DNA Recombination Research Articles

Expression patterns of Arabidopsis thaliana RecQ-like (AtRecQl) genes and the roles of AtRecQl2 and AtRecQl3 in response to abiotic stress.

Helicases are involved in almost every nucleic acid metabolism process. Within this family, RecQ helicase proteins protect genome integrity across all organisms through DNA recombination, repair, and replication. This study focused on five Arabidopsis thaliana RecQ-like (AtRecQl) genes with diverse functionalities. Analysis of ProAtRecQl:GUS expression during vegetative and reproductive development stages revealed organ- and tissue-specific patterns. Changes in AtRecQls transcript levels in response to abiotic stressors suggest their involvement in diverse stimuli responses. Notably, germination and growth rates were lower in atrecql2 and atrecql3 mutants under various salt concentrations and cold conditions. These findings indicate that AtRecQl2 and AtRecQl3 act as positive regulators of abiotic stress tolerance during the germinative and postgerminative phases.

Disparate requirements for RAD54L in replication fork reversal.

RAD54L is a DNA motor protein with multiple roles in homologous recombination DNA repair. In vitro, RAD54L was shown to also catalyze the reversal and restoration of model replication forks. In cells, however, little is known about how RAD54L may regulate the dynamics of DNA replication. Here, we show that RAD54L restrains the progression of replication forks and functions as a fork remodeler in human cancer cell lines and non-transformed cells. Analogous to HLTF, SMARCAL1and FBH1, and consistent with a role in fork reversal, RAD54L decelerates fork progression in response to replication stress and suppresses the formation of replication-associated ssDNA gaps. Interestingly, loss of RAD54L prevents nascent strand DNA degradation in both BRCA1/2- and 53BP1-deficient cells, suggesting that RAD54L functions in both pathways of RAD51-mediated replication fork reversal. In the HLTF/SMARCAL1 pathway, RAD54L is critical, but its ability to catalyze branch migration is dispensable, indicative of its function downstream of HLTF/SMARCAL1. Conversely, in the FBH1 pathway, branch migration activity of RAD54L is essential, and FBH1 engagement is dependent on its concerted action with RAD54L. Collectively, our results reveal disparate requirements for RAD54L in two distinct RAD51-mediated fork reversal pathways, positing its potential as a future therapeutic target.

Prevalence and spectrum of germline pathogenic variants in cancer susceptibility genes among mexican patients with exocrine pancreatic cancer

BackgroundAlthough universal germline genetic testing is recommended for patients with exocrine pancreatic cancer (PC), access to genetic testing remains limited in low- and middle-income countries. This study aims to narrow the gap in our understanding of the spectrum of germline pathogenic and likely pathogenic variants (PVs) in cancer susceptibility genes in the Mexican population. MethodsThe landscape of PVs in cancer susceptibility genes was identified by next-generation sequencing multigene panel assays among patients with PC who were enrolled in the Clinical Cancer Genomics Community Research Network prospective registry in Mexico City. ResultsFrom August 2019 to April 2023, 137 patients underwent genetic testing. The median age at diagnosis was 60 years (range 36–85), 58.4 % were women, and 38.7 % were metastatic at diagnosis. The frequency of germline PVs was 16 % (n = 22): ATM 36.4 % (n = 8), CDKN2A/p16INK4A 27.3 % (n = 6), BRCA2 9.1 % (n = 2), PALB2 9.1 % (n = 2), CHEK2 9.1 % (n = 2), TP53 4.5 % (n = 1), and NF1 4.5 % (n = 1). Additionally, 2 carriers of monoallelic germline variants in MUTYH were identified. No significant differences were observed between carriers and non-carriers in terms of family history of pancreatic cancer. ConclusionsWe identified a significant frequency of actionable germline PVs in Mexicans with PC, wherein the majority were in a broad spectrum of genes associated with the homologous recombination DNA repair mechanism. Most pancreatic cancer associated PVs were detected in non-BRCA genes, so our findings support the recommendation of multigene panel testing for genetic cancer risk assessment of Mexican individuals with PC.

