Abstract
The Su(var)3–9, enhancer of zeste, and trithorax (SET) and really interesting new gene (RING) finger–associated (SRA) protein domain is conserved across bacteria and eukaryota and coordinates extrahelical or “flipped” DNA bases. A functional SRA domain is required for ubiquitin-like with PHD and RING finger domains 1 (UHRF1) E3 ubiquitin ligase activity toward histone H3, a mechanism for recruiting the DNA methylation maintenance enzyme DNA methyltransferase 1 (DNMT1). The SRA domain supports UHRF1 oncogenic activity in colon cancer cells, highlighting that UHRF1 SRA antagonism could be a cancer therapeutic strategy. Here we used molecular dynamics simulations, DNA binding assays, in vitro ubiquitination reactions, and DNA methylation analysis to identify the SRA finger loop as a regulator of UHRF1 ubiquitin targeting and DNA methylation maintenance. A chimeric UHRF1 (finger swap) with diminished E3 ligase activity toward nucleosomal histones, despite tighter binding to unmodified or asymmetric or symmetrically methylated DNA, uncouples DNA affinity from regulation of E3 ligase activity. Our model suggests that SRA domains sample DNA bases through flipping in the presence or absence of a cytosine modification and that specific interactions of the SRA finger loop with DNA are required for downstream host protein function. Our findings provide insight into allosteric regulation of UHRF1 E3 ligase activity, suggesting that UHRF1's SRA finger loop regulates its conformation and function.
Highlights
The Su(var)3–9, enhancer of zeste, and trithorax (SET) and really interesting new gene (RING) finger–associated (SRA) protein domain is conserved across bacteria and eukaryota and coordinates extrahelical or “flipped” DNA bases
We further demonstrated that the SRA finger loop is responsible for selective binding to modified cytosines, regulation of UHRF1 E3 ubiquitin ligase activity, and maintenance of DNA methylation
To define “specific” DNA binding, we will consider the E3 ligases UHRF1 and UHRF2, whose enzymatic activity is linked to the specific recognition of He5mC or hemihydroxymethylated DNA (He5hmC), respectively [17, 20]
Summary
We began our study by evaluating the homology across various SRA domains. Phylogenetic clustering segregated UniProt-reviewed SRA domains by their known or putative enzymatic functions as SET domain– containing methyltransferases or RING-containing E3 ubiquitin ligases (Fig. 1A). The flexible finger loop (i.e. unresolved in crystallography) was a common feature of SRA domains bound to symmetrically modified DNA, as noted previously [12]. These data associated changes in SRA–DNA interactions with unique sequence compositions of SRA finger. The lack of finger-loop resolution for some DNA-bound SRA domains in crystallography points to flexibility. These data led us to hypothesize that the finger loop is dispensable for base flipping but important for the SRA’s selective binding to modified DNA
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