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. 2008 Dec 12;283(50):34490-4.
doi: 10.1074/jbc.C800169200. Epub 2008 Oct 22.

Structure and hemimethylated CpG binding of the SRA domain from human UHRF1

Chengmin Qian  1 , Side Li, Jean Jakoncic, Lei Zeng, Martin J Walsh, Ming-Ming Zhou
Affiliations

Structure and hemimethylated CpG binding of the SRA domain from human UHRF1

Chengmin Qian et al. J Biol Chem. .

Abstract

Human UHRF1 (ubiquitin-like PHD and RING finger 1) functions to maintain CpG DNA methylation patterns through DNA replication by co-localizing with the DNA methyltransferase DNMT1 at chromatin in mammals. Recent studies show that UHRF1 binds selectively to hemimethylated CpG via its conserved SRA (SET- and RING finger-associated) domain. However, the underlying molecular mechanism is not known. Here, we report a 1.95 A resolution crystal structure of the SRA domain of human UHRF1. Using NMR structure-guided mutagenesis, electrophoretic mobility shift assay, and fluorescence anisotropy analysis, we determined key amino acid residues for methyl-DNA binding that are conserved in the SRA domain.

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Figures

FIGURE 1.
FIGURE 1.
Three-dimensional crystal structure of the SRA domain of human UHRF1. a, the ribbon structure of the SRA domain shown in a side view (left panel) and a front view (right panel). The latter depicts key protein residues that interact with bound sulfate anions and glycerol and those that constitute a positively charged patch on the protein surface. b, surface electrostatic potential representation of the protein, shown in the front view in the right panel in a. c, two-dimensional 1H-15N heteronuclear single quantum coherence spectra showing chemical shift changes of the protein amide resonances between its free form (black) and in the presence of a 13-nucleotide hemimethylated DNA duplex (red) (supplemental Table 1). d, surface of the SRA domain highlighting residues color-coded according to chemical shift perturbations or line broadening effects upon binding to a 13-nucleotide hemimethylated DNA duplex. The residues are indicated in a gray-to-red colored gradient according to weighted 1H and 15N chemical shift changes of the protein induced by methylated DNA binding. The residues showing line broadening effects were treated the same as the residues that had most profound chemical shifts perturbations.
FIGURE 2.
FIGURE 2.
Molecular determinants of SRA domain binding to hemimethylated DNA. a, EMSA showing SRA domain (15 μg) binding to a non-methylated or singly or doubly hemimethylated DNA duplex of 13 nucleotides(10ng)(lanes 1–3) and forming a stable complex with hemimethylated DNA(2ng) in a protein concentration-dependent manner (0, 6, 7.5, 9, 10.5, 12, 13.5, and 15 μg, respectively, in lanes 4–11). b, EMSA assessing the binding of the wild-type and mutant SRA domain proteins (15 μg) to a 20-nucleotide hemimethylated DNA (5 ng). Note that the free DNA was run out of the gel in this EMSA experiment. c, fluorescence anisotropy curves of the binding of the wild-type and mutant SRA domain proteins to a 5′-fluorescein-labeled 13-nucleotide hemimethylated DNA (5 nm). The faction of SRA-bound DNA is plotted as a function of the protein concentration (from 5 nm to 0.5 mm) to determine dissociation constants. The DNA sequences are listed in supplemental Table 1.
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