Intein splicing efficiency and RadA levels can control the mode of archaeal DNA replication.

Inteins (intervening proteins), mobile genetic elements removed through protein splicing, often interrupt proteins required for DNA replication, recombination, and repair. An abundance of in vitro evidence implies that inteins may act as regulatory elements, whereby reduced splicing inhibits production of the mature protein lacking the intein, but in vivo evidence of regulatory intein excision in the native host is absent. The model archaeon Thermococcus kodakarensis encodes 15 inteins, and we establish the impacts of intein splicing inhibition on host physiology and replication in vivo. We report that a decrease in intein splicing efficiency of the recombinase RadA, a Rad51/RecA homolog, has widespread physiological consequences, including a general growth defect, increased sensitivity to DNA damage, and a switch in the mode of DNA replication from recombination-dependent replication toward origin-dependent replication.

Abstract B029: Repurposing Digital PCR to Diagnose and Create a Novel Genomic Signature for Homologous Recombination Deficiency in Pancreatic Adenocarcinoma

Abstract Introduction: Germline mutations in DNA repair genes are often associated with increased risk of cancer. Mutations that affect the homologous recombination (HR) DNA repair pathway ( e.g., BRCA 1 and BRCA2) are known drivers of pancreatic adenocarcinoma. Early detection relies on identification of pathogenic mutations in patients with a family history of cancer. Next generation sequencing panels often use a select set of pathogenic variants for diagnosis, meaning that individuals with variants of uncertain significance may be missed, limiting testing of other family members for the potentially disease-causing variant. Here we describe a digital PCR assay developed to detect BRCA1 and BRCA2 variant that may eventually serve as a rapid, cost-effective alternative to sequencing that costs less than $5.00 per sample with results in 24 hours. This study highlights the need for better diagnostic tools, which will become critically important as we begin to study pathogenic mutations in more diverse populations. Methods: We have collected blood from 4 BRCA1 mutants and 5 BRCA2 mutants who have already undergone germline sequencing from commercially available assays. Clinicopathologic data has been collected as well. We designed two fluorescently labelled TaqMan probes, one that recognizes the wild-type BRCA alleles and one specific for mutant BRCA alleles. Briefly, 1 ng of genomic DNA is mixed with amplicon primers and fluorescent Taq-Man probes, then partitioned into 20,000 separate 517 picoliter wells. Quantification and detection are achieved based upon the fluorescent signal measured from each well. Results: We have successfully confirmed pathologic BRCA1 and BRCA2 mutations previously identified with next generation sequencing in 100% of samples analyzed. All three BRCA2 mutations generate a premature stop codon leading to a truncated protein and are known to be pathogenic. Two related patients have the same BRCA1 frameshift mutation that results in a premature stop codon, and one patient has a duplicated cytosine that leads to a frameshift mutation that is one of three characterized Ashkenazi Jewish founder mutations that increases cancer risk in this population . Conclusions: Here we report an innovative assay using digital PCR (dPCR) that provides a cost-effective, time-efficient method to identify MMR mutants and characterize VUS in patients and their families. Citation Format: Jennifer Brooke Valerin, Sophie Hasson, Valeria Rangel, Nicholas Pannunzio. Repurposing Digital PCR to Diagnose and Create a Novel Genomic Signature for Homologous Recombination Deficiency in Pancreatic Adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B029.

Structural Basis for Dimerization and Activation of UvrD-family Helicases.

UvrD-family helicases are superfamily 1A motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD family monomers translocate along single stranded (ss) DNA but need to be activated by dimerization to unwind DNA in the absence of force or accessory factors. However, prior structural studies have only revealed monomeric complexes. Here, we report the first structures of a dimeric UvrD-family helicase, Mycobacterium tuberculosis UvrD1, both free and bound to a DNA junction. In each structure, the dimer interface occurs between the 2B subdomains of each subunit. The apo UvrD1 dimer is observed in symmetric compact and extended forms indicating substantial flexibility. This symmetry is broken in the DNA-bound dimer complex with leading and trailing subunits adopting distinct conformations. Biochemical experiments reveal that the E. coli UvrD dimer shares the same 2B-2B interface. In contrast to the dimeric structures, an inactive, auto-inhibited UvrD1 DNA-bound monomer structure reveals 2B subdomain-DNA contacts that are likely inhibitory. The major re-orientation of the 2B subdomains that occurs upon UvrD1 dimerization prevents these duplex DNA interactions, thus relieving the auto-inhibition. These structures reveal that the 2B subdomain serves a major regulatory role rather than participating directly in DNA unwinding.

