Double-stranded DNA binding function of RAD51 in DNA protection and its regulation by BRCA2.

RAD51 and the breast cancer suppressor BRCA2 have critical functions in DNA double-strand (dsDNA) break repair by homologous recombination and the protection of newly replicated DNA from nucleolytic degradation. The recombination function of RAD51 requires its binding to single-stranded DNA (ssDNA), whereas binding to dsDNA is inhibitory. Using reconstituted MRE11-, EXO1-, and DNA2-dependent nuclease reactions, we show that the protective function of RAD51 unexpectedly depends on its binding to dsDNA. The BRC4 repeat of BRCA2 abrogates RAD51 binding to dsDNA and accordingly impairs the function of RAD51 in protection. The BRCA2 C-terminal RAD51-binding segment (TR2) acts in a dominant manner to overcome the effect of BRC4. Mechanistically, TR2 stabilizes RAD51 binding to dsDNA, even in the presence of BRC4, promoting DNA protection. Our data suggest that RAD51's dsDNA-binding capacity may have evolved together with its function in replication fork protection and provide a mechanistic basis for the DNA-protection function of BRCA2.

DNA End Resection: Mechanism and Control.

DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5'→3' nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.

Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand cross-links.

DNA interstrand cross-links (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, result in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of programmed DNA double-strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA-binding domain (DBD) mutants (c.8488-1G>A and c.8524C>T) discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for the protection of DNA at ICLs. Cells carrying BRCA2 DBD mutations are sensitive to ICL-inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease that is implicated in the resection of HU-induced stalled replication forks. Our results also indicate that unlike the processing at HU-induced stalled forks, the function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.

RPA Phosphorylation Inhibits DNA Resection.

Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom's syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR.

CtIP-Mediated Fork Protection Synergizes with BRCA1 to Suppress Genomic Instability upon DNA Replication Stress.

Protecting stalled DNA replication forks from degradation by promiscuous nucleases is essential to prevent genomic instability, a major driving force of tumorigenesis. Several proteins commonly associated with the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) have been implicated in the stabilization of stalled forks. Human CtIP, in conjunction with the MRE11 nuclease complex, plays an important role in HR by promoting DSB resection. Here, we report an unanticipated function for CtIP in protecting reversed forks from degradation. Unlike BRCA proteins, which defend nascent DNA strands from nucleolytic attack by MRE11, we find that CtIP protects perturbed forks from erroneous over-resection by DNA2. Finally, we uncover functionally synergistic effects between CtIP and BRCA1 in mitigating replication-stress-induced genomic instability. Collectively, our findings reveal a DSB-resection- and MRE11-independent role for CtIP in preserving fork integrity that contributes to the survival of BRCA1-deficient cells.

Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling.

The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic-phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of "free" Mec1 activation domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator proteins, "free" MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1's role in genome maintenance is separable from a previously unappreciated proreplicative function. Both Mec1's functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action.

Loss of Dna2 fidelity results in decreased Exo1-mediated resection at DNA double-strand breaks.

A DNA double-strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination or nonhomologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of homologous recombination, and it is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harboring nuclease-dead dna2-1 compared with dna2Δ pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harboring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater resection defect compared with cells where DNA2 was deleted. Both the resection defect and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change; however, Nej1 increased as did the frequency of alt-end joining/microhomology-mediated end-joining repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability.

Abro1 maintains genome stability and limits replication stress by protecting replication fork stability.

Protection of the stalled replication fork is crucial for responding to replication stress and minimizing its impact on chromosome instability, thus preventing diseases, including cancer. We found a new component, Abro1, in the protection of stalled replication fork integrity. Abro1 deficiency results in increased chromosome instability, and Abro1-null mice are tumor-prone. We show that Abro1 protects stalled replication fork stability by inhibiting DNA2 nuclease/WRN helicase-mediated degradation of stalled forks. Depletion of RAD51 prevents the DNA2/WRN-dependent degradation of stalled forks in Abro1-deficient cells. This mechanism is distinct from the BRCA2-dependent fork protection pathway, in which stable RAD51 filament formation prevents MRE11-dependent degradation of the newly synthesized DNA at stalled forks. Thus, our data reveal a new aspect of regulated protection of stalled replication forks that involves Abro1.

