Expansion of CAG/CTG trinucleotide repeats is associated with certain familial neurological disorders, including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism contributes to their expansion. However, the molecular mechanism(s) by which cells remove CAG/CTG hairpins remain unknown. Here, we demonstrate that human cell extracts can catalyze error-free repair of CAG/CTG hairpins in a nick-directed manner. The repair system specifically targets CAG/CTG tracts for incisions in the nicked DNA strand, followed by DNA resynthesis using the continuous strand as a template, thereby ensuring CAG/CTG stability. PCNA is required for the incision step of the hairpin removal, which utilizes distinct endonuclease activities for individual CAG/CTG hairpins depending on their strand locations and/or secondary structures. The implication of these data for understanding the etiology of neurological diseases and trinucleotide repeat instability is discussed.

Studies in knockout mice provide evidence that MSH2–MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2–MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2–MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion.

Background: Expansion of CAG/CTG repeats causes familial neurological disorders, but the molecular basis is unknown. Results: DNA polymerases ␤ and ␦ effectively incorporate nucleotides to a CAG/CTG hairpin primer, leading to hairpin retention. Conclusion: Coordinated actions by polymerases ␤ and ␦ on hairpin primers during DNA synthesis promotes CAG/CTG repeat expansions. Significance: The work discovers a novel mechanism for CAG/CTG repeat expansions.

Background: DNA hairpin repair (HPR) is an important genome maintenance system, but the components involved in HPR are unknown. Results: WRN unwinds (CTG) n hairpins and facilitates HPR and polymerase ␦-catalyzed DNA synthesis using a (CTG) 25 hairpin as a template. Conclusion: WRN is involved in (CTG) n hairpin removal by resolving the hairpin, promoting DNA synthesis. Significance: This work provides useful information to elucidate HPR and repeat instability-caused diseases.

Polychlorinated biphenyls (PCBs) are persistent and ubiquitous environmental chemicals that bioaccumulate and have hepatic tumor promoting activity in rodents. The present study examined the effect of deleting the p50 subunit of NF-κB on the hepatic tumor promoting activity of 2,2',4,4', 5,5'-hexachlorobiphenyl (PCB-153) in mice. Both wild-type and p50−/− male mice were injected i.p. with diethylnitrosamine (DEN, 90 mg/kg) and then subsequently injected biweekly with 20 i.p. injections of PCB-153 (300 µmol/kg/injection). p50 deletion decreased the tumor incidence in both PCB-and vehicle-treated mice, whereas PCB-153 slightly (P = 0.09) increased the tumor incidence in wild-type and p50−/− mice. PCB-153 increased the total tumor volume in both wild-type and p50 −/− mice, but the total tumor volume was not affected by p50 deletion in either PCB-or vehicletreated mice. The volume of tumors that were positive for glutamine synthetase (GS), which is indicative of mutations in the beta-catenin gene, was increased in both wild-type and p50−/− mice administered PCB-153 compared to vehicle controls and inhibited in p50 −/− mice compared to wildtype mice (in both PCB-and vehicle-treated mice). The volume of tumors that were negative for GS was increased in p50 −/− mice compared to wild-type mice but was not affected by PCB-153. PCB-153 increased cell proliferation in normal hepatocytes in wild-type but not p50−/− mice; this increase was inhibited in p50 −/− mice. In hepatic tumors, the rate of cell proliferation was much higher than in normal hepatocytes, but was not affected by PCB treatment or p50 deletion. The rate of apoptosis, as measured by the TUNEL assay, was not affected by PCB-153 or p50 deletion in normal hepatocytes. In hepatic tumors, the rate of apoptosis was lower than in normal hepatocytes; PCB-153 slightly (P = 0.10) increased apoptosis in p50−/− but not wild-type mice; p50 deletion had no effect. Taken together, these data indicate that the absence of the NF-κB p50 subunit inhibits the Corresponding Author: Howard P. Glauert, Ph.D., University of Kentucky, Graduate Center for Nutritional Sciences, 222 Funkhouser Building, Lexington, KY 40506-0054, USA. Telephone (859) 257-7789, fax: (859) 323-0061, e-mail: hglauert@uky.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of Interest StatementThere are no conflicts of interest. promoting activity of PCB-153 and alters the proliferative and apoptotic changes in mouse liver in the response to PCBs. NIH Public Access

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The ability of antibodies and Fc-fusion proteins to bind multiple targets cooperatively is limited by their topology. Here we describe our discovery that ACE2 Fc-fusion proteins spontaneously cross-dimerize, forming topologically distinct “superdimers” that demonstrate extraordinary SARS-CoV-2 intra-spike cooperative binding and potently neutralize Omicron B.1.1.529 at least 100-fold better than eight clinically authorized antibodies. We also exploited cross- dimerization to topologically engineer novel superdimeric antibodies and Fc-fusion proteins with antibody-like plasma half-lives to address cancer and infectious disease therapy. These include bispecific ACE2-antibody superdimers that potently neutralize all major SARS-CoV-2 variants, and bispecific anti-cancer and anti-viral antibody superdimers that are more potent than two-antibody cocktails. Superdimers are efficiently produced from single cells, providing a new therapeutic approach to many disease indications.

Trinucleotide repeat (TNR) expansion is the genetic basis for certain neurological and neurodegenerative disorders, including Huntington's disease. Since TNR sequences, e.g., (CAG)n, form a loop or hairpin structure, DNA loop repair is considered a major mechanism for TNR stability. However, the molecular mechanisms of DNA loop repair and protein components involved in this pathway are poorly understood. Using a biochemical approach, where human nuclear extracts were examined for their ability to process a plasmid DNA containing a (CAG)25 or (CTG) 25 loop, we show here that repair of CAG or CTG loop in human cells occurs in a nick‐ or loop‐directed manner. The repair is initiated by an incision at the top of the loop, followed by additional incision and excision, limited repair DNA synthesis, and ligation. Several biochemical mutants derived from fractionations allow us to demonstrate that PCNA, RPA, and FEN1 are essential components involved in TNR loop repair. We also show that MutSβ, a mismatch recognition protein that was previously thought to promote CAG expansion in transgenic mouse model by binding to the CAG loop and inhibiting its repair, has no inhibitory effect on TNR loop repair in human extracts, suggesting that the transgenic animal model may not reflect TNR instability in human cells. Our findings provide molecular insight into the mechanism by which cells maintain TNR stability.