Witte, S. J. | Search bioRxiv

bioRxiv2024

The Mlh1-Pms1 endonuclease uses ATP to preserve DNA discontinuities as strand discrimination signals to facilitate mismatch repair

In eukaryotic post-replicative mismatch repair, MutS homologs (MSH) detect mismatches and recruit MLH complexes to nick the newly replicated DNA strand upon activation by the replication processivity clamp, PCNA. This incision enables mismatch removal and DNA repair. Biasing MLH endonuclease activity to the newly replicated DNA strand is crucial for repair. In reconstituted in vitro assays, PCNA is loaded at pre-existing discontinuities and orients the major MLH endonuclease Mlh1-Pms1/MLH1-PMS2 (yeast/human) to nick the discontinuous strand. In vivo, newly replicated DNA transiently contains discontinuities which are critical for efficient mismatch repair. How these discontinuities are preserved as strand discrimination signals during the window of time where mismatch repair occurs is unknown. Here, we demonstrate that yeast Mlh1-Pms1 uses ATP binding to recognize DNA discontinuities. This complex does not efficiently interact with PCNA, which partially suppresses ATPase activity, and prevents dissociation from the discontinuity. These data suggest that in addition to initiating mismatch repair by nicking newly replicated DNA, Mlh1-Pms1 protects strand discrimination signals, aiding in maintaining its own strand discrimination signposts. Our findings also highlight the significance of Mlh1-Pms1s ATPase activity for inducing DNA dissociation, as mutant proteins deficient in this function become immobilized on DNA post-incision, explaining in vivo phenotypes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=48 SRC="FIGDIR/small/598860v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@b1f8f6org.highwire.dtl.DTLVardef@4ff252org.highwire.dtl.DTLVardef@ae1e12org.highwire.dtl.DTLVardef@13851f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

A Non-redundant Role for T cell-derived IL-22 in Antibacterial Defense of Colonic Crypts

IL-22 is a key cytokine in immune defense against pathogens at barrier sites. In response to enteric attaching and effacing bacteria, IL-22 produced by type 3 innate lymphoid cells (ILC3s) is thought to be important early for induction of antimicrobial peptides (AMPs) that protect intestinal epithelial cells (IECs) in advance of T cell-derived IL-22 that arises later. Yet, the basis for a requirement for both innate and adaptive IL-22-producing immune cells in protecting the intestinal mucosa is unknown. Here, using novel mice that both report IL-22 expression and can be targeted for its lineage-specific deletion, we show that mice with deficiency of IL-22 targeted to innate immune cells, including ILC3s, have impaired STAT3 activation of surface colonic IECs colonized by bacteria early in infection. In contrast, mice with IL-22 deficiency limited to T cells have complete loss of STAT3 activation in IECs lining colonic crypts and fail to protect the crypts from bacterial invasion late despite ongoing production of IL-22 from ILC3s. T cell-derived IL-22 is required for upregulation of many host-protective genes by crypt IECs, including those encoding AMPs, neutrophil-recruiting chemokines, and mucins and mucin-related molecules, while also restricting pro-inflammatory genes downstream of IFN{gamma} and TNF signals. Thus, T cell-derived IL-22 is indispensable for antibacterial defense and damage control of intestinal crypts.

immunology↗