Polymerase η Recruits DHX9 Helicase to Promote Replication across Guanine Quadruplex Structures

Abstract

DNA polymerase η (Pol η) catalyzes accurate bypass of ultraviolet light-induced cyclobutane pyrimidine dimers, and it also functions in several other related processes, including bypassing DNA with unusual structures. Here, we performed unbiased proteome-wide profiling of Pol η-interacting proteins by using two independent approaches, i.e., proximity labeling and affinity pull-down followed by LC-MS/MS analysis. We identified several helicases, including DHX9, as novel Pol η-interacting proteins. Additionally, ChIP-Seq analysis showed that Pol η is enriched at guanine quadruplex (G4) structure sites in chromatin. Moreover, Pol η promotes the recruitment of DHX9 to G4 structure loci in chromatin and facilitates DHX9-mediated unwinding of G4 structures. Deficiency in Pol η or DHX9 leads to attenuated replication across G4 regions in genomic DNA. Together, we unveiled the interaction between Pol η and DHX9 and demonstrated that the interaction promotes the replicative bypass of G4 structures in chromatin.

Figure 1.

APEX labeling and affinity pull-down for revealing the Pol η interactome. (a, b) Volcano plots displaying enriched proteins (highlighted in red) for Pol η-APEX versus the control (NLS-APEX) obtained from APEX labeling and LC-MS/MS analysis (a) or anti-Flag pull-down using CRISPR-engineered Pol η-Flag cells versus parental HEK293T cells (b). Proteins with a > 1.5-fold signal over control and a p-value < 0.05 are considered enriched. (c) Venn diagram illustrating the candidate Pol η-interaction proteins obtained from the two proteomic approaches. (d) Western blot results showing Flag pull-down using lysates of Pol η-Flag cells and parental HEK293T cells.

Figure 2.

ChIP-Seq revealed enrichment of Pol η at G4 structure loci in chromatin. (a) Relative enrichment of Pol η peaks in different genomic elements. The dashed line represents an enrichment fold of 1.0. Error bars represent standard deviations from two biological replicates. (b) Venn diagram showing the overlap between Pol η ChIP-seq and BG4 ChIP-Seq peaks. (c) Average signal of Pol η ChIP-Seq against the center of the BG4 ChIP-Seq peaks and (d) vice versa. (e) IGV plots showing the comparison of Pol η occupancy and G4 structure loci in the indicated genomic region.

Figure 3.

Pol η recruits DHX9 to promote the replication across G4 structure sites in DNA. Representative IGV plots showing signal tracks for Pol η and BG4 ChIP-Seq results for PMS2 (a), NEAT1 (b), CLSPN (c), and JUN (d) genes in HEK293T cells. (e) ChIP-qPCR analyses at the G4/non-G4 loci using DHX9 antibody. (f) Amount of BrdU incorporation at the G4/non-G4 regions of the indicated genes with or without knocking down DHX9 in XPV and XPV + Pol η cell lines. The data represent mean ± SD from three independent experiments. The p values were calculated by using two-tailed, unpaired Student’s t-test: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4.

Model of Pol η’s function in promoting replicative bypass of G4 structures through DHX9.