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1. Msh2-Msh3 DNA-binding is not sufficient to promote trinucleotide repeat expansions in Saccharomyces cerevisiae

   https://doi.org/10.1093/genetics/iyae222  

   Casazza, Katherine M; Williams, Gregory M; Johengen, Lauren; Twoey, Gavin; Surtees, Jennifer A (January 2025, GENETICS)

   Argueso, J L (Ed.)

   Abstract Mismatch repair (MMR) is a highly conserved DNA repair pathway that recognizes mispairs that occur spontaneously during DNA replication and coordinates their repair. In Saccharomyces cerevisiae, Msh2-Msh3 and Msh2-Msh6 initiate MMR by recognizing and binding insertion or deletion (in/del) loops up to ∼17 nucleotides (nt.) and base–base mispairs, respectively; the 2 complexes have overlapping specificity for small (1–2 nt.) in/dels. The DNA-binding specificity for the 2 complexes resides in their respective mispair binding domains (MBDs) and has distinct DNA-binding modes. Msh2-Msh3 also plays a role in promoting CAG/CTG trinucleotide repeat (TNR) expansions, which underlie many neurodegenerative diseases such as Huntington's disease and myotonic dystrophy type 1. Models for Msh2-Msh3's role in promoting TNR tract expansion have invoked its specific DNA-binding activity and predict that the TNR structure alters its DNA binding and downstream activities to block repair. Using a chimeric Msh complex that replaces the MBD of Msh6 with the Msh3 MBD, we demonstrate that Msh2-Msh3 DNA-binding activity is not sufficient to promote TNR expansions. We propose a model for Msh2-Msh3-mediated TNR expansions that requires a fully functional Msh2-Msh3 including DNA binding, coordinated ATP binding, and hydrolysis activities and interactions with Mlh complexes that are analogous to those required for MMR. 

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2. Computationally exploring the mechanism of bacteriophage T7 gp4 helicase translocating along ssDNA

   https://doi.org/10.1073/pnas.2202239119  

   Jin, Shikai; Bueno, Carlos; Lu, Wei; Wang, Qian; Chen, Mingchen; Chen, Xun; Wolynes, Peter G.; Gao, Yang (August 2022, Proceedings of the National Academy of Sciences)

   Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 5′-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 5′-triphosphate (ATP) at the subunit interface stabilizes the subunit–subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 5′-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein–protein and protein–DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein–ssDNA force field and elucidate the molecular basis of replicative helicase translocation. 

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3. Single-molecule visualization of human BLM helicase as it acts upon double- and single-stranded DNA substrates

   https://doi.org/10.1093/nar/gkz810  

   Xue, Chaoyou; Daley, James M; Xue, Xiaoyu; Steinfeld, Justin; Kwon, Youngho; Sung, Patrick; Greene, Eric C (September 2019, Nucleic Acids Research)

   Abstract Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70–80 base pairs per second) unwinding extensive tracts (∼8–10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair. 

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4. Ustilago maydis Trf2 ensures genome stability by antagonizing Blm-mediated telomere recombination: Fine-tuning DNA repair factor activity at telomeres through opposing regulations

   https://doi.org/10.1371/journal.pgen.1011515  

   Syed, Shahrez; Aloe, Sarah; Sutherland, Jeanette H; Holloman, William K; Lue, Neal F (December 2024, PLOS Genetics)

   Zappulla, David C (Ed.)

   TRF2 is an essential and conserved double-strand telomere binding protein that stabilizes chromosome ends by suppressing DNA damage response and aberrant DNA repair. Herein we investigated the mechanisms and functions of the Trf2 ortholog in the basidiomycete fungusUstilago maydis, which manifests strong resemblances to metazoans with regards to the telomere and DNA repair machinery. We showed thatUmTrf2 binds to Blmin vitroand inhibits Blm-mediated unwinding of telomeric DNA substrates. Consistent with a similar inhibitory activityin vivo, over-expression of Trf2 induces telomere shortening, just like deletion ofblm, which is required for efficient telomere replication. While the loss of Trf2 engenders growth arrest and multiple telomere aberrations, these defects are fully suppressed by the concurrent deletion ofblmormre11(but not other DNA repair factors). Over-expression of Blm alone triggers aberrant telomere recombination and the accumulation of aberrant telomere structures, which are blocked by concurrent Trf2 over-expression. Together, these findings highlight the suppression of Blm as a key protective mechanism of Trf2. Notably,U.maydisharbors another double-strand telomere-binding protein (Tay1), which promotes Blm activity to ensure efficient replication. We found that deletion oftay1partially suppresses the telomere aberration of Trf2-depleted cells. Our results thus point to opposing regulation of Blm helicase by telomere proteins as a strategy for optimizing both telomere maintenance and protection. We also show that aberrant transcription of both telomere G- and C-strand is a recurrent phenotype of telomere mutants, underscoring another potential similarity between double strand breaks and de-protected telomeres. 

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5. Viral packaging ATPases utilize a glutamate switch to couple ATPase activity and DNA translocation

   https://doi.org/10.1073/pnas.2024928118  

   Pajak, Joshua; Atz, Rockney; Hilbert, Brendan J.; Morais, Marc C.; Kelch, Brian A.; Jardine, Paul J.; Arya, Gaurav (April 2021, Proceedings of the National Academy of Sciences)

   Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors. 

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