RPA Accumulation during Class Switch Recombination Represents 5′–3′ DNA-End Resection during the S–G2/M Phase of the Cell Cycle

Genomics & Immunity, NIAMS, National Institutes of Health, Bethesda, MD 20892, USA.
Cell Reports (Impact Factor: 8.36). 01/2013; 3(1). DOI: 10.1016/j.celrep.2012.12.006
Source: PubMed


Activation-induced cytidine deaminase (AID) promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA)-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR), such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG), or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S-G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S-G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S-G2/M phase of the cell cycle.

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    • "Since recombination occurred at high frequency between the RSV repeats, which are upstream of the transcription start site and hence likely to be hundreds of bases away from the peak of AID-induced cytidine deamination (Saribasak et al., 2006), both models invoke substantial DNA resection to expose single stranded DNA distant from the nicks (Figure 10C,I). The fact that RPA (Hakim et al., 2012) as well as RPA together with RAD51 (Yamane et al., 2013) accumulated at multiple chromosomal positions within activated murine B cells in an AID-dependent fashion is consistent with the formation of recombination competent nucleoprotein filaments. Later stages of the models postulate heteroduxplex DNA and Holliday junctions (Figure 10E,I), which are consistent with the sequence analysis of homeologous repeat recombinants (Figure 7). "
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    ABSTRACT: The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells. DOI: http://dx.doi.org/10.7554/eLife.03110.001
    eLife Sciences 07/2014; 3(3):e03110. DOI:10.7554/eLife.03110 · 9.32 Impact Factor
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    • "This is reminiscent of the role of RAG proteins in orchestrating V(D)J recombination by generating DSBs and efficiently channeling them to productive recombination, keeping translocation risks at bay (109–111). Similar to RAG-dependent coordination of break induction and repair, AID phosphorylated at S38 not only facilitates break formation, but also interacts with the ssDNA-binding protein, replication protein A, and likely enforces DNA repair pathways during CSR (112, 113). "
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    ABSTRACT: Secondary diversification of the antibody repertoire upon antigenic challenge, in the form of immunoglobulin heavy chain (IgH) class-switch recombination (CSR) endows mature, naïve B cells in peripheral lymphoid organs with a limitless ability to mount an optimal humoral immune response, thus expediting pathogen elimination. CSR replaces the default constant (CH) region exons (Cμ) of IgH with any of the downstream CH exons (Cγ, Cε, or Cα), thereby altering effector functions of the antibody molecule. This process depends on, and is orchestrated by, activation-induced deaminase (AID), a DNA cytidine deaminase that acts on single-stranded DNA exposed during transcription of switch (S) region sequences at the IgH locus. DNA lesions thus generated are processed by components of several general DNA repair pathways to drive CSR. Given that AID can instigate DNA lesions and genomic instability, stringent checks are imposed that constrain and restrict its mutagenic potential. In this review, we will discuss how AID expression and substrate specificity and activity is rigorously enforced at the transcriptional, post-transcriptional, post-translational, and epigenetic levels, and how the DNA-damage response is choreographed with precision to permit targeted activity while limiting bystander catastrophe.
    Frontiers in Immunology 03/2014; 5:120. DOI:10.3389/fimmu.2014.00120
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    • "Direct measurement of DSBs has obvious advantages over foci-based methods in that quantitative analysis of the actual resection products can be performed; however, we note that this is limited by the requirement for sequence specificity of the cut site. Other methods such as RPA ChIP-Seq have also been used to address the need for resection assays at random or unknown DNA damage sites in the mammalian genome (62,63). A combination of these methods may be necessary to quantitatively measure ssDNA at nuclease-accessible as well as inaccessible sites and to assess resection efficiency in response to different types of DNA damage. "
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    ABSTRACT: 5' strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5' strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle-dependent manner. Depletion of CtIP, Mre11, Exo1 or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of Brca1 does not, suggesting that the role of Brca1 in HR is primarily to promote Rad51 filament formation, not to regulate end resection.
    Nucleic Acids Research 12/2013; 42(3). DOI:10.1093/nar/gkt1309 · 9.11 Impact Factor
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