DNA End Processing Research Articles

BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle.

Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the two prominent pathways responsible for the repair of DNA double-strand breaks (DSBs). NHEJ is not restricted to a cell-cycle stage, whereas HR is active primarily in the S/G2 phases suggesting there are cell cycle-specific mechanisms that play a role in the choice between NHEJ and HR. Here we show NHEJ is attenuated in S phase via modulation of the autophosphorylation status of the NHEJ factor DNA-PKcs at serine 2056 by the pro-HR factor BRCA1. BRCA1 interacts with DNA-PKcs in a cell cycle-regulated manner and this interaction is mediated by the tandem BRCT domain of BRCA1, but surprisingly in a phospho-independent manner. BRCA1 attenuates DNA-PKcs autophosphorylation via directly blocking the ability of DNA-PKcs to autophosphorylate. Subsequently, blocking autophosphorylation of DNA-PKcs at the serine 2056 phosphorylation cluster promotes HR-required DNA end processing and loading of HR factors to DSBs and is a possible mechanism by which BRCA1 promotes HR.

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

The 5'-3' resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5'-3' DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.

Structural insights into NHEJ: building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time.

Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: (1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), (2) lesion processing, and (3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation.

Quantitation of DNA double-strand break resection intermediates in human cells

5′ strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5′ strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle–dependent manner. Depletion of CtIP, Mre11, Exo1 or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of Brca1 does not, suggesting that the role of Brca1 in HR is primarily to promote Rad51 filament formation, not to regulate end resection.

Non‐homologous end joining‐mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date.

Abstract P3-04-06: Damage-induced BRCA1 phosphorylation contributes to the timing of end resection

Abstract Germline mutations of BRCA1 predispose women to breast and ovarian cancers. BRCA1 functions as a tumor suppressor. A wealth of evidence has established that BRCA1 plays an important role in DNA repair and cell cycle checkpoint control. Efficient repair of double-strand-breaks (DSBs) by BRCA1 is essential to its tumor suppression function. DSBs can be repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ). BRCA1 is hyperphosphorylated upon DSBs. Although phosphorylation of BRCA1 is among the earliest events documented upon DNA damage, the role of BRCA1 phosphorylation in DNA repair remains unclear after more than a decade. It has been proposed that BRCA1 serves as a scaffolding protein that recruits multiple repair proteins to the break sites. One of the BRCA1-containing complexes, MRN (Mre11-Rad50-Nbs1)-BRCA1-CtIP, promotes HR repair by initiating DNA end-resection, the first step in the HR pathway. Mre11 possesses intrinsic nuclease activity, which likely facilitates initial processing of DNA ends for subsequent extensive end-resection. Believed to stimulate the nuclease activity of the MRN complex, CtIP is recruited to the complex by BRCA1. In spite of these useful molecular models, the precise function of BRCA1 in DSB repair, especially the role of its phosphorylation, remains elusive. BRCA1 is phosphorylated by Chk2 kinase upon g-irradiation at serine 988 (human) or serine 971 (mouse). Our data shows that a point mutation in mouse BRCA1 (S971A) that abolishes the Chk2 phosphorylation site impairs end-resection after DSBs. The S971A mutation does not appear to affect the assembly of MRN and CtIP at DSBs, but specifically delays dispersion of BRCA1 from DSBs. Interestingly, BRCA1 is ubiquitinated and degraded by SCFSkp2, and loss of Chk2 phosphorylation at S988 of human BRCA1 impairs its ubiquitination. Previous work has revealed BRCA1 as a scaffold protein for the assembly of HR proteins at DSBs. Paradoxically, BRCA1 also inhibits Mre11 nuclease activity. Our study suggests that BRCA1 plays a dual role in HR. Although BRCA1 is required for initial assembly of the MRN-BRCA1-CtIP complex at DSBs, the exit of BRCA1 from the MRN-BRCA1-CtIP complex is required to relieve its inhibitory effect on Mre11 nuclease activity. Mechanistically, this critical “exit” of BRCA1 from the MRN-BRCA1-CtIP complex is regulated through phosphorylation catalyzed by Chk2 and then degraded by SCFSkp2, which is expressed at S/G2 phases, thus ensuring the initiation of end resection/HR to S/G2 phases of the cell cycle. Our study explains two seemingly paradoxical observations: BRCA1 is required for assembly of the MRN-BRCA1-CtIP complex, but it inhibits Mre11 nuclease activity required for end-resection. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-06.

Visualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET

The Mre11/Rad50/Nbs1 (MRN) complex initiates and coordinates DNA repair and signaling events at double-strand breaks. The interaction between MRN and DNA ends is critical for the recruitment of DNA-processing enzymes, end tethering, and activation of the ATM protein kinase. Here we visualized MRN binding to duplex DNA molecules using single-molecule FRET, and found that MRN unwinds 15-20 base pairs at the end of the duplex, holding the branched structure open for minutes at a time in an ATP-dependent reaction. A Rad50 catalytic domain mutant that is specifically deficient in this ATP-dependent opening is impaired in DNA end resection in vitro and in resection-dependent repair of breaks in human cells, demonstrating the importance of MRN-generated single strands in the repair of DNA breaks.

Coordination and Processing of DNA Ends During Double-Strand Break Repair: The Role of the Bacteriophage T4 Mre11/Rad50 (MR) Complex

The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of double-strand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections.

Treatment with suboptimal doses of raltegravir leads to aberrant HIV-1 integrations

Integration of the DNA copy of the HIV-1 genome into a host chromosome is required for viral replication and is thus an important target for antiviral therapy. The HIV-encoded enzyme integrase (IN) catalyzes two essential steps: 3' processing of the viral DNA ends, followed by the strand transfer reaction, which inserts the viral DNA into host DNA. Raltegravir binds to IN and blocks the integration of the viral DNA. Using the Rous sarcoma virus-derived vector RCAS, we previously showed that mutations that cause one viral DNA end to be defective for IN-mediated integration led to abnormal integrations in which the provirus had one normal and one aberrant end, accompanied by rearrangements in the host genome. On the basis of these results, we expected that suboptimal concentrations of IN inhibitors, which could block one of the ends of viral integration, would lead to similar aberrant integrations. In contrast to the proviruses from untreated cells, which were all normal, ∼10-15% of the proviruses isolated after treatment with a suboptimal dose of raltegravir were aberrant. The aberrant integrations were similar to those seen in the RCAS experiments. Most of the aberrant proviruses had one normal end and one aberrant end and were accompanied by significant rearrangements in the host genome, including duplications, inversions, deletions and, occasionally, acquisition of sequences from other chromosomes. The rearrangements of the host DNA raise concerns that these aberrant integrations might have unintended consequences in HIV-1-infected patients who are not consistent in following a raltegravir-containing treatment regimen.

Track structure based modelling of chromosome aberrations after photon and alpha-particle irradiation

A computational model of radiation-induced chromosome aberrations in human cells within the PARTRAC Monte Carlo simulation framework is presented. The model starts from radiation-induced DNA damage assessed by overlapping radiation track structures with multi-scale DNA and chromatin models, ranging from DNA double-helix in atomic resolution to chromatin fibre loops, heterochromatic and euchromatic regions, and chromosome territories. The repair of DNA double-strand breaks via non-homologous end-joining is followed. Initial spatial distribution and complexity, diffusive motion, enzymatic processing, synapsis and ligation of individual DNA ends from the breaks are simulated. To enable scoring of different chromosome aberration types resulting from improper joining of DNA fragments, the repair module has been complemented by tracking the chromosome origin of the ligated fragments and the positions of centromeres. The modelled motion of DNA ends has sub-diffusive characteristics and corresponds to measured chromatin mobility within time-scales of a few hours. The calculated formation of dicentrics after photon and α-particle irradiation in human fibroblasts is compared to experimental data (Cornforth et al., 2002, Radiat Res 158, 43). The predicted yields of dicentrics overestimate the measurements by factors of five for γ-rays and two for α-particle irradiation. Nevertheless, the observed relative dependence on radiation dose is correctly reproduced. Calculated yields and size distributions of other aberration types are discussed. The present work represents a first mechanistic approach to chromosome aberrations and their kinetics, combining full track structure simulations with detailed models of chromatin and accounting for the kinetics of DNA repair.

