During an immune response, mature B-lymphocytes undergo CSR, a deletional-recombination reaction that exchanges the C
µ constant region gene (CH
) of the expressed Igh
for one of a set of downstream constant CH
genes, such as C
ε or C
α. The B cell thus alters from producing IgM to one expressing a different effector antibody molecule such as IgG, IgE or IgA1
. CSR occurs between transcribed, repetitive 1–12 kb long DNA elements termed switch (S) regions that precede each of the CH
. Activation-induced cytidine deaminase (AID), an essential enzyme for CSR2,3
, deaminates cytidines to uridines within transcribed S regions to initiate a cascade of reactions that generates staggered DSBs4
. Synapsis and end-joining of DSBs between two distinct S regions completes CSR.
The end-joining phase of CSR utilizes general DNA repair processes5
. C-NHEJ, which seals DNA ends with little (1–3 nucleotides) or no homology, is a major DSB repair pathway in somatic cells6
and was thought to be essential for CSR7
. However, recent studies have shown that mutations in several core C-NHEJ components including Ku70, DNA ligase IV and XRCC4 still allowed substantial CSR8–11
. The mutant cells though had a striking alteration in the nature of the switch junctions. While the majority of junctions in normal B cells were either blunt or had 1–3 base pairs of microhomology, those in the C-NHEJ mutants displayed a significant trend towards increased microhomology8–11
. Thus, CSR proceeds through a robust A-NHEJ pathway that displays a significant bias towards microhomology joins.
In addition to CSR, A-NHEJ has also been observed in a few other instances. First, several reporter substrates that measure joining of microhomologous DNA sequences have revealed the existence of A-NHEJ12–15
. Second, while C-NHEJ is essential for end-joining of DSBs generated by RAG proteins during V(D)J recombination, certain RAG mutations unmasked an A-NHEJ reaction that utilized microhomologous sequences for end-joining of reporter recombination substrates16,17
. Finally, interchromosomal translocations involving the Igh
locus frequently observed in C-NHEJ mutant B cells appear to use the A-NHEJ pathway9,18,19
. This process is predicted to involve a DNA end resection step to expose short single-stranded DNA stretches homologous to the other end being joined. Whether all or a subset of microhomology-mediated end joining constitute A-NHEJ is a matter of debate20
but it is clear that A-NHEJ preferably utilizes microhomology sequences. In this study, we have referred to all microhomology-mediated end-joining as A-NHEJ.
The factors required for A-NHEJ have not been elucidated; however, the end-processing proteins Mre11 and CtIP are thought to be involved12–15,21,22
. CtIP was originally identified as an interactor of the transcriptional co-repressor molecule CtBP and was thus thought to modulate transcription23
. It was subsequently shown to participate in cell cycle checkpoint control23
and DNA repair by homologous recombination (HR) through its ability to bind BRCA1 in a phosphorylation-dependent fashion13,24–26
. Additionally, CtIP and its yeast functional homologue Sae227
have been shown to be involved in resection of DSBs during homologous recombination, either acting directly as a nuclease and/or enhancing the nuclease activity of Mre1125,26,28–32
. Recently, studies using reporter substrates have demonstrated that CtIP participates in A-NHEJ12,13,15
, although the role of CtIP in HR and A-NHEJ are distinct as unlike that for HR, A-NHEJ does not require phosphorylation-dependent interaction with BRCA113
The overall model that emerged from these studies is that CtIP promotes processing of DSBs to reveal segments of homology that could be utilized for HR-based repair or stretches of microhomology for A-NHEJ. However, the majority of these studies, especially those that examined the role of CtIP in A-NHEJ, relied on the use of artificial substrates, which could potentially have a dominant effect on the nature of the end-joining reaction20
. In this study, we have used CSR as a physiological reaction to query the role of CtIP in A-NHEJ and demonstrate that CtIP plays a major role in microhomology mediated end-joining in normal as well as in C-NHEJ deficient cells.