FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC

Abstract

FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER.

Fig 1. FACT is implicated in the repair of oxidatively damaged DNA.

(A) FACT is recruited to the sites of oxidative DNA lesions. HeLa cells expressing either OGG1-EGFP (left) or DsRed-SSRP1 (the smaller subunit of FACT, right) were locally irradiated with 405-nm laser in the absence (upper two panels) or presence (lower two panels) of photosensitizer Ro-19-8022 and the accumulation of the fusions at the bleached sites (indicated with white arrow) was observed 5 minutes post irradiation (lower panels). (B) Same as (A), but for Hela cells expressing either Ku80-EGFP or DsRed-SSRP1. (C) Treatment of cells with H₂O₂ results in release of FACT from transcribed chromatin and in binding to chromatin regions associated with both DNA repair proteins and chromatin remodeling factors. Stable Hela cell lines expressing a fusion of HA with SSRP1, treated or not with H₂O₂, were used to immunopurify the chromatin bound FACT complexes. Upper panel shows the silver stained SDS gel of the proteins associated with either control FACT chromatin bound complex (-) or with the FACT chromatin bound complex isolated from H₂O₂ treated cells (+). The lower panel shows the association of the indicated proteins identified by Western blotting in the respective complexes. (D) Mass spectrometry identification of the polypeptides associated with control FACT chromatin bound complex (-) or with the FACT chromatin bound complex, isolated from H₂O₂ treated cells (+). Proteins present in the e-SSRP1.com together with the number of identified peptides are indicated. Proteins involved in transcription are shown in red. DNA repair proteins and chromatin remodelers are shown in blue.

Fig 2. The simultaneous presence of both FACT and RSC, but not FACT alone, is required for efficient UDG excision of uracil at nucleosomal DNA sites oriented towards the histone octamer.

Centrally positioned nucleosomes were reconstituted by using ³²P 5’-labeled 255 bp 601 DNA fragment containing randomly incorporated uracil residues. Lanes 1–6: analysis of the UDG enzymatic activity within the nucleosomal DNA. The nucleosome solution was incubated with the indicated increasing (nine-fold step) amount of UDG for 60 minutes at 30°C and the cleavage pattern of the isolated DNA was analyzed using PAGE under denaturing conditions; lane 2: no UDG added; lanes 1, 25: •OH footprinting of the nucleosomes. Lanes 7–12: RSC induces a highly efficient UDG-mediated excision of uracil at inward facing sites within the nucleosome. Nucleosomes were incubated with increasing (two-fold step) amount of RSC (units) for 50 min at 30°C, and after arresting the reaction they were treated with 1.2x10^-2 units of UDG and the isolated cleaved DNA analyzed on denaturing PAGE; lane 7, control with no RSC added in the reaction. Lanes 13–19: FACT facilitates the RSC-dependent UDG excision of uracil at inward facing sites within the nucleosome. Nucleosomes were incubated with increasing (2-fold step) amount of FACT in the presence of 0.2 units of RSC and, after arresting the reaction they were treated with 1.2x10^-2 units of UDG. The cleaved purified DNA was analyzed on denaturing PAGE; lane 13, control containing 1.6 pmol of FACT with no RSC added. Note that the excision of uracil by UDG is unaffected at this highest concentration of FACT used in the experiment. Lanes 20–24: UDG cleavage pattern of the naked 255 bp 601 fragment. The experiment was carried out as described in Lines 2–6, but with nine-fold smaller concentration of UDG on each respective point; on the left is shown schematics of the nucleosome.

Fig 3. FACT facilitates both RSC-induced remodeling and mobilization of nucleosomes.

