The percentage of bound fraction of DNA was plot-ted against protein concentration using GraphPad Prism version 5. The K d for curve fitting of individual plots was determined by the software working on the principle of the least squares method, applying the formula, Y ϭ B max ϫ [X]/K d ϩ [X], where [X] is the protein concentration, and Y is the bound fraction as described earlier (43). To determine the log equilibrium dissociation constant (log K i ) of nonphosphorylated and phosphorylated DrRecA, the competition assay was performed, where the binding of 0.2 pmol of ssDNA/dsDNA was challenged with either unlabeled homologous ssDNA and dsDNA (2.5-40 M) or unlabeled heterologous dsDNA and ssDNA (2.5-40 M), respectively.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of Deinococcus radiodurans RecA Regulates Its Activity and May Contribute to Radioresistance

Rajpurohit

Bihani

Waldor

et al. 2016

Journal of Biological Chemistry

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Deinococcus radiodurans has a remarkable capacity to survive exposure to extreme levels of radiation that cause hundreds of DNA double strand breaks (DSBs). DSB repair in this bacterium depends on its recombinase A protein (DrRecA). DrRecA plays a pivotal role in both extended synthesis-dependent strand annealing and slow crossover events of DSB repair during the organism's recovery from DNA damage. The mechanisms that control DrRecA activity during the D. radiodurans response to ␥ radiation exposure are unknown. Here, we show that DrRecA undergoes phosphorylation at Tyr-77 and Thr-318 by a DNA damage-responsive serine threonine/tyrosine protein kinase (RqkA). Phosphorylation modifies the activity of DrRecA in several ways, including increasing its affinity for dsDNA and its preference for dATP over ATP. Strand exchange reactions catalyzed by phosphorylated versus unphosphorylated DrRecA also differ. In silico analysis of DrRecA structure support the idea that phosphorylation can modulate crucial functions of this protein. Collectively, our findings suggest that phosphorylation of DrRecA enables the recombinase to selectively use abundant dsDNA substrate present during post-irradiation recovery for efficient DSB repair, thereby promoting the extraordinary radioresistance of D. radiodurans.Deinococcus radiodurans has a remarkable capacity to survive extreme doses of radiations and other DNA-damaging agents. Studies aimed at unraveling the molecular bases for these unusual properties have revealed that D. radiodurans encodes mechanisms for highly efficient DNA double strand break (DSB) 2 repair and oxidative stress management (1-3). DSB repair in this Gram-positive bacterium is accomplished in two phases during post-irradiation recovery (PIR); phase I is dominated by extended synthesis-dependent strand annealing (ESDSA) processes, whereas phase II involves slow crossover events in homologous recombination leading to the repair and re-establishment of the multipartite D. radiodurans genome structure (4). Despite the fact that the two phases of PIR have DNA substrates of different structures and topologies, D. radiodurans RecA (DrRecA) is required throughout DSB repair during PIR (5). Biochemical characterization of recombinant DrRecA revealed that it can form a filament on singlestranded DNA (ssDNA), exhibit co-protease activity, and utilize ATP or dATP for its energy requirements, akin to other bacterial RecA proteins (6), but it also has unusual properties. In contrast to most bacterial RecA proteins, DrRecA promotes inverse strand exchange reactions (7). Also, DrRecA promotes DNA degradation during the early phase of ESDSA repair (5), which is opposite to the function observed with Escherichia coli RecA. Transcription of DrRecA is induced in response to ␥ radiation (8, 9). However, the mechanisms by which ␥ radiation induces DrRecA expression are unusual. Inactivation of both D. radiodurans lexA genes does not attenuate ␥ radiation induction of DrRecA expression (10, 11). Thus, in contrast to many bacteria, LexA...

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“…The consensus boxes similar to the typical P1 element were identified by using Bioedit, version 7, software. The functional motif search was carried out by using standard online bioinformatics tools, as reported previously (10). In brief, the amino acid sequences of DR_0012 and DR_0013, annotated as putative ParB and ParA proteins, respectively, on chromosome I of this bacterium were subjected to a PSI-BLAST search with the Swiss-Prot database, with "genome partitioning proteins" as key words.…”

Section: Methodsmentioning

confidence: 99%

“…, where [X] is the protein concentration, Y is the bound fraction, and B max is the maximum binding capacity, as described previously (10).…”

Section: Expression and Purification Of Recombinant Parb1 And Para1mentioning

confidence: 99%

Functional Characterization of the Role of the Chromosome I Partitioning System in Genome Segregation in Deinococcus radiodurans

Charaka

Misra

2012

J Bacteriol

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Deinococcus radiodurans, a radiation-resistant bacterium, harbors a multipartite genome. Chromosome I contains three putative centromeres (segS1, segS2, and segS3), and ParA (ParA1) and ParB (ParB1) homologues. The ParB1 interaction with segS was sequence specific, and ParA1 was shown to be a DNA binding ATPase. The ATPase activity of ParA1 was stimulated when segS elements were coincubated with ParB1, but the greatest increase was observed with segS3. ParA1 incubated with the segS-ParB1 complex showed increased light scattering in the absence of ATP. In the presence of ATP, this increase was continued with segS1-ParA1B1 and segS2-ParA1B1 complexes, while it decreased rapidly after an initial increase for 30 min in the case of segS3. D. radiodurans cells expressing green fluorescent protein (GFP)-ParB1 produced foci on nucleoids, and the ⌬parB1 mutant showed growth retardation and ϳ13%-higher anucleation than the wild type. Unstable mini-F plasmids carrying segS1 and segS2 showed inheritance in Escherichia coli without ParA1B1, while segS3-mediated plasmid stability required the in trans expression of ParA1B1. Unlike untransformed E. coli cells, cells harboring pDAGS3, a plasmid carrying segS3 and also expressing ParB1-GFP, produced discrete GFP foci on nucleoids. These findings suggested that both segS elements and the ParA1B1 proteins of D. radiodurans are functionally active and have a role in genome segregation.

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Characterization of DRA0282 from Deinococcus radiodurans for its role in bacterial resistance to DNA damage

Cited by 16 publications

References 44 publications

Mycobacterium smegmatis Ku binds DNA without free ends

Mycobacterium smegmatis Ku binds DNA without free ends

Phosphorylation of Deinococcus radiodurans RecA Regulates Its Activity and May Contribute to Radioresistance

Functional Characterization of the Role of the Chromosome I Partitioning System in Genome Segregation in Deinococcus radiodurans

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