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Biochemical characterization of the placeholder nucleosome for DNA hypomethylation maintenance.

ABSTRACT: DNA methylation functions as a prominent epigenetic mark, and its patterns are transmitted to the genomes of offspring. The nucleosome containing the histone H2A.Z variant and histone H3K4 mono-methylation acts as a "placeholder" nucleosome for DNA hypomethylation maintenance in zebrafish embryonic cells. However, the mechanism by which DNA methylation is deterred by the placeholder nucleosome is poorly understood. In the present study, we reconstituted the placeholder nucleosome containing histones H2A.Z and H3 with the Lys4 mono-methylation. The thermal stability assay revealed that the placeholder nucleosome is less stable than the canonical nucleosome. Nuclease susceptibility assays suggested that the nucleosomal DNA ends of the placeholder nucleosome are more accessible than those of the canonical nucleosome. These characteristics of the placeholder nucleosome are quite similar to those of the H2A.Z nucleosome without H3K4 methylation. Importantly, the linker histone H1, which is reportedly involved in the recruitment of DNA methyltransferases, efficiently binds to all of the placeholder, H2A.Z, and canonical nucleosomes. Therefore, the characteristics of the H2A.Z nucleosome are conserved in the placeholder nucleosome without synergistic effects on the H3K4 mono-methylation.

SUBMITTER: Hirano R

PROVIDER: S-EPMC6458450 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Biochemical characterization of the placeholder nucleosome for DNA hypomethylation maintenance.

Hirano Rina R Kujirai Tomoya T Negishi Lumi L Kurumizaka Hitoshi H

Biochemistry and biophysics reports 20190410

DNA methylation functions as a prominent epigenetic mark, and its patterns are transmitted to the genomes of offspring. The nucleosome containing the histone H2A.Z variant and histone H3K4 mono-methylation acts as a "placeholder" nucleosome for DNA hypomethylation maintenance in zebrafish embryonic cells. However, the mechanism by which DNA methylation is deterred by the placeholder nucleosome is poorly understood. In the present study, we reconstituted the placeholder nucleosome containing hist ...[more]

PMID: 31008378

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Project description:Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase ? is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme.The coding sequences of DNA polymerase ? catalytic subunit (PfPol?-cat), DNA polymerase ? small subunit (PfPol?S) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC-MS/MS. PfPol?-cat was biochemically characterized. The roles of PfPol?S and PfPCNA in PfPol?-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPol?-cat activity and on in vitro parasite growth using SYBR Green I assay.The purified recombinant protein PfPol?-cat, PfPol?S and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPol?-cat and PfPol?S had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPol?-cat possessed both DNA polymerase and 3'-5' exonuclease activities. It used both Mg(2+) and Mn(2+) as cofactors and was inhibited by high KCl salt (>200 mM). PfPol?S stimulated PfPol?-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPol?-cat, namely, butylphenyl-dGTP (BuPdGTP; IC50 of 38 µM) and 7-acetoxypentyl-(3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC50 of 55 µM). The latter compound showed higher inhibition on parasite growth (IC50 of 4.1 µM).Recombinant PfPol?-cat, PfPol?S and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPol?-cat. The high sensitivity of PfPol? to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase.

Dataset Information

Biochemical characterization of the placeholder nucleosome for DNA hypomethylation maintenance.

Publications

Biochemical characterization of the placeholder nucleosome for DNA hypomethylation maintenance.

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