A shared paradigm of mismatch repair (MMR) across biology depicts extensive exonucleasedriven 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. Here we visualize the complete strand-specific excision process and find that long-lived EcMutL sliding clamps capture EcUvrD helicase near the ssDNA break, significantly increasing its unwinding processivity. EcSSB modulates the EcMutL-EcUvrD unwinding dynamics, which is rarely accompanied by extensive ssDNA exonuclease digestion. Together these observations are consistent with an exonuclease-independent MMR strand excision mechanism that relies on EcMutL-EcUvrD helicase-driven displacement of ssDNA segments between adjacent EcMutH-GATC incisions.

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. The purified human (Homo sapien or Hs) 5′-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5′ → 3′ exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5′-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5′ MMR.is a highly conserved strand-specific excision-resynthesis process that corrects nucleotide misincorporation errors during replication and nucleotide mismatches arising from recombination between heteroallelic parents or physical damage to the DNA (for review see ref. 1). Mutation of core MMR components results in elevated mutation rates and susceptibility to a variety of cancers (2).MMR has been reconstituted with purified Escherichia coli, Saccharomyces cerevisae, and human proteins (3-6). The core MutS homologs (MSH) and MutL homologs (MLH/PMS) components direct a strand-specific excision reaction, whereas resynthesis appears to be uniquely performed by the replicative polymerase complex (1). In all organisms the excision process is initiated at a single-strand DNA scission (ssDNA/S) that may be located either 3′ or 5′ and hundreds to thousands of base pairs distant from the mismatch (4, 7). An ssDNA/S positioned on the newly replicated strand ensures accurate correction of replication misincorporation errors (1).Excision directionality in γ-proteobacteria (E. coli) is linked to the choice of 3′ or 5′ exonucleases that specifically degrade ssDNA generated by the EcUvrD helicase in concert with EcMutS and EcMutL (1). The lack of a helicase distinguishes yeast and human MMR from γ-proteobacteria. Moreover, the eukaryotic 3′-and 5′-excision reactions require different core MMR components and likely occur by different mechanisms (1). For example, the 3′-MMR excision requires the replicative processivity factor PCNA to activate a cryptic MLH/PMS endonuclease activity (8), whereas 5′ MMR uses the only known MMR exonuclease EXOI (3, 5, 6). Unlike the E. coli ssDNA exonucleases, EXOI will initiate 5′ excision from a ssDNA/S in the absence of a helicase (9). Whereas the purified 5′-MMR reaction does not require MLH/PMS or PCNA, complementation studies with cellular extracts displayed a substantial requirement for...

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. Here, we show that CTCF is essential for RNA polymerase II (Pol II)-mediated chromatin interactions, which occur as hyperconnected spatial clusters at super-enhancers. We also demonstrate that CTCF clustering, unlike Pol II clustering, is independent of liquid-liquid phase-separation and resistant to perturbation of transcription. Interestingly, clusters of Pol II, BRD4, and MED1 were found to dissolve upon CTCF depletion, but were reinstated upon restoration of CTCF, suggesting a potent instructive function for CTCF in the formation of transcriptional condensates. Overall, we provide evidence suggesting that CTCF-mediated chromatin looping acts as an architectural prerequisite for the assembly of phase-separated transcriptional condensates.

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. We used single-molecule assays to determine the excision rate and processivity of the β-associated Pol III core, and observed that both properties are enhanced by mutational strengthening of the interaction between ɛ and β. Thus, the ɛ-β contact is maintained in both the synthesis and proofreading modes. Remarkably, single-molecule real-time fluorescence imaging revealed the dynamics of transfer of primer-template DNA between the polymerase and proofreading sites, showing that it does not involve breaking of the physical interaction between ɛ and β.

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