Rodriguez Y et al. (2017), Unencumbered Pol β lyase activity in nucleosome...

XB-ART-54138

Nucleic Acids Res 2017 Sep 06;4515:8901-8915. doi: 10.1093/nar/gkx593.

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Unencumbered Pol β lyase activity in nucleosome core particles.

Rodriguez Y , Howard MJ , Cuneo MJ , Prasad R , Wilson SH .

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Packaging of DNA into the nucleosome core particle (NCP) is considered to exert constraints to all DNA-templated processes, including base excision repair where Pol β catalyzes two key enzymatic steps: 5'-dRP lyase gap trimming and template-directed DNA synthesis. Despite its biological significance, knowledge of Pol β activities on NCPs is still limited. Here, we show that removal of the 5'-dRP block by Pol β is unaffected by NCP constraints at all sites tested and is even enhanced near the DNA ends. In contrast, strong inhibition of DNA synthesis is observed. These results indicate 5'-dRP gap trimming proceeds unperturbed within the NCP; whereas, gap filling is strongly limited. In the absence of additional factors, base excision repair in NCPs will stall at the gap-filling step.

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Species referenced: Xenopus laevis
Genes referenced: h2ac21 h2bc21 utrn

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	Figure 1. Structural features of designed 5â²-dRP lesions and single nucleotide gaps in the nucleosome core particle (NCP). The color designation of the histones is shown at the top left and bottom. The template and lesion-containing strands of DNA are in cyan and magenta, respectively. Incorporation of dU within the nick at different locations allowed for strategic positioning of the lesions (red spheres) when reconstituted with the histone octamer so that they encompass the major structural parameters that regulate BER in the NCP: rotational and translational positioning of the DNA lesion. A total of four substrates containing either a 5â²-dRP group or a single-nucleotide gap were used in this study: two outwardly oriented O (+10) and O (â35), one inwardly oriented I (â49), and a lesion with no definitive rotational orientation located only 13 nt from the DNA ends (â60). As shown in the diagram at the bottom, the central 60 bp are organized by the histone H3âH4 tetramer; whereas, the 30 nt on either side are in contact with the H2A-H2B heterodimer, and the remaining 13 nt are loosely associated with the histones. The difference in histoneâDNA interactions in the central region vs. the ends gives rise to intrinsic differences in accessibility where regions bound by the H2BâH2A heterodimers are intrinsically more dynamic as described by others (47).
	Figure 2. Pol Î² binding to damaged and undamaged DNA and NCPs. Schematic representations of substrates are illustrated on top of each panel. (A) Fluorescently labeled Pol Î² V303C (5 nM) was titrated with NCPs containing 5â²-phosphorylated tetrahydrofuran (5â²-P-THF) at the 5â²-end of a DNA nick located 35 nucleotides from the dyad [NCP-5â²-P-THF-O (â35)] mimicking a single-nucleotide gap with a stable 5â²-dRP group. The 5â²-ends were blocked with biotin (bio) to prevent binding of Pol Î² to 5â²-phosphates. A representative curve from duplicate anisotropy experiments is shown. (B) DNA and NCP substrates containing 5â²-P-THF at position -35 were incubated with 0â6 Î¼M of Pol Î² on ice for 10 min. A representative image of a non-denaturing 6% polyacrylamide gel is shown in panel (D). (C) Undamaged (UND) DNA and NCP substrates were similarly incubated with varying amounts of Pol Î² as described in (B) with a representative image of a non-denaturing 6% polyacrylamide gel shown in (E). The data points and error bars shown in panels B & C represent the mean of three independent experiments Â± SD. The solid line corresponds to the fits of the data as described in Materials and Methods using a modified Hill binding equation from which the apparent dissociation constants were derived (Table 1).
	Figure 3. Pol Î² dRP lyase activity assay. (A) DNA oligo (15-mer) was 5â²-end labeled with Î³-32P-ATP. Experiments were conducted as described in Materials and Methods. The reaction with Pol Î² was initiated â¼30 s after UDG incubation. A negative control was included to subtract the spontaneous loss of the 5â²-dRP. A heat control (H), as described in Materials and Methods, was included to determine the actual dRP substrate generated after the 5.5-min UDG incubation. Full length Pol Î² (FL) or the N-terminal domain of Pol Î² (8 kDa) were used to determine dRP lyase activity. A representative phosphor image of a 20% polyacrylamide denaturing gel of the replicates plotted in panel (B) is shown. (B) NCP crystal structure pdb file 3LZ0 (37) was modified to show the structural location of the 5â²-dRP group (black spheres). This is the back view of that shown in Figure 1. Data represent the mean of three independent experiments Â± SD. Kinetic parameters are provided in Table 2 (full length Pol Î²) and Table 3 (8 kDa domain of Pol Î²). As shown in Table 2, the ratio (DNA/NCP) of the enzymatic rates for this 5â²-end labeled substrate is comparable to the ratio found by the 3â²-end labeling assay (Supplementary Figure S3). The rate of spontaneous loss, estimated from the âPol Î² control, for the NCP is â¼3.2 nM/min, which is 8-fold faster than the rate in DNA of 0.4 nM/min.
	Figure 4. Pol Î² dRP lyase activity on inwardly- and outwardly-oriented 5â²-dRP intermediate near the DNA ends. The procedure presented in Figure 3A was employed for these experiments. The crystal structure pdb file 3LZ0 was modified to show the structural location of the 5â²-dRP group (black spheres) in (A) dRP-O (â35) and in (B) dRP-I (â49). Experiments were conducted as described in Materials and Methods. The data represent the mean of three independent experiments Â± SD. Comparisons of the kinetic parameters are shown in Tables 2 and 3. The rate of spontaneous loss, estimated from the âPol Î² control, was the same in free DNA and corresponding NCP substrate with 1.4 nM/min for dRP-O (â35) and 0.4 nM/min for dRP-I (â49).
	Figure 5. The effect of rotational and translational positioning of a single-nucleotide gap on the single-turnover kinetics of Pol Î² catalyzed nucleotide insertion in NCPs. (A) Schematic illustrating the generation of the substrate and determination of the product by the appearance of 1-nt slower migrating band for gO (+10) substrates. (B) Structural location of the single-nucleotide gaps (black spheres). (C) Representative phosphor image of an 8% denaturing polyacrylamide sequencing gel for DNA-gO (+10) and NCP-gO (+10) showing the appearance of the product (P) as 1-nt slower migrating band relative to the substrate (S). Representative plots of three independent experiments are shown: (D) gO (+10), (E) gO (â35) and (F) gI (â49). The time- course for DNA was from 0â8 s and for NCP from 0â190 min. The final concentration for all single turnover experiments was 10 nM DNA, or 100 nM NCP with 1 Î¼M Pol Î² and 50 Î¼M dNTP. Data analysis and kinetic fits are described in Materials and Methods, and average rate constants from three independent experiments are summarized in Table 4.
	Figure 6. Steady-state kinetic analysis of Pol Î² catalyzed nucleotide insertion. The crystal structure of the 601 NCP pdb file 3LZ0 (37) was modified to highlight the position of the 1nt gap containing NCP substrates (black spheres). (A) Representative time-course for Pol Î² catalyzed nucleotide insertion with NCP-g (-60) and corresponding DNA. A substrate concentration of 500 nM and 50 nM Pol Î² for both NCP-g (â60) and DNA-g (â60) were used with 50 Î¼M dNTP. The average rate constant from three independent experiments is 0.6 Â± 0.12 minâ1 and 4.8 Â± 0.21 minâ1 for NCP and DNA, respectively. This shows an 8-fold inhibition at NCP-g (-60). B) Representative time-course for Pol Î² catalyzed nucleotide insertion with NCP-gO (+10) and corresponding DNA. Final substrate concentration of DNA-gO (+10) and NCP-gO (+10) was 500 nM with 1 nM and 50 nM Pol Î², respectively, and 50 Î¼M dNTP. Rate constants derived from the kinetic fits were 0.05 Â± 0.004 minâ1 for NCP-gO (+10) compared to 8.9 Â± 0.8 minâ1 for DNA-gO (+10), illustrating a 178-fold inhibition. Data were analyzed and fitted as described in Materials and Methods.
	Figure 7. Model for modulation of Polymerase Î² enzymatic activities by the structural constraints of the nucleosome core particle. (A) The structure of the NCP pdb file 1KX5 was modified to display b-factor values, color-coded as blue (rigid) and red (flexible).The bottom figure is a top view of the top figure rotated 180Â° counter clockwise. Lesions are shown in black. (B) Near the dyad, outwardly oriented 5â²-dRP lesions (black spheres) are repaired efficiently by Pol Î²; however, DNA synthesis is strongly inhibited despite the DNA gap being outwardly-oriented. This is due to occlusion of the templating base (red spheres) and the inability to stably engage the 31-kDa domain in a rigid region as shown in (A). Some DNA synthesis near the dyad occurs due to rotational flexibility, where the DNA rotates along its longitudinal axis on the surface of the histone octamer (dashed black lines). Near the DNA ends, 5â²-dRP groups are repaired efficiently, and inhibition of DNA synthesis is overall decreased. This is due to increased DNA flexibility and DNA unwrapping, although DNA gap-filling is nonetheless inhibited in a site-specific manner, indicating that local structural features as well as rotational orientation of the lesion play an important role for DNA gap-filling by Pol Î². Together, these results indicate external factors capable of remodeling the NCP structure are needed for efficient DNA synthesis.

References [+] :

Admiraal, Reactivity and Cross-Linking of 5'-Terminal Abasic Sites within DNA. 2017, Pubmed