Human polymerase θ helicase positions DNA microhomologies for double-strand break repair
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RECQL4 requires PARP1 for recruitment to DNA damage, and PARG dePARylation facilitates its associated role in end joining
RecQ helicases, highly conserved proteins with pivotal roles in DNA replication, DNA repair and homologous recombination, are crucial for maintaining genomic integrity. Mutations in RECQL4 have been associated with various human diseases, including Rothmund–Thomson syndrome. RECQL4 is involved in regulating major DNA repair pathways, such as homologous recombination and nonhomologous end joining (NHEJ). RECQL4 has more prominent single-strand DNA annealing activity than helicase activity. Its ability to promote DNA damage repair and the precise role of its DNA annealing activity in DNA repair are unclear. Here we demonstrate that PARP1 interacts with RECQL4, increasing its single-stranded DNA strand annealing activity. PARP1 specifically promoted RECQL4 PARylation at both its N- and C-terminal regions, promoting RECQL4 recruitment to DNA double-strand breaks (DSBs). Inhibition or depletion of PARP1 significantly diminished RECQL4 recruitment and occupancy at specific DSB sites on chromosomes. After DNA damage, PARG dePARylated RECQL4 and stimulated its end-joining activity. RECQL4 actively displaced replication protein A from single-stranded DNA, promoting microhomology annealing in vitro. Furthermore, depletion of PARP1 or RECQL4 substantially impacted classical-NHEJ- and alternative-NHEJ-mediated DSB repair. Consequently, the combined activities of PARP1, PARG and RECQL4 modulate DNA repair.
Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands
BsmI, a thermophilic Type IIS restriction endonuclease from Bacillus stearothermophilus, presents a unique structural composition, housing two distinct active sites within a single monomer. Recognition of the non-symmetrical 5’-GAATGC-3’ sequence enables precise cleavage of the top and bottom DNA strands. Synthetic biology interventions have led to the transformation of BsmI into Nb.BsmI, a nicking endonuclease. Here we introduce Nt*.BsmI, tailored for top-strand cleavage, which is inactive on standard double-stranded DNA, but active on bottom-strand nicked DNA, suggesting a sequential cleavage mechanism. Crystallographic structures of pre- and post-reactive complexes with cognate DNA show one major conformational change, a retractable loop possibly governing sequential active site accessibility. The x-ray structures reveal the position of the divalent metal ions in the active sites and the DNA:protein interactions, while the models predicted by Alphafold3 are incorrect. This comprehensive structural and functional study lays a foundation for rational enzyme redesign and potential applications in biotechnology.
Structural basis for intrinsic strand displacement activity of mitochondrial DNA polymerase
Members of the Pol A family of DNA polymerases, found across all domains of life, utilize various strategies for DNA strand separation during replication. In higher eukaryotes, mitochondrial DNA polymerase γ relies on the replicative helicase TWINKLE, whereas the yeast ortholog, Mip1, can unwind DNA independently. Using Mip1 as a model, we present a series of high-resolution cryo-EM structures that capture the process of DNA strand displacement. Our data reveal previously unidentified structural elements that facilitate the unwinding of the downstream DNA duplex. Yeast cells harboring Mip1 variants defective in strand displacement exhibit impaired oxidative phosphorylation and loss of mtDNA, corroborating the structural observations. This study provides a molecular basis for the intrinsic strand displacement activity of Mip1 and illuminates the distinct unwinding mechanisms utilized by Pol A family DNA polymerases.
KorB switching from DNA-sliding clamp to repressor mediates long-range gene silencing in a multi-drug resistance plasmid
Examples of long-range gene regulation in bacteria are rare and generally thought to involve DNA looping. Here, using a combination of biophysical approaches including X-ray crystallography and single-molecule analysis for the KorB–KorA system in Escherichia coli, we show that long-range gene silencing on the plasmid RK2, a source of multi-drug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We show that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters up to 1.5 kb away. We resolved the tripartite crystal structure of a KorB–KorA–DNA co-complex, revealing that KorA latches KorB into a closed clamp state. DNA-bound KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Together, our findings explain the mechanistic basis for KorB role switching from a DNA-sliding clamp to a co-repressor and provide an alternative mechanism for long-range regulation of gene expression in bacteria.
Apaf-1 is an evolutionarily conserved DNA sensor that switches the cell fate between apoptosis and inflammation
Apoptotic protease activating factor 1 (Apaf-1) was traditionally defined as a scaffold protein in mammalian cells for assembling a caspase activation platform known as the ‘apoptosome’ after its binding to cytochrome c. Although Apaf-1 structurally resembles animal NOD-like receptor (NLR) and plant resistance (R) proteins, whether it is directly involved in innate immunity is still largely unknown. Here, we found that Apaf-1-like molecules from lancelets, fruit flies, mice, and humans have conserved DNA sensing functionality. Mechanistically, mammalian Apaf-1 recruits receptor-interacting protein 2 (RIP2, also known as RIPK2) via its WD40 repeat domain and promotes RIP2 oligomerization to initiate NF-κB-driven inflammation upon cytoplasmic DNA recognition. Furthermore, DNA binding of Apaf-1 determines cell fate by switching the cellular processes between intrinsic stimuli-activated apoptosis and inflammation. These findings suggest that Apaf-1 is an evolutionarily conserved DNA sensor and may serve as a cell fate checkpoint, which determines whether cells initiate inflammation or undergo apoptosis by distinct ligand binding.
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