The chromatin remodeler ERCC6 and the histone chaperone NAP1 are involved in apurinic/apyrimidinic endonuclease-mediated DNA repair

Abstract

During base excision repair (BER), the apurinic or apyrimidinic (AP) site serves as an intermediate product following base excision. In plants, APE-redox protein (ARP) represents the major AP site of cleavage activity. Despite the well-established understanding that the nucleosomal structure acts as a barrier to various DNA-templated processes, the regulatory mechanisms underlying BER at the chromatin level remain elusive, especially in plants. In this study, we identified plant chromatin remodeler Excision Repair Cross-Complementing protein group 6 (ERCC6) and histone chaperone Nucleosome Assembly Protein 1 (NAP1) as interacting proteins with ARP. The catalytic ATPase domain of ERCC6 facilitates its interaction with both ARP and NAP1. Additionally, ERCC6 and NAP1 synergistically contribute to nucleosome sliding and exposure of hindered endonuclease cleavage sites. Loss-of-function mutations in Arabidopsis (Arabidopsis thaliana) ERCC6 or NAP1 resulted in arp-dependent plant hypersensitivity to 5-fluorouracil, a toxic agent inducing BER, and the accumulation of AP sites. Furthermore, similar protein interactions are also found in yeast cells, suggesting a conserved recruitment mechanism employed by the AP endonuclease to overcome chromatin barriers during BER progression.

Figure 1.

ARP interacts with chromatin remodeler CHR24 and histone chaperone NAP1 proteins. A) co-IP was performed to detect the interactions between YFP-NAP1;2, CHR24-FLAG, and MYC-ARP in planta. Total protein extracts from the mesophyll protoplasts expressing tagged proteins were first immune-precipitated with or without antibodies against GFP, FLAG, and MYC, respectively, followed by immunoblot analysis of resulting fractions. The arrowheads indicate the target protein bands, taking into consideration potential protein degradation or nonspecific bands observed in the immunoblot results. B) Purified MBP and MBP–ARP proteins in Coomassie Brilliant Blue (CBB)-stained SDS–PAGE gel. C) Immobilized MBP–ARP and MBP (control) were mixed with protein extracts from mesophyll protoplasts expressing YFP-NAP1;2 and CHR24-FLAG for binding assays. D) A schematic diagram of CHR24 was presented along with three truncated parts: CHR24-N, CHR24-ATPase, and CHR24-C. The replacement of GLGKT site by DEDEV was shown as CHR24-ATPase^mut. E) GST and GST-tagged truncated CHR24 proteins (upper panel) were purified for pulldown assay examined by antibody against GFP (YFP-NAP1;2, middle panel) or against MYC (MYC-ARP, lower panel).

Figure 2.

CHR24 and NAP1 can exert synergistic activity in sliding nucleosomes. A) The in vitro ATPase activity assay was conducted using GST-CHR24-ATPase^mut or GST-CHR24-ATPase protein (0.5 or 1 nmol). Mean values were shown with error bars indicating ±Sd from three biological replicate measurements. B) Schematic representation of nucleosome remodeling, where local nucleosome sliding exposed the inaccessible DpnII site (double-strand) or AP site (single-strand). C) The core histones and naked 225-bp DNA used in the nucleosome reconstitution. D) The ATP-dependent nucleosome remodeling was performed for indicated hours (h), with upper arrows representing intact nucleosomal DNA and the lower arrows indicating mobility of fragment cleaved by DpnII after remodeling. GST protein was used as negative control. E) Adding of His-NAP1; 2 greatly enhanced the remodeling activity of GST-CHR24. F) The ATP-dependent remodeling of AP-containing nucleosome (right panel) was performed for 1 h. AP-containing naked DNA (left panel) served as the negative control. To ensure resolution of single-stranded ARP-cleaved fragments, all DNA samples were purified and resolved in denatured PAGE gel. The intact DNA is indicated by the upper arrow, while the mobility of the fragment cleaved by ARP is highlighted by the lower arrow.

Figure 3.

Synergistic action of ERCC6 and NAP1 members in plant resistance to 5-FU. A) Phylogenetic tree depicting ERCC6 members and selected plant remodelers. ERCC6 members include CHR8/24 (in bold), yeast RAD26, and human ERCC6^CSB. Bootstrap values are indicated along branches. B) Images of 12-d-old plants grown on the medium with or without 5-FU. Bar = 10 mm. C) Fresh weight of five seedlings grown on the medium without 5-FU (Mock treatment) as one biological replicate. Mean values were shown together with error bars indicating ±Sd from 10 biologically independent replicates. Asterisks indicate significant differences between the wild type and mutant(s) (P < 0.05, t-test, two-tail). D) Comparison of plant sensitivities to 5-FU by normalizing fresh weights of five seedlings grown with 5-FU to those of plants grown under mock treatment conditions (as one biological replicate). Mean values were shown together with error bars indicating ±Sd from 10 biologically independent replicates. Statistically significant differences between different genotypes are denoted by distinct lowercase letters (P < 0.05, one-way ANOVA). E) Schematic diagram illustrating AP detection method using aldehyde reactive probe for biotin tagging at AP site, followed by recognition through Streptavidin-Horseradish Peroxidase (HRP) conjugate catalyzing substrate into colored product formation. F) Comparison of AP level in the genomic DNA extracted from plants in (B). Mean values were shown together with error bars indicating ±Sd from three biologically independent replicates. Statistically significant differences between different genotypes are denoted by distinct lowercase letters (P < 0.05, one-way ANOVA).

Figure 4.

The conserved interaction of Apn1 with Nap1 and Rad26 in yeast. A) co-IP was performed to detect interactions between FLAG-Apn1, HA-Rad26, and Nap1-MYC. Total protein extracts from the yeast cells expressing tagged proteins were first immunoprecipitated with or without antibodies against FLAG, HA, and MYC, respectively, and the resulting fractions were then analyzed by immunoblotting. The arrows indicate the target protein bands, taking into consideration potential protein degradation or nonspecific bands observed in the immunoblot results. Notably, Nap1-MYC exhibits two closely sized bands in immunoblot analysis, and we speculate that small fragment may undergo splicing in the corresponding transcript within host cells. B) Spot assay of yeast strains (W303-1a as wild type) in the presence of 15 or 30 μg/mL 5-FU.