The UHRF1 Protein Stimulates the Activity and Specificity of the Maintenance DNA Methyltransferase DNMT1 by an Allosteric Mechanism

The ubiquitin-like, containing PHD and RING finger domains protein 1 (UHRF1) is essential for maintenance DNA methylation by DNA methyltransferase 1 (DNMT1). UHRF1 has been shown to recruit DNMT1 to replicated DNA by the ability of its SET and RING-associated (SRA) domain to bind to hemimethylated DNA. Here, we demonstrate that UHRF1 also increases the activity of DNMT1 by almost 5-fold. This stimulation is mediated by a direct interaction of both proteins through the SRA domain of UHRF1 and the replication focus targeting sequence domain of DNMT1, and it does not require DNA binding by the SRA domain. Disruption of the interaction between DNMT1 and UHRF1 by replacement of key residues in the replication focus targeting sequence domain led to a strong reduction of DNMT1 stimulation. Additionally, the interaction with UHRF1 increased the specificity of DNMT1 for methylation of hemimethylated CpG sites. These findings show that apart from the targeting of DNMT1 to the replicated DNA UHRF1 increases the activity and specificity of DNMT1, thus exerting a multifaceted influence on the maintenance of DNA methylation.     

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: II. The shape of the GyrA subunit C-terminal domain (CTD) is not a sole determinant for controlling supercoiling efficiency

DNA topoisomerases are essential enzymes that can overwind, underwind, and disentangle double-helical DNA segments to maintain the topological state of chromosomes. Nearly all bacteria utilize a unique type II topoisomerase, gyrase, which actively adds negative supercoils to chromosomes using an ATP-dependent DNA strand passage mechanism; however, the specific activities of these enzymes can vary markedly from species to species. Escherichia coli gyrase is known to favor supercoiling over decatenation (Zechiedrich, E. L., Khodursky, A. B., and Cozzarelli, N. R. (1997) Genes Dev. 11, 2580-2592), whereas the opposite has been reported for Mycobacterium tuberculosis gyrase (Aubry, A., Fisher, L. M., Jarlier, V., and Cambau, E. (2006) Biochem. Biophys. Res. Commun. 348, 158-165). Here, we set out to understand the molecular basis for these differences using structural and biochemical approaches. Contrary to expectations based on phylogenetic inferences, we find that the dedicated DNA wrapping domains (the C-terminal domains) of both gyrases are highly similar, both architecturally and in their ability to introduce writhe into DNA. However, the M. tuberculosis enzyme lacks a C-terminal control element recently uncovered in E. coli gyrase (see accompanying article (Tretter, E. M., and Berger, J. M. (2012) J. Biol. Chem. 287, 18636-18644)) and turns over ATP at a much slower rate. Together, these findings demonstrate that C-terminal domain shape is not the sole regulatory determinant of gyrase activity and instead indicate that an inability to tightly couple DNA wrapping to ATP turnover is why M. tuberculosis gyrase cannot supercoil DNA to the same extent as its γ-proteobacterial counterpart. Our observations demonstrate that gyrase has been modified in multiple ways throughout evolution to fine-tune its specific catalytic properties.