Transcriptomic analysis of meiotic genes during the mitosis-to-meiosis transition in Drosophila females.

Germline cells produce gametes, which are specialized cells essential for sexual reproduction. Germline cells first amplify through several rounds of mitosis before switching to the meiotic program, which requires specific sets of proteins for DNA recombination, chromosome pairing, and segregation. Surprisingly, we previously found that some proteins of the synaptonemal complex, a prophase I meiotic structure, are already expressed and required in the mitotic region of Drosophila females. Here, to assess if additional meiotic genes were expressed earlier than expected, we isolated mitotic and meiotic cell populations to compare their RNA content. Our transcriptomic analysis reveals that all known meiosis I genes are already expressed in the mitotic region; however, only some of them are translated. As a case study, we focused on mei-W68, the Drosophila homolog of Spo11, to assess its expression at both the mRNA and protein levels and used different mutant alleles to assay for a premeiotic function. We could not detect any functional role for Mei-W68 during homologous chromosome pairing in dividing germ cells. Our study paves the way for further functional analysis of meiotic genes expressed in the mitotic region.

Optogenetic control of early embryos labeling using photoactivatable Cre recombinase 3.0.

Establishing a highly efficient photoactivatable Cre recombinase PA-Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development. This study examined the blue light-mediated optical regulation of Cre-loxP recombination using PA-Cre3.0 transgenic early mouse pre-implantation embryos. We found that inducing PA-Cre3.0 expression in the heterozygous state did not show detectable recombination activation with blue light. Conversely, in homozygous embryos, DNA recombination by PA-Cre3.0 was successfully induced by blue light and resulted in the activation of the red fluorescent protein reporter gene, while almost no leaks of Cre recombination activity were detected in embryos without light illumination. Thus, we characterize the conditions under which the PA-Cre3.0 system functions efficiently in early mouse embryos. These results are expected to provide a new optogenetic tool for certain biological studies, such as developmental process analysis and lineage tracing in early mouse embryos.

Structural and biochemical characterization of the C-terminal region of the human RTEL1 helicase.

RTEL1 is an essential DNA helicase which plays an important role in various aspects of genome stability, from telomere metabolism to DNA replication, repair and recombination. RTEL1 has been implicated in a number of genetic diseases and cancer development, including glioma, breast, lung and gastrointestinal tumors. RTEL1 is a FeS helicase but, in addition to the helicase core, it comprises a long C-terminal region which includes a number of folded domains connected by intrinsically disordered loops and mediates RTEL1 interaction with factors involved in pivotal cellular pathways. However, information on the architecture and the function of this region is still limited. We expressed and purified a variety of fragments encompassing the folded domains and the unstructured regions. We determined the crystal structure of the second repeat, confirming that it has a fold similar to the harmonin homology domains. SAXS data provide low-resolution information on all the fragments and suggest that the presence of the RING domain affects the overall architecture of the C-terminal region, making the structure significantly more compact. NMR data provide experimental information on the interaction between PCNA and the RTEL1 C-terminal region, revealing a putative low-affinity additional site of interaction. A biochemical analysis shows that the C-terminal region, in addition to a preference for telomeric RNA and DNA G-quadruplexes, has a high affinity for R-loops and D-loops, consistent with the role played by the RTEL1 helicase in homologous recombination, telomere maintenance and preventing replication-transcription conflicts. We further dissected the contribution of each domain in binding different substrates.

Genomic Landscape of Osteosarcoma of Bone in an Older-Aged Patient Population and Analysis of Possible Etiologies Based on Molecular Signature.

