HELQ promotes RAD51 paralogue-dependent repair to avert germ cell loss and tumorigenesis

Abstract

Repair of interstrand crosslinks (ICLs) requires the coordinated action of the intra-S-phase checkpoint and the Fanconi anaemia pathway, which promote ICL incision, translesion synthesis and homologous recombination (reviewed in refs 1, 2). Previous studies have implicated the 3'-5' superfamily 2 helicase HELQ in ICL repair in Drosophila melanogaster (MUS301 (ref. 3)) and Caenorhabditis elegans (HELQ-1 (ref. 4)). Although in vitro analysis suggests that HELQ preferentially unwinds synthetic replication fork substrates with 3' single-stranded DNA overhangs and also disrupts protein-DNA interactions while translocating along DNA, little is known regarding its functions in mammalian organisms. Here we report that HELQ helicase-deficient mice exhibit subfertility, germ cell attrition, ICL sensitivity and tumour predisposition, with Helq heterozygous mice exhibiting a similar, albeit less severe, phenotype than the null, indicative of haploinsufficiency. We establish that HELQ interacts directly with the RAD51 paralogue complex BCDX2 and functions in parallel to the Fanconi anaemia pathway to promote efficient homologous recombination at damaged replication forks. Thus, our results reveal a critical role for HELQ in replication-coupled DNA repair, germ cell maintenance and tumour suppression in mammals.

Figure 1. A mouse model of HELQ deficiency

a, HelQ genomic locus; base pairs are indicated above; introns are not to scale; exons are roughly to scale with black bar indicating 1 kb; location of the β-geo trap and genotyping primers are shown. b, HELQ domain architecture: amino acids are indicated, red bar spans epitope recognized by the HELQ antibody used for western blotting. c, HelQ genotype PCR. d, Lysates from ear fibroblasts of HelQ mice probed for HELQ. e, The number of litters (black) and pups (pink) generated per 21-day gestational interval. f, HelQ testis images and weights. g, Histological sections of HelQ gonads. Atrophic tubules (A); developing ovarian follicles (*). h, 5-day-old neonatal testes labelled with the Sertoli marker WT1 (brown) and hematoxylin (blue), to reveal spermatogonia (*); quantification of spermatogonia (SG). i, Epithelial and stromal tumour free survival of HelQ mice. j, Frequency of mice with 2 or more primary tumours, and k, female-specific pathology.

Figure 2. HelQ damage sensitivity and HelQ/FancD2 double mutant phenotypes

a-d, Clonogenic survival assays of immortalized cells exposed to the indicated damaging agents. e, Metaphase chromosomes (meta’s) from immortalized cells treated with 5 ng/ml MMC for 16 hr; frequency of metaphases with radials ± MMC are indicated. f, Testis weights of wild-type, single, and HelQ/FancD2 double mutant mice. g, Histological sections of testes. Atrophic (A); Sertoli cell only (S). h, i, Clonogenic survival assays as in (a). j, Metaphase aberrations from immortalized cells treated ± 10 ng/ml MMC for 16 hr.

Figure 3. HELQ interacts with DNA replication stress response factors

a, Unique and total peptides identified by MS analysis of HELQ-FLAG co-IPs isolated from 293 cells. b, HELQ interaction network based on the MS results (coloured lines) and reported interactions from BIOGRID, STRING and MINT databases (dashed lines). c, Western blots of input and FLAG IPs from HELQ-FLAG and FLAG control samples. d, Reciprocal IP of endogenous RAD51C with HELQ-FLAG. e, Endogenous HELQ and RAD51C IPs from 293T cells. f, Purified, HIS-tagged BCDX2 complexes (top panel) were incubated with FLAG-complexed beads (bottom panels) to test for a direct interaction. FLAG and ALC1-FLAG are shown as negative controls. g, Western blot analysis of whole cells extracts (WCE) and chromatin fractions (Chr) from NIH3T3 HELQ-GFP expressing cells treated ± 100 ng/ml MMC for 24 hr.

Figure 4. HELQ influences DNA repair and HR efficiency

a, Lysates from immortalized mouse cells, grown under physiological O₂ and treated ± 3 μM APH for 6 hr, were probed for FANCD2. Wild-type (WT); HELQ-deficient (HQ); unmodified (S) and ubiquitinated (L) forms of FANCD2. b, Chromatin fractions from immortalized mouse cells, probed for RAD51, histone H3 and α-tubulin at the indicated time points (hr) following treatment with ± 100 ng/ml MMC. c, Left panel, representative images of RAD51 focus formation in immortalized mouse cells at the indicated time points (hr) following treatment with 1 μM MMC. Right panel, quantification of RAD51 foci at the indicated time points. d, Pulsed field gel electrophoresis of genomic DNA from immortalized cells treated ± 1 μM MMC for 1 hr and recovered for the indicated number of hr. Undamaged (un). Wells: intact DNA; arrow: band containing large chromosomal fragments (10-0.45 Mb); below arrow: smaller fragments, resolved by size. e, HR frequencies in DR-GFP reporter cells treated with the indicated siRNAs. f, Clonogenic survival assays of immortalized mouse cells exposed to PARPi.