DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

Abstract

18The DNA-dependent protein kinase (DNA-PK), composed of the KU heterodimer and the 19 large catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) 20 factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with 21 different isotypes by joining two DNA double-strand breaks at different switching regions via 22 the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair 23 phase of CSR. To initiate cNHEJ, KU binds to DNA ends, and recruits and activates DNA-PK. 24 DNA-PKcs is the best-characterized substrate of DNA-PK, which phosphorylates DNA-PKcs at 25 both the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation 26 sensitivity, suggesting a role of T2609 phosphorylation in DNA repair. Using the DNA-PKcs 5A 27 mouse model carrying an alanine substitution at the T2609 cluster, here we show that loss of 28 T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR 29 junctions recovered from DNA-PKcs 5A/5A B cells reveal increased chromosomal translocation, 30 excess end-resection, and preferential usage of micro-homologyall signs of the alternative 31 end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation 32 in promoting cNHEJ repair pathway choice during CSR. 33 34 Key points 35 Loss of T2069 cluster phosphorylation of DNA-PKcs promotes Alt-EJ-mediated CSR.36 37 58 recombination. Correspondingly, DNA-PKcs -/-(null) or Artemis -/mice are born of normal size at 59 the expected ratio but develop severe combined immunodeficiency (SCID) (4-6). Patients with 60 4hypomorphic mutations in DNA-PKcs or Artemis also develop SCID (12, 13). Mature B cells 61 undergo CSR, which replace the initially expressed IgM constant region with another 62 downstream constant region encoding a different isotype, to generate antibodies with different 63 effector functions. The cNHEJ pathway plays an important role in CSR. But in cells lacking a 64 core cNHEJ factor (e.g., Lig4 or Xrcc4), up to 25-50% of CSR can be achieved by the alternative 65 end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. 66 Consistent with DNA-PKcs and Artemis being dispensable for direct end-ligation, DNA-PKcs -/-67 B cells only have moderate defects in CSR (14, 15). Nevertheless, in recent high-throughput 68 sequence analyses, we found that CSR junctions recovered from DNA-PKcs -/-B cells contained 69 increased chromosomal translocations and extensive end-resection, and preferentially used MHs 70 (16), suggesting that the seemingly robust CSR achieved in DNA-PKcs -/cells is primarily 71 mediated by the Alt-EJ pathway like those in Lig4 -/or Xrcc4 -/-B cells (17, 18). Accordingly, 72 human patients with spontaneous mutations in DNA-PKcs show severe defects in both V(D)J 73 recombination and CSR and increased MH in the residual CSR junctions (12, 19). 74Active DNA-PK phosphorylates many substrates, among which, DNA-PKcs is the best-75 characterized...

The recent advances in non-homologous end-joining through the lens of lymphocyte development

“…Loss of Alt-EJ factors, including XRCC1, an obligatory partner of Lig3 in the nucleus 171 , PARP1, or POLQ, all did not compromise CSR efficiency alone 165,169,172,173 . Loss of DNA-PKcs [103][104][105]161,174 or DNA-PKcs phosphorylation at the T2609 cluster 89 has a mild effect on IgG1 CSR efficiency in vitro. But the CSR junctions recovered from the DNA-PKcs −/− or DNA-PKcs 5A/5A mice are highly enriched for MH 89,106 .…”

“…Consistent with this hypothesis, Xlf-deficiency substantially reduced plasmids based V(D)J recombination 30,81,82 , while largely dispensable for chromosomal V(D)J recombination [83][84][85] . Similarly, loss of DNA-PKcs or the T2609 cluster phosphorylation of DNA-PKcs have a significant impact on the end-ligation of plasmid substrates 65,70,86 , but not chromosomal substrates 87,88 or in vivo 51,77,78,89 . Strikingly, in both Xlf-deficient or DNA-PKcs-deficient cells, loss of ATM kinase activity or chromatin bounded ATM substrates -H2AX, MDC1, and 53BP1 abolish the residual end-ligation on chromosomal susbstrate 85,87,88,[90][91][92][93][94] , supporting a critical role of chromatin and DNA damage response in promoting end-ligation.…”

“…Second, IR sensitivity vs defects in V(D)J recombination. From a DNA repair point of view, RAG cleavage generates clean DNA ends that might be less vulnerable to end-ligation defects than complex DNA lesions including oxidized bases generated during IR 89,[103][104][105][106] . Consistent with this hypothesis, Artemis is not required for SJ formation during chromosomal V(D)J recombination, but clear responsible for ~10% of complex ends generated by IR 33,53,107 .…”

A novel assay for screening WIP1 phosphatase substrates in nuclear extracts

“…Autophosphorylation of DNA‐PK was originally proposed to facilitate NHEJ repair by regulating the dynamics of DNA‐PK at DNA double‐strand breaks [56]. Whereas importance of phosphorylation of the T2609 cluster for classical nonhomologous end‐joining has recently been confirmed, function of DNA‐PK‐pS2056 remains elusive [57–59]. Development of novel assays will be necessary for addressing a functional relevance of DNA‐PK phosphorylation mediated by ATM and removed by WIP1.…”

Stretch and Twist of HEAT Repeats Leads to Activation of DNA-PK Kinase

“…S5) and speculate that the displacement of ABCDE from inside to outside the M-HEAT ring is correlated with DNA-end binding. Being near the kinase active site in the complexes with DNA, ABCDE may be phosphorylated in cis and facilitate downstream events in NHEJ (Crowe et al, 2020).…”