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Homology and enzymatic requirements of microhomology-dependent alternative end joining.

Sharma S, Javadekar SM, Pandey M, Srivastava M, Kumari R, Raghavan SC - Cell Death Dis (2015)

Bottom Line: Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory.Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1.Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.

ABSTRACT
Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.

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Related in: MedlinePlus

Evaluation of different experimental conditions for the development of a cell-free repair system to assess microhomology-mediated alternative DNA end joining. (a) Evaluation of MMEJ in presence of increasing concentrations of cell-free extracts. Testicular extracts (0, 0.025, 0.050, 0.075, 0.1, 0.2, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 μg) were incubated with DNA substrates (4 nM) 10 nt microhomology for 2 h at 30 °C. Lane 1 indicates no protein control. (b) Time kinetics of MMEJ on a 10-nt microhomology-containing DNA substrates. Rat testicular extracts (1 μg) were incubated with DNA substrates for 0, 5, 15 and 30 min, and 1, 2, 4, 6, 8, 10 and 12 h, and products were analyzed on 8% denaturing PAGE. (c) MMEJ assay at increasing incubation temperatures. A unit of 1.0 μg of extract was incubated with 10 nt microhomology-containing DNA substrates in rat testicular extracts for 2 h at 4, 16, 25, 30, 37 and 40 °C. Lane 1 is no protein control. (d) Effect of MgCl2 on MMEJ catalyzed by cell-free extracts. Rat testicular extracts (1 μg) were incubated with microhomology substrates and increasing concentrations of MgCl2 at 30 °C. Lane 1 is no protein control. Lanes 2–7 indicate MMEJ in the presence of 0, 1, 2, 5, 10 and 20 mM of MgCl2, respectively. (e) Effect of ATP on MMEJ catalyzed by cell-free extracts. Rat testicular extracts (1 μg) was incubated with microhomology substrates and increasing concentrations of ATP for 2 h. Lanes 2–8 indicate MMEJ in the presence of 0, 0.1, 0.25, 0.5, 1, 2 and 4 mM of ATP, respectively. In panels a–e, bar diagram showing quantification based on at least three independent experiments are provided. MMEJ products are indicated by an arrow, while C-NHEJ products are bracketed. In each case, lower panel serves as the loading control for equal DNA, indicated as ‘input DNA'. M' and M indicate 60 nt marker and 50 nt ladder, respectively. PSLU in y axis of bar diagram indicates photostimulated luminescence units.*P<0.05; **P<0.01; ***P<0.001. Error bars represent S.E.M.
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fig2: Evaluation of different experimental conditions for the development of a cell-free repair system to assess microhomology-mediated alternative DNA end joining. (a) Evaluation of MMEJ in presence of increasing concentrations of cell-free extracts. Testicular extracts (0, 0.025, 0.050, 0.075, 0.1, 0.2, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 μg) were incubated with DNA substrates (4 nM) 10 nt microhomology for 2 h at 30 °C. Lane 1 indicates no protein control. (b) Time kinetics of MMEJ on a 10-nt microhomology-containing DNA substrates. Rat testicular extracts (1 μg) were incubated with DNA substrates for 0, 5, 15 and 30 min, and 1, 2, 4, 6, 8, 10 and 12 h, and products were analyzed on 8% denaturing PAGE. (c) MMEJ assay at increasing incubation temperatures. A unit of 1.0 μg of extract was incubated with 10 nt microhomology-containing DNA substrates in rat testicular extracts for 2 h at 4, 16, 25, 30, 37 and 40 °C. Lane 1 is no protein control. (d) Effect of MgCl2 on MMEJ catalyzed by cell-free extracts. Rat testicular extracts (1 μg) were incubated with microhomology substrates and increasing concentrations of MgCl2 at 30 °C. Lane 1 is no protein control. Lanes 2–7 indicate MMEJ in the presence of 0, 1, 2, 5, 10 and 20 mM of MgCl2, respectively. (e) Effect of ATP on MMEJ catalyzed by cell-free extracts. Rat testicular extracts (1 μg) was incubated with microhomology substrates and increasing concentrations of ATP for 2 h. Lanes 2–8 indicate MMEJ in the presence of 0, 0.1, 0.25, 0.5, 1, 2 and 4 mM of ATP, respectively. In panels a–e, bar diagram showing quantification based on at least three independent experiments are provided. MMEJ products are indicated by an arrow, while C-NHEJ products are bracketed. In each case, lower panel serves as the loading control for equal DNA, indicated as ‘input DNA'. M' and M indicate 60 nt marker and 50 nt ladder, respectively. PSLU in y axis of bar diagram indicates photostimulated luminescence units.*P<0.05; **P<0.01; ***P<0.001. Error bars represent S.E.M.

