CTCF-mediated chromatin looping provides a topological framework for the formation of phase-separated transcriptional condensates.

CTCF is crucial to the organization of mammalian genomes into loop structures. According to recent studies, the transcription apparatus is compartmentalized and concentrated at super-enhancers to form phase-separated condensates and drive the expression of cell-identity genes. However, it remains unclear whether and how transcriptional condensates are coupled to higher-order chromatin organization.

MutL sliding clamps coordinate exonuclease-independent Escherichia coli mismatch repair.

A shared paradigm of mismatch repair (MMR) across biology depicts extensive exonuclease-driven strand-specific excision that begins at a distant single-stranded DNA (ssDNA) break and proceeds back past the mismatched nucleotides. Historical reconstitution studies concluded that Escherichia coli (Ec) MMR employed EcMutS, EcMutL, EcMutH, EcUvrD, EcSSB and one of four ssDNA exonucleases to accomplish excision. Recent single-molecule images demonstrated that EcMutS and EcMutL formed cascading sliding clamps on a mismatched DNA that together assisted EcMutH in introducing ssDNA breaks at distant newly replicated GATC sites.

Multiplexed single-molecule flow-stretching bead assay for DNA enzymology.

Single-molecule techniques have been used successfully to visualize real-time enzymatic activities, revealing transient complex properties and heterogeneity of various biological events. Especially, conventional force spectroscopy including optical tweezers and magnetic tweezers has been widely used to monitor change in DNA length by enzymes with high spatiotemporal resolutions of ∼nanometers and ∼milliseconds. However, DNA metabolism results from coordination of a number of components during the processes, requiring efficient monitoring of a complex of proteins catalyzing DNA substrates.

Dynamics of Proofreading by the E. coli Pol III Replicase.

The αɛθ core of Escherichia coli DNA polymerase III (Pol III) associates with the β sliding clamp to processively synthesize DNA and remove misincorporated nucleotides. The α subunit is the polymerase while ɛ is the 3' to 5' proofreading exonuclease. In contrast to the polymerase activity of Pol III, dynamic features of proofreading are poorly understood.

Dynamic control of strand excision during human DNA mismatch repair.

Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic.