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1. Pol V produced RNA facilitates transposable element excision site repair in Arabidopsis

   Renken, Kaili; Mendoza, Sarah M.; Diaz, Stephanie; Slotkin, R. Keith; Hancock, C. Nathan (May 2023, microPublication biology)

   The plant-specific RNA Polymerase V (Pol V) plays a key role in gene silencing, but its role in repair of double stranded DNA breaks is unclear. Excision of the transposable element mPing creates double stranded breaks that are repaired by NHEJ. We measured mPing excision site repair in multiple DNA methylation mutants including pol V using an mPing : GFP reporter. Two independent mutant alleles of pol V showed less GFP expression, indicating that the Pol V protein plays a role in excision site repair. Sequence analysis of the pol V excision sites indicated an elevated rate of large deletions consistent with less efficient repair. These results clarify the role of Pol V, but not other RNA-directed DNA methylation proteins (Pol IV) or maintenance DNA methylation pathways ( MET1 ), in the repair of double-strand DNA breaks. 

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2. CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

   https://doi.org/10.7554/eLife.55143  

   Cofsky, Joshua C; Karandur, Deepti; Huang, Carolyn J; Witte, Isaac P; Kuriyan, John; Doudna, Jennifer A (June 2020, eLife)

   Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3′ side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems. 

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3. Elf1 promotes Rad26’s interaction with lesion-arrested Pol II for transcription-coupled repair

   https://doi.org/10.1073/pnas.2314245121  

   Sarsam, Reta D; Xu, Jun; Lahiri, Indrajit; Gong, Wenzhi; Li, Qingrong; Oh, Juntaek; Zhou, Zhen; Hou, Peini; Chong, Jenny; Hao, Nan; et al (January 2024, Proceedings of the National Academy of Sciences)

   Transcription-coupled nucleotide excision repair (TC-NER) is a highly conserved DNA repair pathway that removes bulky lesions in the transcribed genome. Cockayne syndrome B protein (CSB), or its yeast ortholog Rad26, has been known for decades to play important roles in the lesion-recognition steps of TC-NER. Another conserved protein ELOF1, or its yeast ortholog Elf1, was recently identified as a core transcription-coupled repair factor. How Rad26 distinguishes between RNA polymerase II (Pol II) stalled at a DNA lesion or other obstacles and what role Elf1 plays in this process remains unknown. Here, we present cryo-EM structures of Pol II-Rad26 complexes stalled at different obstacles that show that Rad26 uses a common mechanism to recognize a stalled Pol II, with additional interactions when Pol II is arrested at a lesion. A cryo-EM structure of lesion-arrested Pol II-Rad26 bound to Elf1 revealed that Elf1 induces further interactions between Rad26 and a lesion-arrested Pol II. Biochemical and genetic data support the importance of the interplay between Elf1 and Rad26 in TC-NER initiation. Together, our results provide important mechanistic insights into how two conserved transcription-coupled repair factors, Rad26/CSB and Elf1/ELOF1, work together at the initial lesion recognition steps of transcription-coupled repair. 

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4. An siRNA-guided ARGONAUTE protein directs RNA polymerase V to initiate DNA methylation

   https://doi.org/10.1038/s41477-021-01008-7  

   Sigman, Meredith J.; Panda, Kaushik; Kirchner, Rachel; McLain, Lauren L.; Payne, Hayden; Peasari, John Reddy; Husbands, Aman Y.; Slotkin, R. Keith; McCue, Andrea D. (November 2021, Nature Plants)

   Abstract In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing. 

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5. Molecular model of TFIIH recruitment to the transcription-coupled repair machinery

   https://doi.org/10.1038/s41467-025-57593-0  

   Paul, Tanmoy; Yan, Chunli; Yu, Jina; Tsutakawa, Susan_E; Tainer, John_A; Wang, Dong; Ivanov, Ivaylo (March 2025, Nature Communications)

   Abstract Transcription-coupled repair (TCR) is a vital nucleotide excision repair sub-pathway that removes DNA lesions from actively transcribed DNA strands. Binding of CSB to lesion-stalled RNA Polymerase II (Pol II) initiates TCR by triggering the recruitment of downstream repair factors. Yet it remains unknown how transcription factor IIH (TFIIH) is recruited to the intact TCR complex. Combining existing structural data with AlphaFold predictions, we build an integrative model of the initial TFIIH-bound TCR complex. We show how TFIIH can be first recruited in an open repair-inhibited conformation, which requires subsequent CAK module removal and conformational closure to process damaged DNA. In our model, CSB, CSA, UVSSA, elongation factor 1 (ELOF1), and specific Pol II and UVSSA-bound ubiquitin moieties come together to provide interaction interfaces needed for TFIIH recruitment. STK19 acts as a linchpin of the assembly, orienting the incoming TFIIH and bridging Pol II to core TCR factors and DNA. Molecular simulations of the TCR-associated CRL4^CSAubiquitin ligase complex unveil the interplay of segmental DDB1 flexibility, continuous Cullin4A flexibility, and the key role of ELOF1 for Pol II ubiquitination that enables TCR. Collectively, these findings elucidate the coordinated assembly of repair proteins in early TCR. 

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