Mentions: The steps involved in assaying MMEJ are outlined (Figure 1a). Two double-stranded oligomers of different lengths, containing 10 (or 22) nt microhomology were designed such that joining by utilizing microhomology results in shortening of the product (62 or 76 nt), which can be detected using radioactive PCR (Figures 1b and c). Similarly, joining by C-NHEJ could result in reaction products of varying sizes. In order to optimize assay conditions for MMEJ, various parameters were tested. Increasing concentrations of rat testicular extracts were incubated with DNA substrates containing 10 nt microhomology for 2 h, heat inactivated, PCR amplified using radiolabelled primers and resolved on 8% denaturing polyacrylamide gel. Results showed efficient MMEJ (62 nt) from 200 ng protein onwards, whereas C-NHEJ (~97 nt) was detectable only at protein concentration of 500 ng onwards (Figure 2a). Time kinetics analysis showed MMEJ and C-NHEJ from 5 min onwards. Interestingly, kinetics of C-NHEJ was faster compared with MMEJ (Figure 2b). Besides, testicular extracts showed optimum MMEJ at 30 ºC, the physiological temperature (Figure 2c). An enhancement in the efficiency of MMEJ was observed with an increase in concentrations of Mg2+, a co-factor, optimum being 10 mM (Figure 2d). Efficiency of MMEJ was maximum at 0.5 mM adenosine triphosphate (ATP; Figure 2e). Sensitivity of MMEJ assay was determined by serially diluting DNA and detecting the joining from 100 pM onwards, which exponentially increased till 4 nM (Supplementary Figure 1). Thus, we were successful in establishing a cell-free repair assay system to study MMEJ.


Homology and enzymatic requirements of microhomology-dependent alternative end joining.

Sharma S, Javadekar SM, Pandey M, Srivastava M, Kumari R, Raghavan SC - Cell Death Dis (2015)

Evaluation of different experimental conditions for the development of a cell-free repair system to assess microhomology-mediated alternative DNA end joining. (a) Evaluation of MMEJ in presence of increasing concentrations of cell-free extracts. Testicular extracts (0, 0.025, 0.050, 0.075, 0.1, 0.2, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 μg) were incubated with DNA substrates (4 nM) 10 nt microhomology for 2 h at 30 °C. Lane 1 indicates no protein control. (b) Time kinetics of MMEJ on a 10-nt microhomology-containing DNA substrates. Rat testicular extracts (1 μg) were incubated with DNA substrates for 0, 5, 15 and 30 min, and 1, 2, 4, 6, 8, 10 and 12 h, and products were analyzed on 8% denaturing PAGE. (c) MMEJ assay at increasing incubation temperatures. A unit of 1.0 μg of extract was incubated with 10 nt microhomology-containing DNA substrates in rat testicular extracts for 2 h at 4, 16, 25, 30, 37 and 40 °C. Lane 1 is no protein control. (d) Effect of MgCl2 on MMEJ catalyzed by cell-free extracts. Rat testicular extracts (1 μg) were incubated with microhomology substrates and increasing concentrations of MgCl2 at 30 °C. Lane 1 is no protein control. Lanes 2–7 indicate MMEJ in the presence of 0, 1, 2, 5, 10 and 20 mM of MgCl2, respectively. (e) Effect of ATP on MMEJ catalyzed by cell-free extracts. Rat testicular extracts (1 μg) was incubated with microhomology substrates and increasing concentrations of ATP for 2 h. Lanes 2–8 indicate MMEJ in the presence of 0, 0.1, 0.25, 0.5, 1, 2 and 4 mM of ATP, respectively. In panels a–e, bar diagram showing quantification based on at least three independent experiments are provided. MMEJ products are indicated by an arrow, while C-NHEJ products are bracketed. In each case, lower panel serves as the loading control for equal DNA, indicated as ‘input DNA'. M' and M indicate 60 nt marker and 50 nt ladder, respectively. PSLU in y axis of bar diagram indicates photostimulated luminescence units.*P<0.05; **P<0.01; ***P<0.001. Error bars represent S.E.M.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385936&req=5

