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Here the authors reveal how magnesium regulates the conformational dynamics and activity of Type IA topoisomerases by reconfiguring salt-bridge networks in the protein. This simple, conserved mechanism may explain magnesium regulation of other proteins.

Nucleotide level resolution genome-wide mapping of DNA breaks reveals that different topoisomerase 2 poisons target distinct cleavage complexes, likely driven by the different local sequence preference, genomic context and chromatin microenvironment.

Guan, Ocampo and colleagues report the discovery and mechanistic dissection of Al3Cas12f, a metagenome-derived miniature nuclease that retains notable genome-editing capacity. They engineer an RKK variant, which boosts editing and helps overcome the potency threshold that has limited compact editors.

Wang, Guo, Zhang and colleagues obtain four cryo-electron microscopy snapshots that show how IscB is kept off by two RNA lids, with a car-pedal-like guide shift activating cleavage after ~11-nt pairing. They also engineer hinge regions that boost flexibility and improve genome editing in cells.

Zheng et al. show that extending the guide RNA restores the reduced activity of a high-fidelity CRISPR–Cas9 enzyme while preserving accuracy, revealing how strengthened PAM-distal interactions can improve genome-editing performance.

DNA polymerases are molecular machines that copy genetic material using a template. Here, authors characterize the ability of diverse polymerases to synthesize DNA without a template and show that environmental conditions can alter sequence composition, enabling guided kilobasescale DNA synthesis.

I-motifs are non-canonical secondary DNA structures that form dynamically in certain C-rich DNA regions. Here, the authors show that PCBP1 binds and unfolds i-motifs in a protonation- and structure-dependent manner, controlling their formation during the cell cycle to maintain genomic stability.

Smc5/6 association with DNA junctions can support genomic functions. Here, the authors show that Smc5/6 junction polarity preferences, targeting, and dwell times are determined by its structural modules as well as the RPA and PCNA genomic factors.

Breakage of both chromosomal DNA strands creates unique problems for DNA repair. Here, Luthman et al. show that for some broken ends, the two strand breaks are repaired in parallel, while for other ends one strand must be repaired before the other.

Telomere length is tightly controlled in normal cellular function and the human population. Here, the authors reveal that telomere synthesis is bidirectionally constrained by cellular nucleotide pools, in a manner which may be therapeutically manipulated.

Toxin-antitoxin systems make up a branch of the bacterial anti-phage immune system. Here, Johannesman et al demonstrate that some phages have co-opted an orphan antitoxin to counter a DNA ADP-ribosyltransferase defense system.

Synthesis and maturation of Okazaki fragments is crucial for lagging strand DNA replication. Here the authors find that the ubiquitin-specific protease Ubp10 works with the DNA synthesis machinery to promote Okazaki fragments joining by removal of PCNA through deubiquitylation of PCNA-K164.

Superoxide oxidizes the iron–sulfur cluster in the DNA demethylase ROS1 and extensively participates in the establishment of active DNA demethylation in plants to maintain stem cell fate.

Horizontal gene transfer in bacteria can occur through mechanisms such as conjugation, transduction and transformation, which facilitate the passage of DNA across the cell wall. Here, Kapteijn et al. show that cell wall-deficient bacteria can take up DNA and other extracellular materials via an endocytosis-like process.

DNA:RNA hybrids are linked to genetic instability in several pathological situations. Here, the authors report that various types of DNA lesions can trigger hybrid formation in yeast and humans, calling for a re-examination of the causality between these structures and genetic instability.

BrxX methylase in complex with SAM cofactor mediates foreign DNA recognition by the BREX system. BrxX can be engineered to modify BREX specificity and enhance defense. BREX defense and methylation require assembly of supramolecular BrxBCXZ complex.

5-Formylcytosine (5fC), produced by TET-mediated oxidation of 5-methylcytosine, is considered an intermediate in active DNA demethylation. Labeling studies and LC/MS analysis across mouse developmental stages reveals that 5fC modifications are more persistent in the genome and may have other functional roles.

Drug-resistance mutations provide a classical means to identify biological targets of small molecules. A combination of next-generation DNA sequencing with CRISPR-Cas9 genome editing confirms the targets of 6-thioguanine and triptolide and offers a general approach for target identification in cells.

G-quadruplexes (G4s) are stable structures that can disrupt genome stability and are dissolved by helicases. Here the authors present the structure of the yeast helicase Pif1 bound to a G4 and reveal a conserved G4 interacting region.

The single stranded DNA binding protein RPA plays a pivotal role in DNA replication. The archaeal RPA hosts a WH domain that interacts with the DNA primase and the replicative polymerase PolD. Here the authors provide a molecular understanding of the regulatory activity of archaeal RPA.

By leveraging structural and biochemical methods, the authors show that the interbacterial deaminase toxin DddA, a potential gene-editing tool, uses a tandem displacement mechanism to catalyze cytosine-to-uracil conversion in double-stranded DNA.

The Bloom syndrome helicase (BLM) unwinds a variety of complex DNA structures including G quadruplex. Here the authors report RNF111-ARKL1-dependent ubiquitination of BLM in PML NBs, which limits BLM protein levels and maintains G quadruplex abundance in the nucleus.

TDRD3 is a key interaction partner of TOP3B. Here the authors provide molecular mechanisms by which TDRD3 stabilizes TOP3B protein by recruiting the deubiquitylase USP9X. In addition, they show that TDRD3 protects cells from deleterious TOP3B linked DNA and RNA cleavage complexes.

The authors review the consequences of mutations in genes linked to rare hereditary disorders associated with G-quadruplex (G4)-induced replisome stalling, as well as the molecular functions and cellular pathways of these G4-interacting proteins.