Relationship of DNA degradation by Saccharomyces cerevisiae Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection

Homologous recombination is a major pathway for repair of DNA double-strand breaks. This repair process is initiated by resection of the 5′-terminated strand at the break site. In yeast, resection is carried out by three nucleolytic complexes: Mre11-Rad50-Xrs2, which functions at the initial step and also stimulates the two processive pathways, Sgs1-Dna2 and Exonuclease 1 (Exo1). Here we investigated the relationship between the three resection pathways with a focus on Exo1. Exo1 preferentially degrades the 5′-terminal stand of duplex DNA that is single stranded at the 3′ end, in agreement with its role downstream of the Mre11-Rad50-Xrs2 complex. Replication protein A (RPA) stimulates DNA end resection by Exo1 by both preventing nonspecific binding of Exo1 to and preventing degradation of single-stranded DNA. Nucleolytic degradation of DNA by Exo1 is inhibited by the helicase-deficient Sgs1 K706A mutant protein and, reciprocally, the nuclease-deficient Exo1 D173A mutant protein inhibits DNA unwinding by Sgs1. Thus, the activities of Sgs1 and Exo1 at DNA ends are mutually exclusive, establishing biochemically that both machineries function independently in DNA end processing. We also reconstituted Sgs1-Top3-Rmi1-RPA-Dna2 and Exo1 resection reactions both individually and combined, either with or without the Mre11-Rad50-Xrs2 complex. We show that the yeast Sgs1-Dna2 and Exo1 pathways do not stimulate one another and function as independent and separate DNA end-processing machineries, even in the presence of the stimulatory Mre11-Rad50-Xrs2 complex.

Analyzing the effects of sub‐optimal doses of raltegravir on HIV‐1 integration

Integration of the DNA copy of HIV‐1 genome into the host chromosome is required for viral replication and is, for this reason, an important drug target. The HIV‐encoded enzyme integrase (IN) catalyzes two critical steps: 3′ processing of the viral DNA ends, followed by the strand transfer reaction which inserts the viral DNA into host DNA. Raltegravir is the first approved drug that blocks the integration of the viral DNA. Using the Rous Sarcoma Virus (RSV)‐derived vector system RCAS, we previously showed that mutants in which one of the viral DNA ends was not a good substrate for IN were integrated abnormally, leading to rearrangements of the host genome including duplications, inversions and, occasionally, the acquisition of sequences from other chromosomes. Based on these results, we expected that suboptimal concentrations of IN inhibitors, like Raltegravir, which could also block one end of the viral integration reaction, would cause similar aberrant integrations. In contrast to the normal proviruses recovered from untreated cells, all of which were normal, 10–15% proviruses isolated after treatment with a suboptimal dose of Raltegravir were aberrant and these integrations were accompanied by chromosomal defects similar to those seen with the RCAS system. These rearrangements of the host DNA raise concerns for HIV‐1‐infected patients who do not consistently follow their Raltegravir regimen.

Sensing of DNA breaks by the ATM and ATR checkpoint kinases

DNA breaks are commonly induced during genome engineering and are recognized as DNA damage. Understanding how cells recognize and respond to DNA breaks is critically important for developing and improving strategies for genome engineering. In human cells, ATM and ATR are the two master checkpoint kinases that recognize and respond to double‐stranded DNA breaks (DSBs). While both ATM and ATR are activated by DSBs, their DNA damage specificities are distinct and their functions are not redundant. How ATM and ATR are activate by DSBs and how they execute their distinct functions are yet to be fully understood.To understand how ATM and ATR are activated by DSBs, we developed an in vitro biochemical assay in which both ATM and ATR can be activated in a DNA structure‐regulated manner. Using this assay, we found that ATM and ATR are activated by distinct types of DSBs. While ATM is activated by DSBs with blunt ends or short single‐stranded DNA (ssDNA) overhangs, ATR is activated by DSBs carrying long ssDNA overhangs. Our findings suggest that ATM and ATR are sequentially activated during the cellular response to DSBs. When DSBs with blunt ends or short ssDNA overhangs are generated in cells, they will primarily activate ATM. If DNA breaks are engaged in homologous recombination and long ssDNA is generated by nucleolytic processing of DNA ends, ATR will become the primary sensor of these processed DSBs. Using this biochemical assay, we have also characterized which DNA damage sensors are required for the activation of ATM and ATR by DSBs. Surprisingly, the Nijmegen breakage syndrome protein 1 (Nbs1), a known regulator of ATM, is also important for ATR activation. Our results suggest that Nbs1 is a multi‐functional sensor of DSBs that is able to detect different types of DSBs and regulate the activation of both ATM and ATR.