fig2: Evaluation of different experimental conditions for the development of a cell-free repair system to assess microhomology-mediated alternative DNA end joining. (a) Evaluation of MMEJ in presence of increasing concentrations of cell-free extracts. Testicular extracts (0, 0.025, 0.050, 0.075, 0.1, 0.2, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 μg) were incubated with DNA substrates (4 nM) 10 nt microhomology for 2 h at 30 °C. Lane 1 indicates no protein control. (b) Time kinetics of MMEJ on a 10-nt microhomology-containing DNA substrates. Rat testicular extracts (1 μg) were incubated with DNA substrates for 0, 5, 15 and 30 min, and 1, 2, 4, 6, 8, 10 and 12 h, and products were analyzed on 8% denaturing PAGE. (c) MMEJ assay at increasing incubation temperatures. A unit of 1.0 μg of extract was incubated with 10 nt microhomology-containing DNA substrates in rat testicular extracts for 2 h at 4, 16, 25, 30, 37 and 40 °C. Lane 1 is no protein control. (d) Effect of MgCl2 on MMEJ catalyzed by cell-free extracts. Rat testicular extracts (1 μg) were incubated with microhomology substrates and increasing concentrations of MgCl2 at 30 °C. Lane 1 is no protein control. Lanes 2–7 indicate MMEJ in the presence of 0, 1, 2, 5, 10 and 20 mM of MgCl2, respectively. (e) Effect of ATP on MMEJ catalyzed by cell-free extracts. Rat testicular extracts (1 μg) was incubated with microhomology substrates and increasing concentrations of ATP for 2 h. Lanes 2–8 indicate MMEJ in the presence of 0, 0.1, 0.25, 0.5, 1, 2 and 4 mM of ATP, respectively. In panels a–e, bar diagram showing quantification based on at least three independent experiments are provided. MMEJ products are indicated by an arrow, while C-NHEJ products are bracketed. In each case, lower panel serves as the loading control for equal DNA, indicated as ‘input DNA'. M' and M indicate 60 nt marker and 50 nt ladder, respectively. PSLU in y axis of bar diagram indicates photostimulated luminescence units.*P<0.05; **P<0.01; ***P<0.001. Error bars represent S.E.M.
Mentions: The steps involved in assaying MMEJ are outlined (Figure 1a). Two double-stranded oligomers of different lengths, containing 10 (or 22) nt microhomology were designed such that joining by utilizing microhomology results in shortening of the product (62 or 76 nt), which can be detected using radioactive PCR (Figures 1b and c). Similarly, joining by C-NHEJ could result in reaction products of varying sizes. In order to optimize assay conditions for MMEJ, various parameters were tested. Increasing concentrations of rat testicular extracts were incubated with DNA substrates containing 10 nt microhomology for 2 h, heat inactivated, PCR amplified using radiolabelled primers and resolved on 8% denaturing polyacrylamide gel. Results showed efficient MMEJ (62 nt) from 200 ng protein onwards, whereas C-NHEJ (~97 nt) was detectable only at protein concentration of 500 ng onwards (Figure 2a). Time kinetics analysis showed MMEJ and C-NHEJ from 5 min onwards. Interestingly, kinetics of C-NHEJ was faster compared with MMEJ (Figure 2b). Besides, testicular extracts showed optimum MMEJ at 30 ºC, the physiological temperature (Figure 2c). An enhancement in the efficiency of MMEJ was observed with an increase in concentrations of Mg2+, a co-factor, optimum being 10 mM (Figure 2d). Efficiency of MMEJ was maximum at 0.5 mM adenosine triphosphate (ATP; Figure 2e). Sensitivity of MMEJ assay was determined by serially diluting DNA and detecting the joining from 100 pM onwards, which exponentially increased till 4 nM (Supplementary Figure 1). Thus, we were successful in establishing a cell-free repair assay system to study MMEJ.

Bottom Line: Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory.Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1.Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.

ABSTRACT
Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.

Show MeSH
Related in: MedlinePlus