Background: Osteosarcoma (OS), the most common primary malignant bone tumor, occurs mostly in the pediatric and adolescent (P/A) population where it has been subject to intense study whereas OS arising in the older-aged adult population has undergone less scrutiny. Materials and Methods: In this study, we assess the molecular aberrations detected in eight older adult patients (>59 years of age) with OS of bone by whole-exome sequencing (WES) on formalin-fixed, paraffin-embedded tissue and quantified the contributions of endogenous and exogenous mutational processes to tumor mutational burden and to tumorigenesis through computational analysis. Results: We identified 86 clinically significant somatic mutations. TP53 mutations occurred in OSs of three patients and one patient harbored a pathogenic germline mutation of TP53. Loss-of-heterozygosity of DNA-damage repair genes occurred in all six tumors evaluated. Computational analysis of single nucleotide variants within each tumor detected eight distinct mutagenic processes of which age-associated mutational processes, thiopurine chemotherapy, and defective homologous DNA recombination repair contributed the most to both tumor mutation burden and tumor pathogenesis. Conclusion: The genomic landscape of our older OS patients deciphered by WES is extremely diverse with only 15% of mutated somatic genes uncovered in our study previously described in P/A-enriched OS studies. Endogenous age-related mutagenic processes, defective DNA homologous recombination repair, and exogenous effects of chemotherapy are mainly responsible for pathogenic mutations in OS occurring in our cohort.

Triple-Negative Breast Cancer: Molecular Particularities Still a Challenge.

Worldwide, breast cancer (BC) is one of the most common cancers in women and is responsible for the highest number of cancer-related deaths among women, with a special clinical behavior and therapy response. Triple-negative breast cancer (TNBC) is seen as a highly invasive BC, characterized by a short survival, higher mortality, recurrence, and metastasis when it is compared to the other BC subtypes. The molecular subtyping of TNBC based on mRNA expression levels does not accurately reflect protein expression levels, which impacts targeted therapy effectiveness and prognostic predictions. Most TNBC cases exhibit a high frequency of homologous recombination (HR) DNA repair deficiency (HRD) signatures and are associated with a complex genomic profile. Biomarker research in TNBC includes investigating genetic mutations, gene expression patterns, immune system-related markers, and other factors that can provide valuable information for diagnosis, treatment selection, and patient outcomes. Additionally, these biomarkers are often crucial in the development of personalized and precision medicine approaches, where treatments are customized to each patient's unique characteristics. This ongoing research is essential for improving the management and outcomes of TNBC, which is a challenging and heterogeneous form of breast cancer. The findings of this research have practical implications for refining treatment strategies, particularly in selecting appropriate systemic therapies and integrating traditional treatment modalities like surgery and radiotherapy into comprehensive care plans for TNBC patients.

Unveiling the role of uranium in enhancing the transformation of antibiotic resistance genes

Transformation represents one of the most important pathways for the horizontal transfer of antibiotic resistance genes (ARGs), which enables competent bacteria to acquire extracellular ARGs from the surrounding environment. Both heavy metals and irradiation have been demonstrated to influence the bacterial transformation process. However, the impact of ubiquitously occurring radioactive heavy metals on the transformation of ARGs remains largely unknown. Here, we showed that a representative radioactive nuclide, uranium (U), at environmental concentrations (0.005–5 mg/L), improved the transformation frequency of resistant plasmid pUC19 into Escherichia coli by 0.10–0.85-fold in a concentration-dependent manner. The enhanced ARGs transformation ability under U stress was demonstrated to be associated with reactive oxygen species (ROS) overproduction, membrane damage, and up-regulation of genes related to DNA uptake and recombination. Membrane permeability and ROS production were the predominant direct and indirect factors affecting transformation ability, respectively. Our findings provide valuable insight into the underlying mechanisms of the impacts of U on the ARGs transformation process and highlight concerns about the exacerbated spread of ARGs in radioactive heavy metal-contaminated ecosystems, especially in areas with nuclear activity or accidents.

Separation of telomere protection from length regulation by two different point mutations at amino acid 492 of RTEL1.

