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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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Determination of length of microhomology requirement during MMEJ. (a) Depiction of sequence and probable MMEJ product following joining of ds oligomeric substrates possessing 3, 5, 8, 10, 13, 16, 19 and 22 nt of microhomology, which are indicated in red. Restriction enzyme sites generated due to microhomology-mediated joining are also indicated. (b) Comparison of MMEJ catalyzed by testicular extracts when different microhomology regions were used. Rat testicular extracts were incubated with oligomeric DNA substrates harboring 3, 5, 8, 10, 13, 16, 19 and 22 nt microhomology for 2 h at 25 ºC. End-joined products were detected by radioactive PCR. In case of every substrate, reactions are shown with either of the substrate or both. MMEJ products are indicated by arrow, while NHEJ products are bracketed. M and M' are molecular weight markers. (c) Comparison of MMEJ of oligomeric DNA containing DSBs when flanked with different length microhomology catalyzed by Reh cell-free extract. M and M' are markers as indicated
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fig4: Determination of length of microhomology requirement during MMEJ. (a) Depiction of sequence and probable MMEJ product following joining of ds oligomeric substrates possessing 3, 5, 8, 10, 13, 16, 19 and 22 nt of microhomology, which are indicated in red. Restriction enzyme sites generated due to microhomology-mediated joining are also indicated. (b) Comparison of MMEJ catalyzed by testicular extracts when different microhomology regions were used. Rat testicular extracts were incubated with oligomeric DNA substrates harboring 3, 5, 8, 10, 13, 16, 19 and 22 nt microhomology for 2 h at 25 ºC. End-joined products were detected by radioactive PCR. In case of every substrate, reactions are shown with either of the substrate or both. MMEJ products are indicated by arrow, while NHEJ products are bracketed. M and M' are molecular weight markers. (c) Comparison of MMEJ of oligomeric DNA containing DSBs when flanked with different length microhomology catalyzed by Reh cell-free extract. M and M' are markers as indicated

Mentions: In order to understand the extent of microhomology required for MMEJ, oligomeric DNA containing DSBs flanked by various lengths of microhomology (3, 5, 8, 10, 13, 16, 19 or 22 nt) were designed and synthesized in such a way that following MMEJ a restriction enzyme site would be generated (Figure 4a and Supplementary Tables 2 and 4A). Results showed a prominent band due to MMEJ in all the substrates, except for 3 nt microhomology, when incubated with testicular extracts (Figure 4b). In order to corroborate this observation, PCR products obtained from 3, 5, 8 and 13 nt microhomology substrates following MMEJ reaction, were digested with NotI, XmnI and XcmI, respectively. A low-intensity band corresponding to ~55 nt in 5 nt microhomology substrate was detected, which was digestible with NotI indicating that the joining was mediated by MMEJ (Supplementary Figure 4b). Similarly, bands corresponding to ~60 and 65 nt in 8 and 13 nt microhomology substrates were digestible with XmnI and XcmI, respectively (Supplementary Figure 4b). Besides, we could not find any specific band, which was sensitive to NotI digestion when 3 nt microhomology substrate was used (Supplementary Figure 4b). As there was no suitable restriction enzyme site, we could not perform similar analysis for substrates containing 10 nt microhomology. These findings suggest that MMEJ protein machinery recognizes microhomology regions to undergo end joining. Furthermore, a microhomology length of more than 3 nt is essential for its operation.


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)

Determination of length of microhomology requirement during MMEJ. (a) Depiction of sequence and probable MMEJ product following joining of ds oligomeric substrates possessing 3, 5, 8, 10, 13, 16, 19 and 22 nt of microhomology, which are indicated in red. Restriction enzyme sites generated due to microhomology-mediated joining are also indicated. (b) Comparison of MMEJ catalyzed by testicular extracts when different microhomology regions were used. Rat testicular extracts were incubated with oligomeric DNA substrates harboring 3, 5, 8, 10, 13, 16, 19 and 22 nt microhomology for 2 h at 25 ºC. End-joined products were detected by radioactive PCR. In case of every substrate, reactions are shown with either of the substrate or both. MMEJ products are indicated by arrow, while NHEJ products are bracketed. M and M' are molecular weight markers. (c) Comparison of MMEJ of oligomeric DNA containing DSBs when flanked with different length microhomology catalyzed by Reh cell-free extract. M and M' are markers as indicated
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Determination of length of microhomology requirement during MMEJ. (a) Depiction of sequence and probable MMEJ product following joining of ds oligomeric substrates possessing 3, 5, 8, 10, 13, 16, 19 and 22 nt of microhomology, which are indicated in red. Restriction enzyme sites generated due to microhomology-mediated joining are also indicated. (b) Comparison of MMEJ catalyzed by testicular extracts when different microhomology regions were used. Rat testicular extracts were incubated with oligomeric DNA substrates harboring 3, 5, 8, 10, 13, 16, 19 and 22 nt microhomology for 2 h at 25 ºC. End-joined products were detected by radioactive PCR. In case of every substrate, reactions are shown with either of the substrate or both. MMEJ products are indicated by arrow, while NHEJ products are bracketed. M and M' are molecular weight markers. (c) Comparison of MMEJ of oligomeric DNA containing DSBs when flanked with different length microhomology catalyzed by Reh cell-free extract. M and M' are markers as indicated
Mentions: In order to understand the extent of microhomology required for MMEJ, oligomeric DNA containing DSBs flanked by various lengths of microhomology (3, 5, 8, 10, 13, 16, 19 or 22 nt) were designed and synthesized in such a way that following MMEJ a restriction enzyme site would be generated (Figure 4a and Supplementary Tables 2 and 4A). Results showed a prominent band due to MMEJ in all the substrates, except for 3 nt microhomology, when incubated with testicular extracts (Figure 4b). In order to corroborate this observation, PCR products obtained from 3, 5, 8 and 13 nt microhomology substrates following MMEJ reaction, were digested with NotI, XmnI and XcmI, respectively. A low-intensity band corresponding to ~55 nt in 5 nt microhomology substrate was detected, which was digestible with NotI indicating that the joining was mediated by MMEJ (Supplementary Figure 4b). Similarly, bands corresponding to ~60 and 65 nt in 8 and 13 nt microhomology substrates were digestible with XmnI and XcmI, respectively (Supplementary Figure 4b). Besides, we could not find any specific band, which was sensitive to NotI digestion when 3 nt microhomology substrate was used (Supplementary Figure 4b). As there was no suitable restriction enzyme site, we could not perform similar analysis for substrates containing 10 nt microhomology. These findings suggest that MMEJ protein machinery recognizes microhomology regions to undergo end joining. Furthermore, a microhomology length of more than 3 nt is essential for its operation.

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