Long interspersed element-1 (L1) is an autonomous retroelement that is active in the human genome. The proposed mechanism of insertion for L1 suggests that cleavage of both strands of genomic DNA is required. We demonstrate that L1 expression leads to a high level of doublestrand break (DSB) formation in DNA using immunolocalization of γ-H2AX foci and the COMET assay. Similar to its role in mediating DSB repair in response to radiation, ATM is required for L1-induced γ-H2AX foci and for L1 retrotransposition. This is the first characterization of a DNA repair response from expression of a non-long terminal repeat (non-LTR) retrotransposon in mammalian cells as well as the first demonstration that a host DNA repair gene is required for successful integration. Notably, the number of L1-induced DSBs is greater than the predicted numbers of successful insertions, suggesting a significant degree of inefficiency during the integration process. This result suggests that the endonuclease activity of endogenously expressed L1 elements could contribute to DSB formation in germ-line and somatic tissues.

Two RecA homologs, Rad51 and Dmc1, assemble as cytologically visible complexes (foci) at the same sites on meiotic chromosomes. Time course analysis confirms that co-foci appear and disappear as the single predominant form. A large fraction of co-foci are eliminated in a red1 mutant, which is expected as a characteristic of the interhomolog-specific recombination pathway. Previous studies suggested that normal Dmc1 loading depends on Rad51. We show here that a mutation in TID1͞RDH54, encoding a RAD54 homolog, reduces Rad51-Dmc1 colocalization relative to WT. A rad54 mutation, in contrast, has relatively little effect on RecA homolog foci except when strains also contain a tid1͞rdh54 mutation. The role of Tid1͞Rdh54 in coordinating RecA homolog assembly may be very direct, because Tid1͞Rdh54 is known to physically bind both Dmc1 and Rad51. Also, Dmc1 foci appear early in a tid1͞rdh54 mutant. Thus, Tid1 may normally act with Rad51 to promote ordered RecA homolog assembly by blocking Dmc1 until Rad51 is present. Finally, whereas double-staining foci predominate in WT nuclei, a subset of nuclei with expanded chromatin exhibit individual Rad51 and Dmc1 foci side-by-side, suggesting that a Rad51 homo-oligomer and a Dmc1 homo-oligomer assemble next to one another at the site of a single double-strand break (DSB) recombination intermediate.

We show that the Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; Rad52, Rad55, and Rad57 are also required supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes.

Background: Rad51 and Dmc1 are Saccharomyces cerevisiae homologues of the Escherichia coli recombination protein RecA. Mutant analysis has shown that both proteins are required for normal meiotic recombination, for timely and efficient formation of synaptonemal complex and for normal progression out from meiotic prophase.

In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422–603 amino acids) CRISPR–Cas12f nucleases that recognize and cleave dsDNA in a PAM dependent manner. Categorized as class 2 type V-F, they originate from the previously identified Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5′ T- or C-rich PAM sequence triggers dsDNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in Escherichia coli and find that some but not all can. Altogether, our findings show that miniature Cas12f nucleases can protect against invading dsDNA like much larger class 2 CRISPR effectors and have the potential to be harnessed as programmable nucleases for genome editing.