RTEL1 is an essential DNA helicase that plays multiple roles in genome stability and telomere length regulation. A variant of RTEL1 with a lysine at position 492 is associated with short telomeres in Mus spretus , while a conserved methionine at this position is found in M. musculus , which has ultra-long telomeres. In humans, a missense mutation at this position ( Rtel1 M492I ) causes a fatal telomere biology disease termed Hoyeraal-Hreidarsson syndrome (HHS). Introducing the Rtel1 M492K mutation into M. musculus shortened the telomeres of the resulting strain, termed 'Telomouse', to the length of human telomeres. Here, we report on a mouse strain carrying the Rtel1 M492I mutation, termed 'HHS mouse'. The HHS mouse telomeres are not as short as those of Telomice but nevertheless they display higher levels of telomeric DNA damage, fragility and recombination, associated with anaphase bridges and micronuclei. These observations indicate that the two mutations separate critical functions of RTEL1: M492K mainly reduces the telomere length setpoint, while M492I predominantly disrupts telomere protection. The two mouse models enable dissecting the mechanistic roles of RTEL1 and the different contributions of short telomeres and DNA damage to telomere biology diseases, genomic instability, cancer, and aging.

Mechanism of homology search expansion during recombinational DNA break repair in Saccharomyces cerevisiae

Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination (HR). How it operates in cells remains elusive. We developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S.cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembling extensive NPFs. DSB end-tethering promotes coordinated search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology searchthat isorchestrated by chromatin organizers, long-range resection, end-tethering,andspecialized genetic elements andthatexploits the stiff NPF structure conferred by Rad51 oligomerization.

Germline RAD51C and RAD51D Mutations in High-Risk Chinese Breast and/or Ovarian Cancer Patients and Families.

RAD51C and RAD51D are crucial in homologous recombination (HR) DNA repair. The prevalence of the RAD51C and RAD51D mutations in breast cancer varies across ethnic groups. Associations of RAD51C and RAD51D germline pathogenic variants (GPVs) with breast and ovarian cancer predisposition have been recently reported and are of interest. We performed multi-gene panel sequencing to study the prevalence of RAD51C and RAD51D germline mutations among 3728 patients with hereditary breast and/or ovarian cancer (HBOC). We identified 18 pathogenic RAD51C and RAD51D mutation carriers, with a mutation frequency of 0.13% (5/3728) and 0.35% (13/3728), respectively. The most common recurrent mutation was RAD51D c.270_271dupTA; p.(Lys91Ilefs*13), with a mutation frequency of 0.30% (11/3728), which was also commonly identified in Asians. Only four out of six cases (66.7%) of this common mutation tested positive for homologous recombination deficiency (HRD). Taking the family studies in our registry and tumor molecular pathology together, we concluded that this relatively common RAD51D variant showed incomplete penetrance in our local Chinese community. Personalized genetic counseling emphasizing family history for families with this variant, as suggested at the UK Cancer Genetics Group (UKCGG) Consensus meeting, would also be appropriate in Chinese families.

The multifaceted roles of the Ctf4 replisome hub in the maintenance of genome integrity

At the core of cellular life lies a carefully orchestrated interplay of DNA replication, recombination, chromatin assembly, sister-chromatid cohesion and transcription. These fundamental processes, while seemingly discrete, are inextricably linked during genome replication. A set of replisome factors integrate various DNA transactions and contribute to the transient formation of sister chromatid junctions involving either the cohesin complex or DNA four-way junctions. The latter structures serve DNA damage bypass and may have additional roles in replication fork stabilization or in marking regions of replication fork blockage. Here, we will discuss these concepts based on the ability of one replisome component, Ctf4, to act as a hub and functionally link these processes during DNA replication to ensure genome maintenance.

RHNO1: at the crossroads of DNA replication stress, DNA repair, and cancer.