Mutant Ataxin‐3 inhibits 3’ phosphatase activity of human polynucleotide kinase 3′‐phosphatase (PNKP)

Ataxin‐3 (ATXN3), a polyglutamine repeat‐containing protein is mutated to abnormally expanded poly Q tracts in Spinocerebellar Ataxia type 3 SCA3 / Machado‐Joseph Disease (MJD). This disease is one of the most common dominantly inherited Ataxia worldwide; the molecular defect is CAG repeat expansion (from 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is poorly understood. Here, we show that, expanded poly Q form of human Ataxin‐3 (Q72), stably associates and inhibits 3’[prime]‐phosphatase activity of PNKP one of the major DNA end processing enzyme in mammalian cell. Activities of other single strand break repair (SSBR) proteins are not affected. A conditionally expressing pathological ATXN3 cells also showed reduced 3’[prime]‐phophatase activity of PNKP and subsequent accumulation of DNA strand breaks in nuclear genomes. Moreover, LA‐QPCR analysis of human MJD patients’ brain samples showed significant accumulation of DNA strand breaks that supported the cause. In summary, selective inhibition of 3’[prime]‐phosphatase activity of PNKP may be the signature mechanism contributing to accumulated DNA damage and pathophysiology of SCA3/MJD.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the "reaching dimer" previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.

High Frequency Targeted Mutagenesis Using Engineered Endonucleases and DNA-End Processing Enzymes

Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells.

A single strand that links multiple neuropathologies in human disease

The development of the human central nervous system is a complex process involving highly coordinated periods of neuronal proliferation, migration and differentiation. Disruptions in these neurodevelopmental processes can result in microcephaly, a neuropathological disorder characterized by a reduction in skull circumference and total brain volume, whereas a failure to maintain neuronal health in the adult brain can lead to progressive neurodegeneration. Defects in the cellular pathways that detect and repair DNA damage are a common cause of both these neuropathologies and are associated with a growing number of hereditary human disorders. In particular, defects in the repair of DNA single strand breaks, one of the most commonly occurring types of DNA lesion, have been associated with three neuropathological diseases: ataxia oculomotor apraxia 1, spinocerebellar ataxia with neuronal neuropathy 1 and microcephaly, early-onset, intractable seizures and developmental delay. A striking similarity between these three human diseases is that they are all caused by mutations in DNA end processing factors, suggesting that a particularly crucial stage of DNA single strand break repair is the repair of breaks with 'damaged' termini. Additionally all three disorders lack any extraneurological symptoms, such as immunodeficiency and cancer predisposition, which are typically found in other human diseases associated with defective DNA repair. However despite these similarities, two of these disorders present with progressive cerebellar degeneration, whereas the third presents with severe microcephaly. This review discusses the molecular defects behind these disorders and presents several hypotheses based on current literature on a number of important questions, in particular, how do mutations in different end processing factors within the same DNA repair pathway lead to such different neuropathologies?

Mre11 ATLD17/18 mutation retains Tel1/ATM activity but blocks DNA double-strand break repair

The Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structural and in vivo functional studies to uncover key properties of Mre11-W243R, a mutation identified in two pediatric cancer patients with enhanced ataxia telangiectasia-like disorder. Purified human Mre11-W243R retains nuclease and DNA binding activities in vitro. X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 three-dimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions. The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure. W248R differs from other ataxia telangiectasia-like disorder analog alleles by the reduced stability of its interaction with Rad50 in cell lysates. Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions.

Retroviral Integrase Proteins and HIV-1 DNA Integration

Retroviral integrases catalyze two reactions, 3'-processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of integrase-DNA complexes from prototype foamy virus, a member of the Spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinically relevant integrase strand transfer inhibitors work. Underscoring the translational potential of targeting virus-host interactions, small molecules that bind at the host factor lens epithelium-derived growth factor/p75-binding site on HIV-1 integrase promote dimerization and inhibit integrase-viral DNA assembly and catalysis. Here, we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions.

Coupling endonucleases with DNA end–processing enzymes to drive gene disruption

Targeted DNA double-strand breaks introduced by rare-cleaving designer endonucleases can be harnessed for gene disruption applications by engaging mutagenic nonhomologous end-joining DNA repair pathways. However, endonuclease-mediated DNA breaks are often subject to precise repair, which limits the efficiency of targeted genome editing. To address this issue, we coupled designer endonucleases to DNA end-processing enzymes to drive mutagenic break resolution, achieving up to 25-fold enhancements in gene disruption rates.