(A) DNase I footprinting. End-positioned nucleosomes, reconstituted on ³²P 5’-labeled 241 bp 601 DNA fragment, were incubated with 0.2 units of RSC in the absence (lane 3) or in the presence of 1.6 pmol of FACT (lane 4) for 50 min at 30°C; lane 5, same as lane 3, but with 1 unit of RSC; After arresting the remodeling reaction, the samples were digested with 0.1 units of DNase I for 2 min, the cleaved DNA was isolated and run on 8% PAGE under denaturing conditions; lanes 1 and 2, controls showing the DNase I cleavage pattern of nucleosomes (lane 1) alone or incubated with 1.6 pmol FACT under the conditions described above. (B) The presence of FACT increases the efficiency of RSC-induced nucleosome mobilization. Centrally positioned nucleosomes were incubated with 0.2 units of RSC in the presence of increasing amount of FACT, the reaction was arrested and the samples were run on native PAGE. The position of the non-mobilized and the slid end-positioned nucleosomes were indicated; lane 1 control nucleosomes; lane 2, nucleosomes incubated with RSC alone (in the absence of FACT). (C) Quantification of the data presented in (B).

Fig 4. “In gel one pot assay” analysis of the effect of FACT on the DNA accessibility towards HaeIII along the length of nucleosomal DNA in control and RSC treated nucleosomes.

(A, B) Effect of FACT on RSC-induced remosomes generation. (B) Preparative PAGE. Centrally positioned nucleosomes were treated with increasing amount of FACT in the presence of 0.2 units of RSC and after arresting the reaction they were separated on native PAGE; last lane, nucleosomes treated with 5-fold higher amount (1 unit) of RSC, in the absence of FACT; the first three lanes, untreated, and treated with FACT and with 0.2 units of RSC nucleosomes, respectively. The indicated bands (from 1 to 9) were excised from the gel and in-gel digested with 8 units of HaeIII for 10 minutes at 30°C. The cleaved DNA was then isolated and separated in 8% PAGE under denaturing conditions (B). The positions at the cleavage of the different dyads are indicated on the left; the numbers of each lane refers to the respective excised bands from the preparative PAGE (see A); ctrl, control, non-digested DNA; DNA, naked DNA used for reconstitutions of the nucleosomes digested with HaeIII. (C) Quantification of the data presented in (B).

Fig 5. Efficient UDG excision of uracil from RSC-generated remosomes and slid nucleosomes.

³²P 5’-labeled 255 bp 601 DNA fragment containing randomly incorporated uracil residues was used for reconstitution of centrally positioned nucleosomes. The nucleosomes were treated with RSC either in the absence of ATP (control particles) or in the presence of ATP to produce ∼ 50% mobilized particles. The remodeling reaction was arrested and the samples were separated on native PAGE. The end-positioned slid nucleosomes and the non-mobilized nucleosomes (containing the remosome fraction) as well as the control nucleosomes were eluted from the gel slice. The particles were then treated with the indicated increasing concentrations of UDG, the cleaved DNA was isolated and run on 8% PAGE under denaturing conditions; DNA, naked 255 bp 601 DNA fragment digested with UDG; first and last lane, •OH footprinting of native nucleosomes; on the right part of the figure is shown a schematics of the reconstituted nucleosome.

Fig 6. FACT increases the efficiency of nucleosome remodeling by RSC without affecting the ATP hydrolysis.

(A) Nucleosome mobilization assay. Centrally positioned nucleosomes were incubated with 0.3 units of RSC at 120 μM of ATP in either the absence or presence of increasing amount of FACT for 50 minutes at 30°C. After arresting the reaction, the samples were run on native PAGE. The bands corresponding to the centrally and end-positioned nucleosomes are indicated. The lower panel represents the respective quantified data. (B) ATPase hydrolysis assay. Centrally positioned nucleosomes were incubated with RSC (0.3 units) and increasing amount of FACT in the presence of 120 μM of ATP and 3.3 μM of ³²P-γATP. The products of the ATP hydrolysis were analyzed on 15% PAGE under denaturing conditions. Lower panel shows the respective quantified data. Fluctuations of the values are within the experimental error, typically ±10%.