Differential Reaction Kinetics, Cleavage Complex Formation, and Nonamer Binding Domain Dependence Dictate the Structure-Specific and Sequence-Specific Nuclease Activity of RAGs

Naik, Abani Kanta; Raghavan, Sathees C.

doi:10.1016/j.jmb.2011.11.002

Cited by 9 publications

(28 citation statements)

References 55 publications

(73 reference statements)

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“…4A,F,G). This is understandable as RAGs bind to the longer downstream ds DNA sequence to execute their nicking activity, which is consistent with a previous report [13]. Immediate flanking sequence affected RAG nicking at heteroduplex DNA in a sequencedependent manner Previous studies have shown that placing a nonamer of V(D)J recombination on heteroduplex DNA can enhance the efficiency of RAG cleavage significantly, while a single nucleotide change next to the bubble region did not result in any difference in the RAG cleavage efficiency [12,24].…”

Section: Standardization Of Rag Binding and Cleavage On Heteroduplex Dnasupporting

confidence: 92%

“…In addition, two bands due to RAG cleavage were observed in the case of each substrate. Mapping of the cleaved fragments revealed that the first RAG nicking occurred at the cytosine present at the ss-ds DNA 7) and IV (lanes [8][9][10][11][12][13][14] were incubated with different concentrations of cRAGs in a buffer containing 5 mM MgCl 2 for 1 h at 37°C and products were resolved by 15% denaturing PAGE. M represents the single nucleotide ladder.…”

Section: Standardization Of Rag Binding and Cleavage On Heteroduplex Dnamentioning

confidence: 99%

“…In brief, for coexpression of RAG1 and RAG2, 293T cells were transfected with 15 lg each of RAG expression vectors by the calcium phosphate method [12,13,32]. After 48 h of transfection, cells were harvested and resuspended in buffer R (25 mM HEPES, pH 7.4, 150 mM KCl, 10 mM MgCl 2 , 10% glycerol, 2 mM DTT).…”

Section: Expression Of Crags and Purificationmentioning

confidence: 99%

“…Apart from the classical function of RAGs, as a sequence-specific nuclease, recent studies have shown that RAGs possess structure-specific nuclease activity [9][10][11][12][13][14]. It has been shown that RAGs can cleave different altered DNA structures or end configurations in physiological ionic conditions [11][12][13][14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

“…It has been shown that RAGs can cleave different altered DNA structures or end configurations in physiological ionic conditions [11][12][13][14][15][16][17]. Besides, RAGs can also cleave hairpin structures in the presence of Mn 2+ [18,19].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Structure‐specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double‐stranded DNA

Kumari

Raghavan

2014

The FEBS Journal

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RAGs (recombination activating genes) are responsible for the generation of antigen receptor diversity through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. In addition to its physiological property, wherein RAG functions as a sequence-specific nuclease, it can also act as a structure-specific nuclease leading to genomic instability and cancer. In the present study, we investigate the factors that regulate RAG cleavage on non-B DNA structures. We find that RAG binding and cleavage on heteroduplex DNA is dependent on the length of the double-stranded flanking region. Besides, the immediate flanking doublestranded region regulates RAG activity in a sequence-dependent manner. Interestingly, the cleavage efficiency of RAGs at the heteroduplex region is influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights on the regulation of the pathological functions of RAGs.

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Section: Standardization Of Rag Binding and Cleavage On Heteroduplex Dnasupporting

confidence: 92%

Section: Standardization Of Rag Binding and Cleavage On Heteroduplex Dnamentioning

confidence: 99%

Section: Expression Of Crags and Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Structure‐specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double‐stranded DNA

Kumari

Raghavan

2014

The FEBS Journal

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HIV integrase inhibitor, Elvitegravir, impairs RAG functions and inhibits V(D)J recombination

et al. 2017

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Integrase inhibitors are a class of antiretroviral drugs used for the treatment of AIDS that target HIV integrase, an enzyme responsible for integration of viral cDNA into host genome. RAG1, a critical enzyme involved in V(D)J recombination exhibits structural similarity to HIV integrase. We find that two integrase inhibitors, Raltegravir and Elvitegravir, interfered with the physiological functions of RAGs such as binding, cleavage and hairpin formation at the recombination signal sequence (RSS), though the effect of Raltegravir was limited. Circular dichroism studies demonstrated a distinct change in the secondary structure of RAG1 central domain (RAG1 shares DDE motif amino acids with integrases), and when incubated with Elvitegravir, an equilibrium dissociation constant (Kd) of 32.53±2.9 μM was determined by Biolayer interferometry, leading to inhibition of its binding to DNA. Besides, using extrachromosomal assays, we show that Elvitegravir inhibited both coding and signal joint formation in pre-B cells. Importantly, treatment with Elvitegravir resulted in significant reduction of mature B lymphocytes in 70% of mice studied. Thus, our study suggests a potential risk associated with the use of Elvitegravir as an antiretroviral drug, considering the evolutionary and structural similarities between HIV integrase and RAGs.

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Biochemical Characterization of Nonamer Binding Domain of RAG1 Reveals its Thymine Preference with Respect to Length and Position

Raveendran

Raghavan

2016

Sci Rep

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RAG complex consisting of RAG1 and RAG2 is a site-specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves recombination signal sequence (RSS), comprising of conserved heptamer and nonamer. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS. To investigate the DNA binding properties of the domain, NBD of murine RAG1 was cloned, expressed and purified. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS or heteroduplex DNA. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G or T were used. Biolayer interferometry studies showed that poly T binding to NBD was robust and comparable to that of 12RSS. More than 23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C or random sequences. Although NBD is indispensable for sequence specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore its activity, suggesting that the overall domain architecture of RAG1 is important. Therefore, we define the sequence requirements of NBD binding to DNA.

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Differential Reaction Kinetics, Cleavage Complex Formation, and Nonamer Binding Domain Dependence Dictate the Structure-Specific and Sequence-Specific Nuclease Activity of RAGs

Cited by 9 publications

References 55 publications

Structure‐specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double‐stranded DNA

Structure‐specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double‐stranded DNA

HIV integrase inhibitor, Elvitegravir, impairs RAG functions and inhibits V(D)J recombination

Biochemical Characterization of Nonamer Binding Domain of RAG1 Reveals its Thymine Preference with Respect to Length and Position

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