ATM signaling is known to be activated following DNA double-strand breaks. Here, this study provides evidences that ATM-dependent signaling pathway is activated by DNA single-strand breaks and regulated by its direct activator protein APE1.

Homologue juxtaposition during meiosis is examined in real time by imaging of tagged chromosomal loci at high resolution in budding yeast, showing that corresponding loci come together and complete pairing in a very short time.

The cryogenic-electron microscopy structure of the D. thermocuniculi IsrB protein in complex with its cognate ωRNA and a target DNA shows that the RNA-dominant IsrB effector complex shares a common scaffold with the protein-dominant Cas9 effector complex.

A hypercompact adenine base editor termed TaRGET-ABE was developed by fusing a catalytically inactive transposon-associated transposase B guided by an engineered RNA to deaminases, which achieves efficient A-to-G conversions via adeno-associated viral delivery in mammalian genomes.

Using single-molecule visualization and manipulation, Chang et al. show that the eukaryotic Smc5/6 complex preferentially binds to and stabilizes ssDNA-dsDNA junctions, which could serve as the molecular basis for its diverse roles in genome maintenance.

Cryo-electron microscopy structures of Cas9 during mismatch cleavage provide insight into the mechanisms that control off-target effects of Cas9, which will aid in the future design of high-fidelity Cas9 variants with reduced off-target cleavage.

Different proteins localised at telomeres ensure chromosome end stability to prevent double strand-end break recognition. Here the authors provide new insight into how in S. cerevisiae the interaction between Rif2 and Rad50 inhibits MRX functions at telomeres.

Different factors protect cells from harmful R-loops, but the way these are formed is still unclear. Authors show here that R-loops form co-transcriptionally by different manners and cells possess specialized mechanisms to prevent them in each case, a major mechanism being independent of replication and another one being linked to replication.

It has been a longstanding goal to promote the propagation of functional mitochondrial DNAs at the expense of pathological molecules in cells where the two species coexist. Here, the authors show that restricting the availability of glucose and glutamine can achieve this outcome.

Using single-molecule magnetic tweezers and biochemical methods, Naqvi et al. revealed how CRISPR–Cas12a regulates the DNA cleavage rate through a conserved stacking interaction between the R-loop and the W355 residue and the sequence of the CRISPR RNA 3′ end.

Cryo-electron microscopy structures of human telomerase and telomerase in complex with TPP1 provide insights into the interactions of these proteins and their activities.

Extracting functional information from 16S rRNA data surveys would provide a valuable tool for large-scale functional ecology. Here, the authors use PICRUSt2 to infer metabolic functions from bacterial marker gene data across the South Pacific Ocean, and compare them with rate data, biomass estimators and predictions based on shotgun metagenomes.

The cyanophage S-2L incorporates 2-aminoadenine (Z) instead of adenine into its DNA, which still pairs with thymine forming a triple hydrogen bond. Here, the authors identify a third gene mazZ located between purZ and datZ that is required for 2-aminoadenine biosynthesis and determine the crystal structures of MazZ and PurZ. They further show that co-expression of these three genes in E.coli enables 2-aminoadenine incorporation into the bacterial genome.

The cyanophage S-2L incorporates 2-aminoadenine (Z) instead of adenine (A) in its genome. Here, the authors provide an explanation for the absence of A in S-2L genome by identifying and characterising functionally and structurally both the HD phosphohydrolase (datZ) that specifically cleaves dATP, and the sole DNA primase-polymerase of S-2L, nonspecific of dATP or dZTP.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer. This study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their substrate selectivity.

Extrachromosomal circular DNA made of telomeric repeats have been found to have an effect on telomere maintenance. By combining electron microscopy with a telomere purification procedure the authors identify damage-induced i-loops as a key intermediate in telomere circle formation.

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleoside diphosphates to provide dNTP precursors for DNA synthesis. Here the authors show that the availability of dNTPs, DNA replication, and cellular proliferation, are modulated by acetylation and deacetylation of RRM2 by KAT7 and Sirt2 respectively.

Primed adaptation in the CRISPR-Cas system helps recognition of previously encountered sequence elements and promotes the formation of new memories. Here the authors characterized spacer precursors of type I-E and type I-F CRISPR-Cas system using in vivo models.

Bacteriophage single-stranded DNA annealing proteins (SSAPs) interact with the C termini of single-stranded binding proteins in host bacteria, a finding that enables engineering of enhanced SSAP portability and DNA recombineering activities.

The combination of heavy isotope labeling and ultra-high-pressure liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC–MS) is used to quantify modified genomic cytosines in pluripotent stem cells in different states and reveals active turnover of methylcytidine in oxidation-dependent and oxidation-independent manners.

Single-molecule fluorescence resonance energy transfer and real-time confocal laser tracking with fluorescence correlation spectroscopy together characterize how individual lac repressor molecules bypass operator sites while exploring the DNA surface at microsecond timescales.

DNA 5-hydroxymethylcytosine (5hmC) modification is associated with gene transcription and used as a mark of mammalian development. Here the authors report a comprehensive 5hmC tissue map and analysis of 5hmC genomic distributions in 19 human tissues derived from 10 organ systems, thus providing insights into the role of 5hmC in tissue-specific development.

Despite some well-characterized functions in cancer, the impact of most long non-coding RNAs remains unknown. Here, the authors discover the lncRNA lincNMR which is upregulated in cancer and drives cell proliferation by interacting with YBX1 and controlling nucleotide metabolism.

The ATR kinase has essential functions apart from its role in DNA replication stress. Here the authors find that in mouse primary B cells ATR tempers the pace of origin firing during the early S phase to avoid exhaustion of dNTPs and other replication factors.