The MRN complex and topoisomerase IIIa-RMI1/2 synchronize DNA resection motor proteins.

DNA resection-the nucleolytic processing of broken DNA ends-is the first step of homologous recombination. Resection is catalyzed by the resectosome, a multienzyme complex that includes bloom syndrome helicase (BLM), DNA2 or exonuclease 1 nucleases, and additional DNA-binding proteins. Although the molecular players have been known for over a decade, how the individual proteins work together to regulate DNA resection remains unknown. Using single-molecule imaging, we characterized the roles of the MRE11-RAD50-NBS1 complex (MRN) and topoisomerase IIIa (TOP3A)-RMI1/2 during long-range DNA resection. BLM partners with TOP3A-RMI1/2 to form the BTRR (BLM-TOP3A-RMI1/2) complex (or BLM dissolvasome). We determined that TOP3A-RMI1/2 aids BLM in initiating DNA unwinding, and along with MRN, stimulates DNA2-mediated resection. Furthermore, we found that MRN promotes the association between BTRR and DNA and synchronizes BLM and DNA2 translocation to prevent BLM from pausing during resection. Together, this work provides direct observation of how MRN and DNA2 harness the BTRR complex to resect DNA efficiently and how TOP3A-RMI1/2 regulates the helicase activity of BLM to promote efficient DNA repair.

Nej1 interacts with Sae2 at DNA double-stranded breaks to inhibit DNA resection.

The two major pathways of DNA double-strand break repair, nonhomologous end-joining and homologous recombination, are highly conserved from yeast to mammals. The regulation of 5'-DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends, and more recently, it was shown to participate in DNA end-bridging and in the inhibition of 5'-resection mediated by the nuclease/helicase complex Dna2-Sgs1. Here, we show that Nej1 interacts with Sae2 to impact DSB repair in three ways. First, we show that Nej1 inhibits interaction of Sae2 with the Mre11-Rad50-Xrs2 complex and Sae2 localization to DSBs. Second, we found that Nej1 inhibits Sae2-dependent recruitment of Dna2 independently of Sgs1. Third, we determined that NEJ1 and SAE2 showed an epistatic relationship for end-bridging, an event that restrains broken DNA ends and reduces the frequency of genomic deletions from developing at the break site. Finally, we demonstrate that deletion of NEJ1 suppressed the synthetic lethality of sae2Δ sgs1Δ mutants, and that triple mutant viability was dependent on Dna2 nuclease activity. Taken together, these findings provide mechanistic insight to how Nej1 functionality inhibits the initiation of DNA resection, a role that is distinct from its involvement in end-joining repair at DSBs.

Competing interaction partners modulate the activity of Sgs1 helicase during DNA end resection.

DNA double-strand break repair by homologous recombination employs long-range resection of the 5' DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay between them has been shown, it remains unclear whether and how these proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single-molecule level and investigated Sgs1 regulation by Dna2, the single-stranded DNA-binding protein RPA, and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection both proteins form a functional complex and couple their activities. Sgs1 drives DNA unwinding and feeds single-stranded DNA to Dna2 for degradation. RPA was found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We hypothesize that the differential regulation of Sgs1 activity by its protein partners is important to support diverse cellular functions of Sgs1 during the maintenance of genome stability.

MERIT40 cooperates with BRCA2 to resolve DNA interstrand cross-links.

MERIT40 is an essential component of the RAP80 ubiquitin recognition complex that targets BRCA1 to DNA damage sites. Although this complex is required for BRCA1 foci formation, its physiologic role in DNA repair has remained enigmatic, as has its relationship to canonical DNA repair mechanisms. Surprisingly, we found that Merit40(-/-) mice displayed marked hypersensitivity to DNA interstrand cross-links (ICLs) but not whole-body irradiation. MERIT40 was rapidly recruited to ICL lesions prior to FANCD2, and Merit40-null cells exhibited delayed ICL unhooking coupled with reduced end resection and homologous recombination at ICL damage. Interestingly, Merit40 mutation exacerbated ICL-induced chromosome instability in the context of concomitant Brca2 deficiency but not in conjunction with Fancd2 mutation. These findings implicate MERIT40 in the earliest stages of ICL repair and define specific functional interactions between RAP80 complex-dependent ubiquitin recognition and the Fanconi anemia (FA)-BRCA ICL repair network.

