The replication focus targeting sequence (RFTS) domain is a DNA-competitive inhibitor of Dnmt1

Structural Genomics Consortium and Department of Physiology, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2011; 286(17):15344-51. DOI: 10.1074/jbc.M110.209882
Source: PubMed

ABSTRACT Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.

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Available from: Rebecca Lee Fagan, Aug 08, 2015
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    • "In order to avoid runaway methylation , DNMT1 undergoes to several auto - inhibitory mechanisms . In particular , to keep DNMT1 inactive the RFTS domain is positioned and stabilized deep inside the catalytic domain in a task where the hemimethy - lated DNA is expected to fit , thus , masking the catalytic core of the enzyme ( Qin et al . , 2011 ; Syeda et al . , 2011 ; Bashtrykov et al . , 2014 ) . The auto - inhibitory function of the CXXC domain is more controversial . Song et al . ( 2011 ) . reported that CXXC specifically binds to unmethylated CpG , activating the CXXC - BH21 linker , which then occupies the catalytic pocket and interferes with its function . By contrast , Bashtrykov et al . ( 2"
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    ABSTRACT: Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN) and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E) are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5)-methyltransferase 1. DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA) suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is regulated and executed?
    Frontiers in Genetics 01/2015; 6. DOI:10.3389/fgene.2015.00090
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    • "This suggests that Uhrf1 may act to first engage a hemimethylated site which is then subsequently bound by Dnmt1, displacing Uhrf1, before the methylation reaction can occur [95]. A displacement type mechanism may be mediated through the replication foci targeting sequence (RFTS) domain in Dnmt1 that normally functions to inhibit catalytic activity [96] [97]. This is supported by reports that Uhrf1 interacts with Dnmt1 through the RFTS domain [98], and this interaction may be instrumental in relieving the autoinhibitory effect on Dnmt1, making it competent for DNA methylation . "
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    • "This base flipping mechanism has been suggested to create a high-affinity binding site in order to stably target DNMT1 to hemimethylated DNA sites for faithful maintenance methylation [8] [9] [10]. Interaction between the SRA domain and DNMT1 may occur via the latter's SRA binding domain, which overlaps with the RFTS domain of DNMT1 [11] [12]. Recently, the SRA domain has gained even more interest by its ability to bind to hydroxymethylcytosine, the 6th DNA base [13]. "
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