The DNA replication stress (DRS) response is a crucial homeostatic mechanism for maintaining genome integrity in the face of intrinsic and extrinsic barriers to DNA replication. Importantly, DRS is often significantly increased in tumor cells, making tumors dependent on the cellular DRS response for growth and survival. Rad9-Hus1-Rad1 Interacting Nuclear Orphan 1 (RHNO1), a protein involved in the DRS response, has recently emerged as a potential therapeutic target in cancer. RHNO1 interacts with the 9-1-1 checkpoint clamp and TopBP1 to activate the ATR/Chk1 signaling pathway, the crucial mediator of the DRS response. Moreover, RHNO1 was also recently identified as a key facilitator of theta-mediated end joining (TMEJ), a DNA repair mechanism implicated in cancer progression and chemoresistance. In this literature review, we provide an overview of our current understanding of RHNO1, including its structure, function in the DRS response, and role in DNA repair, and discuss its potential as a cancer therapeutic target. Therapeutic targeting of RHNO1 holds promise for tumors with elevated DRS as well as tumors with DNA repair deficiencies, including homologous recombination DNA repair deficient (HRD) tumors. Further investigation into RHNO1 function in cancer, and development of approaches to target RHNO1, are expected to yield novel strategies for cancer treatment.

Accumulation of Large Lineage-Specific Repeats Coincides with Sequence Acceleration and Structural Rearrangement in Plantago Plastomes.

Repeats can mediate rearrangements and recombination in plant mitochondrial genomes and plastid genomes. While repeat accumulations are linked to heightened evolutionary rates and complex structures in specific lineages, debates persist regarding the extent of their influence on sequence and structural evolution. In this study, 75 Plantago plastomes were analyzed to investigate the relationships between repeats, nucleotide substitution rates, and structural variations. Extensive repeat accumulations were associated with significant rearrangements and inversions in the large inverted repeats (IRs), suggesting that repeats contribute to rearrangement hotspots. Repeats caused infrequent recombination that potentially led to substoichiometric shifting, supported by long-read sequencing. Repeats were implicated in elevating evolutionary rates by facilitating localized hypermutation, likely through DNA damage and repair processes. This study also observed a decrease in nucleotide substitution rates for loci translocating into IRs, supporting the role of biased gene conversion in maintaining lower substitution rates. Combined with known parallel changes in mitogenomes, it is proposed that potential dysfunction in nuclear-encoded genes associated with DNA replication, recombination, and repair may drive the evolution of Plantago organellar genomes. These findings contribute to understanding how repeats impact organellar evolution and stability, particularly in rapidly evolving plant lineages.

Pharmacotherapeutic Considerations on Telomere Biology: The Positive Effect of Pharmacologically Active Substances on Telomere Length.

Telomeres are part of chromatin structures containing repeated DNA sequences, which function as protective caps at the ends of chromosomes and prevent DNA degradation and recombination, thus ensuring the integrity of the genome. While telomere length (TL) can be genetically inherited, TL shortening has been associated with ageing and multiple xenobiotics and bioactive substances. TL has been characterised as a reliable biomarker for the predisposition to developing chronic pathologies and their progression. This narrative review aims to provide arguments in favour of including TL measurements in a complex prognostic and diagnostic panel of chronic pathologies and the importance of assessing the effect of different pharmacologically active molecules on the biology of telomeres. Medicines used in the management of cardiovascular diseases, diabetes, schizophrenia, hormone replacement therapy at menopause, danazol, melatonin, and probiotics have been studied for their positive protective effects against TL shortening. All these classes of drugs are analysed in the present review, with a particular focus on the molecular mechanisms involved.

Structural mechanism of bridge RNA-guided recombination

Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes1. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops2. Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle. The IS110 synaptic complex comprises two recombinase dimers, one of which houses the target-binding loop of the bRNA and binds to target DNA, whereas the other coordinates the bRNA donor-binding loop and donor DNA. We uncovered the formation of a composite RuvC–Tnp active site that spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both target and donor DNA. A comparison of the three structures revealed that (1) the top strands of target and donor DNA are cleaved at the composite active sites to form covalent 5′-phosphoserine intermediates, (2) the cleaved DNA strands are exchanged and religated to create a Holliday junction intermediate, and (3) this intermediate is subsequently resolved by cleavage of the bottom strands. Overall, this study reveals the mechanism by which a bispecific RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination.