Histone H3 Lysine 56 Acetylation Enhances AP Endonuclease 1

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Histone H3 lysine 56 acetylation enhances AP endonuclease 1-mediated repair of AP sites in nucleosome core particles Yesenia Rodriguez, Julie K Horton, and Samuel H. Wilson Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00433 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

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Biochemistry

Histone H3 lysine 56 acetylation enhances AP endonuclease 1-mediated repair of AP sites in nucleosome core particles Yesenia Rodriguez, Julie K. Horton and Samuel H. Wilson* Genome Integrity and Structural Biology Laboratory, National Institutes of Health, NIEHS, Research Triangle Park, North Carolina 27709, USA

KEYWORDS: AP endonuclease 1, base excision repair, histone H3 lysine 56 acetylation, nucleosome core particle, DNA damage

ABSTRACT: Deciphering factors modulating DNA repair in chromatin is of great interest since nucleosomal positioning influences mutation rates. H3K56 acetylation (Ac) is implicated in chromatin landscape regulation, impacting genomic stability. Yet, the effect of H3K56Ac on DNA base excision repair (BER) remains unclear. We determined whether H3K56Ac plays a role in regulating AP site incision by AP endonuclease 1 (APE1), an early step in BER. Our in vitro studies of acetylated, well-positioned nucleosome core particles (H3K56Ac-601-NCPs) demonstrate APE1 strand incision is enhanced compared with unacetylated WT-601-NCPs. The high-mobility group box 1 protein enhances APE1 activity in WT-601-NCPs, but this effect is not observed in H3K56Ac-601-NCPs. Therefore, our results suggest APE1 activity on NCPs can be modulated by H3K56Ac.

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INTRODUCTION Damage to DNA occurs continuously as a consequence of its inherent instability and propensity to undergo a myriad of spontaneous and agent-induced chemical modifications . In mammalian 3

cells, many of these genomic stressors give rise to the apurinic/apyrimidinic (AP) lesion in double stranded DNA, resulting in a steady-state level of 50,000-200,000 AP sites . Unrepaired and 3-5

persistent AP sites can have negative biological consequences as they disrupt replication and transcription, potentially leading to cytotoxic strand breaks and mutations . Accumulation of 6

mutations, and other forms of genomic instability, is associated with carcinogenesis, aging, and other neurological disorders . 7

The human AP endonuclease 1 (APE1) protein consists of 318 residues (~37 kDa) including regions involved in both DNA repair processes and redox regulation . In the context of DNA repair, 8

APE1 is responsible for removing more than 95% of the steady-state level of AP lesions by incising the DNA backbone 5´ to the AP site sugar using Mg as a cofactor . APE1 functions in the base 2+

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excision repair (BER) pathway following the activity of lesion-specific DNA glycosylases or spontaneous base loss. The incision product of APE1 following monofunctional glycosylase activity or through spontaneous base loss is a one-nucleotide gap characterized by a 3´-hydroxyl group and a 5´-deoxyribose phosphate (5´-dRP) group at the margins. The activity of APE1 is also required to remove 3´ blocking moieties after the action of bifunctional DNA glycosylases, which also cleave the DNA backbone. In all cases, the generated 3´-OH is subsequently utilized by a DNA polymerase that fills in the missing nucleotide(s). The integrity of DNA is restored after ligation of the DNA backbone by DNA ligase I or the DNA ligase III-XRCC1 complex.


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Biochemistry

APE1 is crucial for cell viability . In fact, APE1-null mice display significant developmental 10

problems and die at ~embryonic day 5.5 . In humans, dysregulated activity of APE1 has been 11

found in many solid tumors, and APE1 over-expression is associated with resistance to some traditional therapeutic treatments . These findings, along with its redox function, involved in 12

transcriptional regulation, have made APE1 an attractive candidate for the development of new therapeutic treatments; however, our understanding on how APE1 functions to remove AP sites is limited to kinetic and structural studies using short free DNA substrates . Recent reports provide 13, 14

some insight on the activity of APE1 in the context of the fundamental unit of chromatin, the nucleosome core particle (NCP). These studies documented reduced APE1 activity in the NCP in a site-specific manner . However, they did not provide the kinetic parameters necessary to better 15, 16

understand the potential mechanisms for this APE1 inhibition. Moreover, NCPs are subject to intrinsic and extrinsic regulation, and how these factors influence AP site removal has not been evaluated. Identifying these parameters is vital, given that AP sites are inherently unstable as they exist in an equilibrium between a closed-ring hemiacetal and an open-ring aldehyde , that is prone 17

to b-elimination reactions promoting DNA-protein crosslinks (DPCs) . DPCs are not repaired 18

efficiently and require alternative repair pathways such as nucleotide excision repair. Importantly, DPCs are likely to be converted into cytotoxic double strand breaks . The alkali lability of AP sites 19

is consequently influenced by the presence and proximity of histone lysine residues that can promote strand scission. Greenberg and colleagues have found AP sites to be almost two orders of magnitude more reactive in nucleosomes than in free DNA, where rotational orientation and 20

proximity to other AP sites play a role in AP site propensity to crosslink . Although these 18

observations are important for the overall biological effect and cellular consequences of AP sites, unraveling the kinetic mechanism with natural AP sites may be challenging due to this crosslinking


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susceptibility. Consequently, to delineate the kinetic parameters of APE1 activity in NCPs, we have utilized tetrahydrofuran (THF), a stable analog of the AP site, as the substrate for APE1 in all of our in vitro assays. NCP stability is influenced by histone posttranslational modifications (PTMs) and action of chromatin-associated factors, which are postulated to influence repair; however, it is still unknown if PTMs exert an effect on APE1 activity. In this study, we investigated the role of H3K56Ac on BER in vitro. It was previously shown that histone acetylation at H3K14 and H3K56 decreased DNA synthesis by DNA polymerase (Pol) b . Although the lesions were outside the region of 21

increased accessibility afforded by H3K56Ac

1, 2, 22, 23

, the negative correlation was unexpected, and it

suggested that histone acetylation in vivo may serve alternative roles than those associated with increased nucleosome dynamics or that this regulation is limited to the structural location of the DNA lesion. H3K56Ac is a unique histone PTM deposited behind the replication fork in newly synthesized H3 molecules. In the absence of DNA damage, SIRT6 can deacetylate this site when the cell enters G2 or later stages of the cell cycle ; however, when DNA damage is present, H3K56Ac levels 24

persist and are believed to promote repair due to charge neutralization at the entry-exit region of the NCP . Despite this apparent concept of increased accessibility potentially leading to increased 25

repair, the levels of H3K56Ac in response to DNA damage are still debatable with conflicting findings . In order to isolate the effects of histone site-specific acetylation on the kinetic 26, 27

parameters of APE1 activity, we generated homogeneously acetylated NCPs at H3K56 and H3K14. Our results with these NCPs as substrates indicate that APE1 activity is significantly hindered in the NCP. Site-specific histone acetylation that promotes nucleosome dynamics, such


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Biochemistry

as H3K56Ac, enhances APE1 activity. However, acetylation at the alternate site H3K14Ac does not have an effect on APE1 activity, suggesting the structural location of histone acetylation is important in mediating repair. HMGB1 can additionally enhance APE1 activity in the absence of H3K56Ac. Our results have uncovered the complexity of an intrinsic and extrinsic regulation of APE1 activity and suggest this regulation is imbedded in the structural position of histone acetylation relative to the lesion and the action of chromatin associated factors (i.e., HMGB1) in BER that had not been previously appreciated in the context of chromatin.

MATERIALS AND METHODS Preparation of DNA substrates containing tetrahydrofuran (THF). The 147-bp 601 nucleosome positioning DNA sequence was modified to introduce a single THF group 11 bp from 28

the 5´ end of the J chain, which is equivalent to 64 nt from the dyad, and designated as THF (+64). The forward primer containing the THF: 5´-FAM-CAC AGG ATG TTHFGA TAT CTG GCC TGG AGA CTA G-3´, the reverse primer: 5´-TGG AGA ATC CCG GTG CCG AGG CCG CTC AAT TG-3´, and the plasmid: pGEM-3Z/601 were used to generate the substrate via the polymerase chain reaction (PCR) listed in Table S1. Following a PCR reaction, the PCR product was concentrated using a standard ethanol precipitation protocol and purified from a 1.2% agarose gel using a DNA agarose gel extraction kit (Qiagen). For the substrates containing the 5S nucleosomal positioning sequence , the 162-bp DNA substrate was generated by first ligating the 21

damaged strand containing a single THF group and 5´-FAM, using a splinter complementary DNA upstream and downstream of the lesion. This ligation reaction contained 110 units of E. coli DNA ligase and 1x ligation buffer provided by the manufacturer (New England Biolabs). The ligated product and the undamaged complementary strand (162-mer) were PAGE purified and


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subsequently annealed by heating to 95°C for 10 min and slow cooling in buffer containing 30 mM Tris, pH 7.5 and 100 mM potassium acetate. The dsDNA substrate is listed in Table S1.

Nucleosome core particle reconstitution. Nucleosome core particles (NCPs) were reconstituted by salt gradient dialysis using recombinant octamer from Xenopus laevis containing all unmodified “wild type” (WT) core histones or histone H3 acetylated at Lys14 or Lys56. All WT core histones were each individually expressed in E. coli (BL21) as previously described . 29

After isolation, the histones were subjected to dialysis in a buffer containing 5 mM 2mercaptoethanol and 0.2 mM PMSF. They were then lyophilized until dry, and the histone octamer was prepared as described by Luger et al. Briefly, the concentration of each unfolded histone 30

protein was determined by A

280 nm

and equimolar amounts of all four histones were mixed and

dialyzed three times in refolding buffer (2 M NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM Na-EDTA, 5 mM 2-mercaptoethanol) at 4°C. Generation of homogeneously acetylated histone octamers containing either H3K14Ac or H3K56Ac was performed as described previously and in Neumann 30

et al. BL21 E. coli were co-transformed with a plasmid (pAcKRS-3) containing the ORF of the 22

gene directing the synthesis of acetyl-lysine-tRNA synthetase from M. barkeri (Mb), which directs insertion of aceyl-lysine residues at amber codons, and plasmid (pCDF PyIT-1) containing the MbtRNA gene and an N-terminally hexahistadine-tagged histone H3, encoding an amber codon CUA

at position 14 or 56, downstream of a T7 promoter (both recombinant modified histone octamers were a generous gift of Dr. Michael Smerdon, Washington State University). Purification and assembly of these site-specifically modified histone octamers has been described previously . Site21

specific acetylated or WT histone octamers were then mixed with DNA containing a single THF group in a 1.2:1 molar ratio via salt gradient dialysis to reconstitute NCPs as described

31, 32

.


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Biochemistry

Reconstitution efficiency was assessed in a 6% native/nondenaturing polyacrylamide gel, where reconstitutions containing £ 5% free DNA were used for biochemical assays.

Purification of WT and H133Y mouse SIRT6 proteins. Bacterial expression plasmids for mouse WT and H133Y were obtained from Addgene (plasmid IDs: 20277, pET28a-Hix6-SIRT6 and 20279, pET28a-Hix6-SIRT6-H133Y). Competent cells BL21-codon plus (DE3) from Agilent (230245) were used to express WT and H133Y SIRT6 following a standard heat-pulse transformation protocol at 42°C with a 23s sonic pulse. Terrific broth containing the appropriate antibiotics (chloramphenicol and kanamycin) was inoculated for an overnight culture and then maintained at 37°C for 1.5 h with vigorous shaking. After this time, the temperature was lowered to 16°C until the OD

600 nm

reached 0.7, at which time cells were induced with IPTG (0.5 mM)

overnight reaching an OD

600 nm

of 2.1, and then cells were harvested. The cell pellet from 1 l culture

was resuspended with 75 ml of low salt lysis buffer (50 mM Tris-Cl, pH 8, 500 mM NaCl, 0.5 mM TCEP and freshly added protease inhibitors (1X): Leupeptin (1 µg/ml), protinin (1 µg/ml), pefabloc (100 µg/ml), and PepstinA (1 µg/ml). Cells were lysed via sonication in a dry ice-ethanol bath at 40% power for 5 min, taking 30 s breaks. Lysed cells were centrifuged using the Beckman Coulter Optima L-100 XP Ultracentrifuge with rotor Ti-45 (41,000 rpm) at 4°C for 1 h. The supernatant fraction was passed through a pre-equilibrated HisTrap HP column: washed with 2 column volumes of 50 mM EDTA, washed with 3 column volumes of dH O, charged with 2 2

column volumes of 100 mM NiCl , washed again with dH O (3 column volumes), followed by 2

