GANP Regulates the Choice of DNA Repair Pathway by DNA-PKcs Interaction in AID-Dependent IgV Region Diversification

Abstract

RNA export factor germinal center–associated nuclear protein (GANP) interacts with activation-induced cytidine deaminase (AID) and shepherds it from the cytoplasm to the nucleus and toward the IgV region loci in B cells. In this study, we demonstrate a role for GANP in the repair of AID-initiated DNA damage in chicken DT40 B cells to generate IgV region diversity by gene conversion and somatic hypermutation. GANP plays a positive role in IgV region diversification of DT40 B cells in a nonhomologous end joining–proficient state. DNA-PKcs physically interacts with GANP, and this interaction is dissociated by dsDNA breaks induced by a topoisomerase II inhibitor, etoposide, or AID overexpression. GANP affects the choice of DNA repair mechanism in B cells toward homologous recombination rather than nonhomologous end joining repair. Thus, GANP presumably plays a critical role in protection of the rearranged IgV loci by favoring homologous recombination of the DNA breaks under accelerated AID recruitment.

GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development

“…D). Moreover, GANP interacts with DNA‐PKcs and is involved in the choice of DNA repair pathway toward higher fidelity homologous recombination by the suppression of error‐prone non‐homologous end‐joining of the camptothecin‐induced double‐strand DNA breaks . These results clearly support that GANP is an essential molecule to counteract at the very early stage of tumor initiation by preventing extensive changes in the genome associated with oncogenic mutations and chromosomal translocations during the growth and development of mammary gland cells.…”

“…GANP has been shown to inhibit DNA recombination in NIH3T3 cells using a β‐galactosidase tandem‐repeat construct for both extrachromosomal and genome‐integrated substrate DNA . GANP interacts with DNA‐dependent protein kinase, catalytic subunit (DNA‐PKcs) and may regulate the DNA repair pathway during homologous recombination, suggesting that this protein plays roles in DNA repair and genomic stability.…”

Integrity of immunoglobulin variable regions is supported by GANP during AID-induced somatic hypermutation in germinal center B cells

“…These changes in the SHM profile are quite similar to those reported previously in the B cells of UNG −/− mice ( 13 ) and in chicken DT40 B cells ( 38 ). Therefore, we re-examined the effects of GANP on the SHM profile in the genomic IgV L segment of DT40 B cells (Supplementary Figure S3a, available at International Immunology Online) ( 39 ). We used an UNG −/− mutant DT40 clone that does not undergo gene conversion in the IgV region, but instead produces SHM Ts mutations in the IgV region upon the introduction of AID (AID R UNG −/− ) ( 39 ).…”

“…Therefore, we re-examined the effects of GANP on the SHM profile in the genomic IgV L segment of DT40 B cells (Supplementary Figure S3a, available at International Immunology Online) ( 39 ). We used an UNG −/− mutant DT40 clone that does not undergo gene conversion in the IgV region, but instead produces SHM Ts mutations in the IgV region upon the introduction of AID (AID R UNG −/− ) ( 39 ). GANP O/E in AID R UNG −/− cells induces a marked 7-fold increase in the SHM mutation frequency (105.5 × 10 −3 ) compared with that in the AID R UNG −/− cells (14.5 × 10 −3 ) (Supplementary Figure S3b, available at International Immunology Online).…”

A double-strand break can trigger immunoglobulin gene conversion

“…AID induces a DNA lesion as an early step of B cell-specific recombination by its cytidine deaminase activity (23,24); however, as the N-terminal of AID is responsible for Ig gene conversion and somatic hypermutation and the C-terminal domain of AID is essential for switch recombination (48,49), AID can further influence the dissected B cell-specific recombination pathways, possibly via co-factors that bind the N- or C-terminus of AID. The germinal center-associated nuclear protein is one of the candidates that affects the fate of DSB in Ig V regions in the presence of AID (50,51). An AID-induced DNA break is supposed to be protected by AID co-factors that direct the DNA lesion to the gene conversion pathway and prevent undesired DNA repair pathways.…”