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.
Highlights
Nonhomologous end joining (NHEJ) (A-NHEJ) has been described to have an important role in double-strand breaks (DSBs) repair.[4,5,6,7] It has been shown that majority of A-NHEJ
It can be concluded that canonical NHEJ (C-NHEJ) requires LIGASE IV–XRCC4 complex, while A-NHEJ is predominant in the absence of C-NHEJ proteins and is mainly characterized by joining utilizing microhomology (MMEJ)
We show that a minimum of 5 nt microhomology is required for microhomology-mediated end joining (MMEJ) and is independent of classical NHEJ proteins such as KU70, KU80 and LIGASE IV
Summary
NHEJ (A-NHEJ) has been described to have an important role in DSB repair.[4,5,6,7] It has been shown that majority of A-NHEJ-. It was shown that MRE11-RAD50-NBS1 complex may be involved in a subset of alternative NHEJ,[5,12,13,14] whereas ATM has a regulatory role.[15] Role of PARP1 in repairing switch regions through a microhomology-mediated pathway leading to IgH/c-MYC translocations during immunoglobulin CSR has been described.[16] Besides, studies have suggested a role for DNA LIGASE IIIα and WRN in A-NHEJ.[17] Interestingly, XRCC1 was shown to be dispensable in A-NHEJ during CSR, whereas functional relevance of Ligase I, III and Pol λ have been established.[18,19,20] it can be concluded that canonical NHEJ (C-NHEJ) requires LIGASE IV–XRCC4 complex, while A-NHEJ is predominant in the absence of C-NHEJ proteins and is mainly characterized by joining utilizing microhomology (MMEJ). We show that MRN complex, XRCC1, FEN1, PARP1 and LIGASE III are the factors responsible for joining mediated through microhomology
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