2

addition of 3 column volumes of low salt lysis buffer with protease inhibitors and 5 mM imidazole). This was followed by a wash with high salt buffer (50 mM Tris-Cl, pH 8, 1M NaCl, 0.5 mM TCEP and freshly added protease inhibitors previously listed. The column was then


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washed again with low salt lysis buffer containing protease inhibitors and 5 mM imidazole. Elution was performed with 50 ml of elution buffer (50 mM Tris-Cl, pH 8, 250 mM NaCl, 0.5 mM TCEP, protease inhibitors, and 400 mM imidazole). The eluted sample was diluted 5X with a buffer containing 50 mM Tris-Cl, pH 8, 0.5 mM TCEP and protease inhibitors to allow the sample to be at the lower salt concentration of 50 mM NaCl. Then, it was loaded onto a pre-equilibrated Hi-trap SP sepharose column with low-salt buffer (50 mM Tris-Cl, pH 8, 50 mM NaCl, 1 mM EDTA, pH 8, and 0.5 mM TCEP). Elution from this column was gradually achieved with a high salt buffer containing 50 mM Tris-Cl, pH8, 1M NaCl, 1 mM EDTA, pH 8, and 0.5 mM TCEP. The final step in the purification was performed using an AKTA HPLC and Superdex 200 column with an elution buffer of 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 1mM EDTA. Samples along the purification steps were fractionated in a 12% Bis-tris SDS gel and Coomassie blue stained with SimplyBlue SafeStain. Deacetylation activity assay of WT and H133Y mouse SIRT6 purified recombinant proteins. To determine the deacetylation activity of both purified SIRT6 proteins, a fluorometric SIRT6 activity assay (Abcam) was used following the manufacturer’s recommendations. In this assay, a fluorophore and quencher are coupled at opposite N- and C-termini of a substrate peptide containing an acetylated lysine. Following the deacetylation activity of SIRT6, the substrate becomes a substrate peptide for a peptidase (which is added simultaneously) that cuts the peptide, and thus allowing the fluorophore to emit fluorescence. Fluorescence intensity was measured at extinction/emission wavelengths of A480/530 nm, respectively.

Western Blotting. SIRT6 deacetylation activity was measured using H3K56Ac-NCP-THF (+64) as a deacetylation substrate. This damaged deacetylation substrate (630 nM) was incubated


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Biochemistry

in a buffer containing 5 mM NAD , 50 mM HEPES, pH 8, 100 mM KCl, 5 mM MgCl , and 1 +

2

mg/ml BSA for 10 and 30 min at 37°C. Proteins were then fractionated in a 12% Bis-tris SDS gel and transferred onto a nitrocellulose membrane (Life Techonology). The membrane was then incubated in 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 1 h, followed by incubation with primary antibody anti-H3K56 Ac (1:1000) from Abcam (ab76307). Goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) was used as secondary antibody, and immobilized horseradish peroxidase activity was detected by enhanced chemiluminescence (Thermo Fisher Scientific). Acetylation of the recombinant octamers was performed with the same antibody for H3K56Ac (1:5000) and for H3K14Ac (Active Motif 39697; 1:1000). Incubation with primary antibodies was performed either for 1 h at room temperature or overnight at 4°C, respectively. Membranes were subsequently washed three times for 10 min and incubated with the appropriate dilution of horseradish peroxidase-conjugated anti-rabbit antibodies for 2 h at room temperature. Blots were then washed with Tris buffered saline and Tween 20 solution (TBST) three times (5 min each) and developed with the ECL system (Amersham Biosciences) according to the manufacturer’s protocols either using Amersham hyperfilm ECL or Amersham Imager 600 system (as indicated in the figure legends).

Steady-state and single-turnover assays. DNA/NCP substrate (amount indicated in each plot) reaction mixtures containing 50 mM HEPES, pH 8, 100 mM KCl, 5 mM MgCl and 0.01 mg/ml 2,

BSA were incubated at 37°C for 5 min prior to the addition of APE1 to initiate the reaction. Time points were taken (6 µl) and quenched with an equal volume of 100 mM EDTA, pH 8. This was followed by a phenol:chloroform:isoamyl alcohol extraction to isolate the DNA. Each sample was then mixed 1:1 (vol:vol) with deionized formamide, boiled, and fractionated in a 20%


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polyacrylamide 7 M urea denaturing gel. The ratio of DNA:enzyme is shown as an insert within the plots, indicating if they are steady-state or single-turnover assays. The summary bar graphs for the calculated apparent rate constants performed under steady-state conditions was as follows:

K = v / [E ], where v is the initial velocity and E is the total APE1 concentration as calculated ss

0

total

0

total

from Bradford assay. For steady-state reactions where data were fitted to a single exponential, the rate constant was calculated as reported previously

Y = C + Ymax (1 - e -kobs t )

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Equation 1

The initial rate of the reaction (v ) was determined by taking the derivative of equation 1 (dY/dt) 0

as t approaches 0. The rate constant, (v /[E ]), was then calculated as follows 0

v 0 /[E total ] =

total

Ymax * kobs [E total ]

Equation 2

For single turnover conditions, k was obtained directly from the fit of the data to a single obs

exponential equation (Equation 1).

RESULTS To better understand H3K56Ac regulation of BER, we conducted an assessment of the acetylation effect on APE1 activity within NCP substrates. The design of our in vitro substrates considers proximity of the AP site DNA lesion to the histone acetylation site. The experimental


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Biochemistry

design includes analysis of the presence of the chromatin associated protein high mobility group box 1 (HMGB1), found to regulate chromatin structure , as well as APE1 activity . 34

35

Histone site-specific acetylation at H3K56 enhances APE1 activity. The in vivo results described by others suggest higher levels of H3K56Ac as a result of SIRT6 absence contribute to genomic instability

36, 37

, possibly by regulating the

chromatin landscape and accessibility to DNA lesions.

Because there are multiple in vivo

parameters that could be at play, we chose to investigate the effect of H3K56Ac on APE1 strand incision activity. Nucleosome core particles (NCPs) were designed containing a strategically located AP site within the well-positioned 601 DNA (Table S1). The NCP reconstitution efficiency was verified for this substrate using native/non-denaturing PAGE. Representative results are shown in Figure S1A, demonstrating that NCP integrity was achieved independent of the presence of the AP site DNA lesion and histone acetylation status (confirmed by

Figure 1. Positioning of tetrahydrofuran (THF) relative to histone H3 site-specific acetylation in reconstituted nucleosomes and its effect on APE1 activity. The NCP crystal structure [Protein Data Bank (PDB) code IKX5] was modified to indicate the location of the THF lesion placed within the 147 bp 601 synthetic DNA sequence. The complete sequence is listed in Table S1 with the chemical structure of the THF highlighted in black in this figure. The two copies of histone H3 are shown in gray, residue H3K14 is shown in orange and H3K56 in red. Because this DNA lesion is near the DNA ends, it does not maintain definitive rotational orientation but it was chosen due to its proximity to H3K56Ac, strategically located within the region of increased nucleosome dynamics and accessibility . Numbers in parenthesis indicate the number of nucleotides from the 5´ end (+) of the dyad center of symmetry, which is designated as translational position 0. 1, 2

western blotting in Figure S1B). As shown in Figure 1, the AP site is in proximity to the H3K56Ac near


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the DNA ends, where DNA unwrapping has been shown to be enhanced by acetylation. The substrate nomenclature has been described previously , where the number in parenthesis indicates 21, 32

the

number

of

nucleotides away from the dyad toward the 5´ end

(designated

as

positive) or toward the 3´

end

(negative).

Because this lesion is near the end, it does not adopt

a

definitive

rotational orientation, as the DNA is more

Figure 2. Incision of 601 NCPs. A) APE1 activity was assessed under single turnover conditions (enzyme is in excess relative to substrate). The mean of at least triplicate experiments was plotted and fitted to a single exponential equation given under Materials and Methods. Acetylation of H3K56 enhances the single turnover rate by ~4-fold in 601 well-positioned NCPs. B) The k were plotted, and an unpaired, two-tailed t-test was performed with asterisks denoting a significant difference (p-value

Histone H3 Lysine 56 Acetylation Enhances AP Endonuclease 1

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