The CST (CTC1–STN1–TEN1) complex mediates critical functions in maintaining telomere DNA and overcoming genome-wide replication stress. A conserved biochemical function of the CST complex is its primase-Pol α (PP) stimulatory activity. In this report, we demonstrate the ability of purified human STN1 alone to promote PP activity in vitro. We show that this regulation is mediated primarily by the N-terminal OB fold of STN1, but does not require the DNA-binding activity of this domain. Rather, we observed a strong correlation between the PP-stimulatory activity of STN1 variants and their abilities to bind POLA2. Remarkably, the main binding target of STN1 in POLA2 is the latter's central OB fold domain. In the substrate-free structure of PP, this domain is positioned so as to block nucleic acid entry to the Pol α active site. Thus the STN1–POLA2 interaction may promote the necessary conformational change for nucleic acid delivery to Pol α and subsequent DNA synthesis. A disease-causing mutation in human STN1 engenders a selective defect in POLA2-binding and PP stimulation, indicating that these activities are critical for the in vivo function of STN1. Our findings have implications for the molecular mechanisms of PP, STN1 and STN1-related molecular pathology.

We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.

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