Hyb4mC: a hybrid DNA2vec-based model for DNA N4-methylcytosine sites prediction.

BACKGROUND: DNA N4-methylcytosine is part of the restrictive modification system, which works by regulating some biological processes, for example, the initiation of DNA replication, mismatch repair and inactivation of transposon. However, using experimental methods to detect 4mC sites is time-consuming and expensive. Besides, considering the huge differences in the number of 4mC samples among different species, it is challenging to achieve a robust multi-species 4mC site prediction performance. Hence, it is of great significance to develop effective computational tools to identify 4mC sites. RESULTS: This work proposes a flexible deep learning-based framework to predict 4mC sites, called Hyb4mC. Hyb4mC adopts the DNA2vec method for sequence embedding, which captures more efficient and comprehensive information compared with the sequence-based feature method. Then, two different subnets are used for further analysis: Hyb_Caps and Hyb_Conv. Hyb_Caps is composed of a capsule neural network and can generalize from fewer samples. Hyb_Conv combines the attention mechanism with a text convolutional neural network for further feature learning. CONCLUSIONS: Extensive benchmark tests have shown that Hyb4mC can significantly enhance the performance of predicting 4mC sites compared with the recently proposed methods.

hDNA2 nuclease/helicase promotes centromeric DNA replication and genome stability.

DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double-strand breaks. Similar such helicase activity for resolving secondary structures and structure-specific nuclease activity are needed during DNA replication to process the chromosome-specific higher order repeat units present in the centromeres of human chromosomes. Here, we show that DNA2 binds preferentially to centromeric DNA The nuclease and helicase activities of DNA2 are both essential for resolution of DNA structural obstacles to facilitate DNA replication fork movement. Loss of DNA2-mediated clean-up mechanisms impairs centromeric DNA replication and CENP-A deposition, leading to activation of the ATR DNA damage checkpoints at centromeric DNA regions and late-S/G2 cell cycle arrest. Cells that escape arrest show impaired metaphase plate formation and abnormal chromosomal segregation. Furthermore, the DNA2 inhibitor C5 mimics DNA2 knockout and synergistically kills cancer cells when combined with an ATR inhibitor. These findings provide mechanistic insights into how DNA2 supports replication of centromeric DNA and give further insights into new therapeutic strategies.

The diagnostic importance of pathogenic variants and variant coexistence determined by NGS-based liquid biopsy approach in patients with lung adenocarcinoma.

BACKROUND: Identification of driver mutations and rapid detection of genetic changes in lung cancer are critical in the management of the disease. Genetic structures of tumor tissues tend to change constantly and the possibility of emergence of new pathogenic variants that will create resistance to treatment. Liquid biopsy analysis has been one of the most effective approaches used to monitor and identify individual genetic changes. METHODS: In this study, TP53, EGFR, MET, ALK, PIK3CA, MAP2K, ERBB2 and ROS genes in cf DNA samples of 324 patients with lung adenocarcinoma were screened for genetic variations by NGS method. Analysis of the data showed that there were a total of 755 variations in 324 patients. RESULTS: Pathogenic and possibly pathogenic variations were identified in 178 patients (54.9%) on TP53, 118 (36.4%) on EGFR, 55 (17.0%) on MET, 46 (14.2%) on ALK, 39 (12.0%) on MAP2K, 6 (1.9%) on ERBB2 and in 2 (0,6%) patients ROS genes. The detailed variant data of the genes included in the study were compared with the patients' stage status, metastasis status, smoking, age distribution and life span data, and the presence of possible significant relationships and candidate biomarkers for the molecular pathogenesis of the disease were investigated. CONCLUSION: As a result of data analysis, genetic changes associated with metastasis and adenocarcinoma formation were identified. It has been shown that variations identified in TP53, PIK3CA, MAP2K1 and EGFR genes can play critical roles in the pathogenesis and development of the disease.

DeepSE: Detecting super-enhancers among typical enhancers using only sequence feature embeddings.

Super-enhancer (SE) is a cluster of active typical enhancers (TE) with high levels of the Mediator complex, master transcriptional factors, and chromatin regulators. SEs play a key role in the control of cell identity and disease. Traditionally, scientists used a variety of high-throughput data of different transcriptional factors or chromatin marks to distinguish SEs from TEs. This kind of experimental methods are usually costly and time-consuming. In this paper, we proposed a model DeepSE, which is based on a deep convolutional neural network model, to distinguish the SEs from TEs. DeepSE represent the DNA sequences using the dna2vec feature embeddings. With only the DNA sequence information, DeepSE outperformed all state-of-the-art methods. In addition, DeepSE can be generalized well across different cell lines, which implied that cell-type specific SEs may share hidden sequence patterns across different cell lines. The source code and data are stored in GitHub (https://github.com/QiaoyingJi/DeepSE).

Limiting homologous recombination at stalled replication forks is essential for cell viability: DNA2 to the rescue.

The disease-associated nuclease-helicase DNA2 has been implicated in DNA end-resection during DNA double-strand break repair, Okazaki fragment processing, and the recovery of stalled DNA replication forks (RFs). Its role in Okazaki fragment processing has been proposed to explain why DNA2 is indispensable for cell survival across organisms. Unexpectedly, we found that DNA2 has an essential role in suppressing homologous recombination (HR)-dependent replication restart at stalled RFs. In the absence of DNA2-mediated RF recovery, excessive HR-restart of stalled RFs results in toxic levels of abortive recombination intermediates that lead to DNA damage-checkpoint activation and terminal cell-cycle arrest. While HR proteins protect and restart stalled RFs to promote faithful genome replication, these findings show how HR-dependent replication restart is actively constrained by DNA2 to ensure cell survival. These new insights disambiguate the effects of DNA2 dysfunction on cell survival, and provide a framework to rationalize the association of DNA2 with cancer and the primordial dwarfism disorder Seckel syndrome based on its role in RF recovery.

Biallelic variants in DNA2 cause microcephalic primordial dwarfism.

Microcephalic primordial dwarfism (MPD) is a group of rare single-gene disorders characterized by the extreme reduction in brain and body size from early development onwards. Proteins encoded by MPD-associated genes play important roles in fundamental cellular processes, notably genome replication and repair. Here we report the identification of four MPD individuals with biallelic variants in DNA2, which encodes an adenosine triphosphate (ATP)-dependent helicase/nuclease involved in DNA replication and repair. We demonstrate that the two intronic variants (c.1764-38_1764-37ins(53) and c.74+4A>C) found in these individuals substantially impair DNA2 transcript splicing. Additionally, we identify a missense variant (c.1963A>G), affecting a residue of the ATP-dependent helicase domain that is highly conserved between humans and yeast, with the resulting substitution (p.Thr655Ala) predicted to directly impact ATP/ADP (adenosine diphosphate) binding by DNA2. Our findings support the pathogenicity of these variants as biallelic hypomorphic mutations, establishing DNA2 as an MPD disease gene.

A DNA nick at Ku-blocked double-strand break ends serves as an entry site for exonuclease 1 (Exo1) or Sgs1-Dna2 in long-range DNA end resection.

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in Saccharomyces cerevisiae and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1-Top3-Rmi1-Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70-Ku80 becomes a suitable substrate for long-range 5'-3' resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes.

Mechanism and regulation of DNA end resection in eukaryotes.

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic degradation of the 5'-terminated strands in a process termed end resection. End resection generates 3'-single-stranded DNA tails, substrates for Rad51 to catalyze homologous pairing and DNA strand exchange, and for activation of the DNA damage checkpoint. The commonly accepted view is that end resection occurs by a two-step mechanism. In the first step, Sae2/CtIP activates the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex to endonucleolytically cleave the 5'-terminated DNA strands close to break ends, and in the second step Exo1 and/or Dna2 nucleases extend the resected tracts to produce long 3'-ssDNA-tailed intermediates. Initiation of resection commits a cell to repair a DSB by HR because long ssDNA overhangs are poor substrates for non-homologous end joining (NHEJ). Thus, the initiation of end resection has emerged as a critical control point for repair pathway choice. Here, I review recent studies on the mechanism of end resection and how this process is regulated to ensure the most appropriate repair outcome.