SAMHD 1‐mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination

Abstract

Sterile alpha motif and histidine‐aspartic acid domain‐containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi–Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c‐Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break‐point junctions is a notable feature in SAMHD1 deficiency during activation‐induced cytidine deaminase‐mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double‐stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1‐mediated dNTP balance regulates dNTP‐sensitive DNA end‐processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.

SAMHD1 restrains aberrant nucleotide insertions at repair junctions generated by DNA end joining

“…As our study reveals that insertions were increased in absence of catalytically active SAMHD1 or in presence of elevated dNTP pools, we propose that super-physiological dNTP levels may increase DNA polymerase resynthesis rates leading to increased refilling of staggered DNA ends and synthesis over inserted DNA templates prior end ligation. Strikingly, our data are in line with a parallel report, addressing SAMHD1 in class switch recombination of antibody constant regions in B cells, which was published during finalization of our manuscript ( 15 ). In this parallel work, it was concomitantly shown that absence of SAMHD1 and high intracellular dNTP levels lead to aberrant insertions at EJ dependent switch junctions.…”

“…In this study, we asked whether aside of involvement in homologous DNA repair, SAMHD1 may have an additional effect on NHEJ. Our own results reveal a novel role of SAMHD1 for DNA DSB repair by NHEJ and give new insights into its implication in genome stability and cancer development, and corroborate a very recent report on a novel role of SAMHD1 in regulating class switch recombination in B lymphocytes ( 15 ).…”

Charting a DNA Repair Roadmap for Immunoglobulin Class Switch Recombination

“…Furthermore, in agreement with the findings in mouse cells, a similar Cas9-based study highlighted the preferential use of 5′ overhangs over 3′ DSBs as substrates for Cas9-mediated CSR and translocations in human cells [73,74], and suggested a differential mode of overhang processing based on break polarity [74]. It has been shown recently that high cellular levels of purine nucleotides shift repair towards an increased use and length of insertions at junctions without affecting microhomology usage, and concomitantly reduce CSR efficiency and Igh translocation frequency [76]. A similar phenotype has been observed during Cas9-mediated CSR of staggered DSBs with 5′ overhangs, but not blunt ends [76].…”

“…It has been shown recently that high cellular levels of purine nucleotides shift repair towards an increased use and length of insertions at junctions without affecting microhomology usage, and concomitantly reduce CSR efficiency and Igh translocation frequency [76]. A similar phenotype has been observed during Cas9-mediated CSR of staggered DSBs with 5′ overhangs, but not blunt ends [76]. Considering that the conversion of AID-generated U:G mispairs generates a considerable portion of staggered DSBs, these findings suggest that insertional repair negatively impacts processing and joining of AID-induced breaks.…”

Locus-Specific Genomic DNA Purification Using the CRISPR System: Methods and Applications

“…A locus of interest can be isolated by insertion of recognition sequences of DNA-binding molecules, expression of the DNA-binding molecules in the cells, fragmentation of chromatin by sonication or other methods, followed by affinity purification of the DNA-binding molecules bound to the target locus. [4][5][6][7][8][9][10][11][12][13][14] Cross-linking can be performed, if necessary. The locus tagged with the recognition sequences of the DNA-binding molecules can also be isolated by cross-linking, if necessary, fragmentation, and incubation with recombinant or synthetic DNA-binding molecules before affinity purification of the DNA-binding molecules bound to the target locus.…”