Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing

Abstract

DNA strand breaks are repaired by DNA synthesis from an exposed DNA end paired with a homologous DNA template. DNA polymerase delta (Pol δ) catalyses DNA synthesis in multiple eukaryotic DNA break repair pathways but triggers genome instability unless its activity is restrained. We show that human HelQ halts DNA synthesis by isolated Pol δ and Pol δ-PCNA-RPA holoenzyme. Using novel HelQ mutant proteins we identify that inhibition of Pol δ is independent of DNA binding, and maps to a 70 amino acid intrinsically disordered region of HelQ. Pol δ and its POLD3 subunit robustly stimulated DNA single-strand annealing by HelQ, and POLD3 and HelQ interact physically via the intrinsically disordered HelQ region. This data, and inability of HelQ to inhibit DNA synthesis by the POLD1 catalytic subunit of Pol δ, reveal a mechanism for limiting DNA synthesis and promoting DNA strand annealing during human DNA break repair, which centres on POLD3.

CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single stranded DNA gaps

“…Recent studies have shown that HELQ interacts with the POLD3 subunit of the DNA replicative polymerase Polο to suppress DNA synthesis during break-induced replication (BIR) and promote DNA strand annealing (He et al, 2023). We thought to investigate if loss of POLD3 has a similar impact as HELQ on ssDNA gap accumulation in PRIMPOL-overexpressing cells.…”

“…Previous studies showed that BRCA-deficient cells accumulate gaps upon treatment with cisplatin and other replication stress-inducing agents, and this may correlate with their chemosensitivity (Cong et al, 2021a;Hale et al, 2023;Panzarino et al, 2021;Quinet et al, 2021;Quinet et al, 2020;Tirman et al, 2021). The surprising gap suppression effect observed upon RAD52 depletion in PRIMPOL-overexpressing cells prompted us to investigate if a similar effect occurs in BRCA-deficient cells.…”

“…RAD52 was also previously suggested to participate, directly or indirectly, in the recruitment of the MRE11 nuclease to stalled forks in BRCA-deficient cells (Mijic et al, 2017;Ronson et al, 2023). Recent studies indicated that MRE11 participates in the exonucleolytic expansion of ssDNA gaps in both BRCA-deficient cells as well as in PRIMPOL-overexpressing cells (Hale et al, 2023;Nusawardhana et al, 2024;Tirman et al, 2021). A reduction in MRE11 recruitment to ssDNA gaps may potentially explain the suppression of ssDNA gap accumulation in PRIMPOLoverexpressing cells.…”

“…It is also possible that the BRCA pathway suppresses the engagement of nucleases on ssDNA structures, similar to its activity in protecting reversed forks against nucleolytic degradation. In line with this, it was recently shown that nucleases including MRE11 and EXO1 are expanding ssDNA gaps in BRCA-deficient cells (Hale et al, 2023;Tirman et al, 2021). Alternatively, ssDNA gaps can also be filled through translesion DNA synthesis (TLS), a process which involves specialized, low-fidelity polymerases able to bypass DNA lesions and extend beyond the lesion on the undamaged template.…”

“…This process leaves behind a single stranded DNA (ssDNA) gap, which needs to be filled at a later time. The BRCA pathway was shown to be important for gap suppression (Cong et al, 2021a;Hale et al, 2023;Panzarino et al, 2021;Quinet et al, 2021;Quinet et al, 2020;Tirman et al, 2021). This potentially reflects a role for BRCA-mediated recombination using the nascent strand of the sister chromatid as template for gap filling.…”