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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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Sequence analysis of MMEJ junctions, when DSBs with different microhomology regions were used. Products due to possible MMEJ (3, 5, 8, 10, 13 and 16 nt microhomology) catalyzed by testicular and thymic extracts were gel purified, cloned and sequenced. The sequences denoted in red color are the deleted nucleotides. (a) MMEJ junctions from rat testicular extracts. (b) MMEJ junctions derived from rat thymic extracts. For other details, refer Figures 1 and 4 legends
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fig5: Sequence analysis of MMEJ junctions, when DSBs with different microhomology regions were used. Products due to possible MMEJ (3, 5, 8, 10, 13 and 16 nt microhomology) catalyzed by testicular and thymic extracts were gel purified, cloned and sequenced. The sequences denoted in red color are the deleted nucleotides. (a) MMEJ junctions from rat testicular extracts. (b) MMEJ junctions derived from rat thymic extracts. For other details, refer Figures 1 and 4 legends

Mentions: In order to confirm the identity of potential MMEJ products, corresponding bands were excised, purified and subjected to DNA sequencing for 3, 5, 8, 10, 13 and 16 nt microhomology-containing substrates. Results confirmed the presence of MMEJ in case of each substrate, when the microhomology was 8 nt or more (Figure 5a). In case of 5 nt microhomology substrates, only few junctions utilized microhomology (based on restriction digestion analysis), whereas majority of the joining was through C-NHEJ. In case of 3 nt microhomology substrates, none of the clones showed usage of microhomology. Unlike C-NHEJ, MMEJ junctions did not show any insertions, although point mutations were rarely seen. Similar results were also seen when MMEJ junctions were sequenced from thymus (Figure 5b). These results suggest that the MMEJ is dependent on the length of microhomology region.


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)

Sequence analysis of MMEJ junctions, when DSBs with different microhomology regions were used. Products due to possible MMEJ (3, 5, 8, 10, 13 and 16 nt microhomology) catalyzed by testicular and thymic extracts were gel purified, cloned and sequenced. The sequences denoted in red color are the deleted nucleotides. (a) MMEJ junctions from rat testicular extracts. (b) MMEJ junctions derived from rat thymic extracts. For other details, refer Figures 1 and 4 legends
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Sequence analysis of MMEJ junctions, when DSBs with different microhomology regions were used. Products due to possible MMEJ (3, 5, 8, 10, 13 and 16 nt microhomology) catalyzed by testicular and thymic extracts were gel purified, cloned and sequenced. The sequences denoted in red color are the deleted nucleotides. (a) MMEJ junctions from rat testicular extracts. (b) MMEJ junctions derived from rat thymic extracts. For other details, refer Figures 1 and 4 legends
Mentions: In order to confirm the identity of potential MMEJ products, corresponding bands were excised, purified and subjected to DNA sequencing for 3, 5, 8, 10, 13 and 16 nt microhomology-containing substrates. Results confirmed the presence of MMEJ in case of each substrate, when the microhomology was 8 nt or more (Figure 5a). In case of 5 nt microhomology substrates, only few junctions utilized microhomology (based on restriction digestion analysis), whereas majority of the joining was through C-NHEJ. In case of 3 nt microhomology substrates, none of the clones showed usage of microhomology. Unlike C-NHEJ, MMEJ junctions did not show any insertions, although point mutations were rarely seen. Similar results were also seen when MMEJ junctions were sequenced from thymus (Figure 5b). These results suggest that the MMEJ is dependent on the length of microhomology region.

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