Human Exonuclease Research Articles

Exonuclease editor promotes precision of gene editing in mammalian cells

BackgroundMany efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging.ResultsHere, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control.ConclusionsTherefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.

Increasing deletion sizes and the efficiency of CRISPR/Cas9-mediated mutagenesis by SunTag-mediated TREX1 recruitment.

Previously, it has been shown that mutagenesis frequencies can be improved by directly fusing the human exonuclease TREX2 to Cas9, resulting in a strong increase in the frequency of smaller deletions at the cut site. Here, we demonstrate that, by using the SunTag system for recruitment of TREX2, the mutagenesis efficiency can be doubled in comparison to the direct fusion in Arabidopsis thaliana. Therefore, we also tested the efficiency of the system for targeted deletion formation by recruiting two other 3'-5' exonucleases, namely the human TREX1 and E. coli ExoI. It turns out that SunTag-mediated recruitment of TREX1 not only improved the general mutation induction efficiency slightly in comparison to TREX2, but that, more importantly, the mean size of the induced deletions was also enhanced, mainly via an increase of deletions of 25 bp or more. EcExoI also yielded a higher amount of larger deletions. However, only in the case of TREX1 and TREX2, the effect was predominately SunTag-dependent, indicating efficient target-specific recruitment. Using SunTag-mediated TREX1 recruitment at other genomic sites, we were able to obtain similar deletion patterns. Thus, we were able to develop an attractive novel editing tool that is especially useful for obtaining deletions in the range from 20 to 40 bp around the cut site. Such sizes are often required for the manipulation of cis-regulatory elements. This feature is closing an existing gap as previous approaches, based on single nucleases or paired nickases or nucleases, resulted in either shorter or longer deletions, respectively.

Exonuclease 1 Drives Growth and Genomic Instability in Multiple Myeloma through Interaction with CAD, a Key Protein Involved in De Novo Pyrimidine Biosynthesis

Human exonuclease 1 (Exo1) plays important roles in DNA replication and repair including the resection of DNA ends during homologous recombination (HR). Based on our previous data demonstrating the role of HR in genomic instability and growth of cancer cells, we investigated role of EXO1 in multiple myeloma (MM). Expression of EXO1 and pRPA32 (a marker of DNA end resection) was elevated in all myeloma cell lines tested (N=10) relative to normal peripheral blood mononuclear (PBMC) cell samples. We developed a fluorescence-based EXO1 activity assay and observed elevated (~1.5- to > 5-fold; p < 0.05) Exo1 activity in all twelve MM cell lines tested relative to normal PBMCs. Moreover., elevated expression of EXO1 correlated with poor overall and event-free survival in MM patients (MMRF dataset; p < 1x10-5). Transgenic suppression of EXO1 in MM cell lines caused a time-dependent loss in cell viability. Evaluation at day 5 after selection indicated that, relative to control cells, EXO1 suppression in RPMI, MM1S, MM1R and U266 cells was associated with ∼95%, 85%, 60% and 40% cell death, respectively. Apoptosis, as evaluated by annexin-labeling, was detected in ~50% of EXO1-knockdown cells compared to 11.9% of control MM1S cells. Conditional knockdown in H929 cells evaluation at 48 h after adding doxycycline demonstrated apoptosis in 18% of EXO1-suppressed cells vs. 4.9% of control cells. To investigate the mechanisms by which EXO1 impacts MM cell viability, the proteins interacting with EXO1 in MM cell lines (MM1S and H929) were identified using Exo1 pull-down and mass spectrometry. Interaction of EXO1 with CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a trifunctional protein involved in the first three steps of de novo pyrimidine biosynthesis, was identified and its binding confirmed with co-immunoprecipitation. We found that EXO1 overexpression increased the growth rate of non-cancerous cell types (HS5 - bone marrow stromal cells and normal fibroblasts), whereas suppression of CAD inhibited the spontaneous and EXO1-mediated increase in growth rate as well as DNA replication in both normal cell types. Consistently, the CAD-knockdown increased apoptosis in HS5 cells by ∼5-fold, whereas EXO1 overexpression reduced it by ~2-fold. Conversely, suppression of CAD also increased apoptosis in EXO1-overexpressing cells by 3-fold. In normal diploid fibroblast cells with a limited lifespan, EXO1 overexpression increased DNA replication by 32%, whereas CAD-knockdown reduced it by ∼65%. Importantly, CAD-knockdown in EXO1-overexpressing cells also reduced DNA replication by 26%. These data indicate that CAD is involved in EXO1-dependent cell proliferation and survival. Investigating the molecular events associated with EXO1, we observed that its suppression in MM cells inhibits DNA end resection, DNA breaks, HR activity and genomic instability (as assessed by a micronucleus assay); whereas overexpression in normal cells increased those activities. Moreover, inhibition of CAD reduced spontaneous as well as EXO1-induced HR activity and genomic instability in both hematopoietic and solid tumor cell types. Since CAD is involved in the first three steps of pyrimidine biosynthesis, our data suggest that interaction with CAD enables EXO1 to drive DNA replication, HR and associated genomic instability. Functional relevance of this interaction and possible regulation of de novo pyrimidine biosynthesis by EXO1 is being further investigated. Inhibitors of CAD may provide a novel therapeutic target in myeloma as well as other Exo1 overexpressing malignancies.

Computational and Biophysical Analysis of RNA/Protein Complexes in Histone mRNA Degradation

Histone mRNA is the only known metazoan mRNA that is not polyadenylated at its 3’ end; instead, it ends in a highly conserved stem‐loop. This stem‐loop and its protein binding partner, the stem‐loop binding protein (SLBP), control every aspect of histone mRNA metabolism. As the cell prepares to degrade histone mRNA in the cytoplasm towards the end of S‐phase, both of these elements are modified; the mRNA is trimmed by 3’ human exonuclease (3’hExo) and uridylated by a terminal uridylyl transferase, while SLBP is dephosphorylated. The trimming and uridylation process creates uridylated intermediates that differ in length as the histone mRNA begins to undergo degradation, while the dephosphorylation of SLBP destabilizes the protein. Both of these events are associated with histone mRNA degradation, their exact roles in histone mRNA metabolism are not fully understood. In this study, we seek to characterize how these changes to SLBP and histone mRNA affect the stability of the ternary SLBP/3’hEXO/mRNA complex using both computational and experimental methods. To computationally characterize the effects of uridylation on stability of the ternary complex, we performed molecular dynamics simulations (1 µs) using AMBER force fields and Nanoscale Molecular Dynamics (NAMD). To experimentally characterize these observations, we used a fluorescence‐based EMSA competition assay to measure the proteins’ difference in affinity for the wild‐type stem‐loop and uridylated intermediates. Through these experiments, we have gained valuable insight into the role of both the uridylation of histone mRNA and the dephosphorylation of SLBP at the beginning of histone mRNA degradation.

Bioinformatics Analysis and Experimental Study of Exonuclease 1 Gene in Lung Adenocarcinoma.

The objective of this study is to examine the role of Human Exonuclease 1(EXO1) gene in the diagnosis and prognosis of lung adenocarcinoma (LUAD), and predict the signal pathways EXO1 involved in. The clinical parameters and EXO1 expression datasets of LUAD patients were obtained from The Cancer Genome Atlas (TCGA), Oncomine and Gene Expression Omnibus (GEO) database. Wilcoxon rank-sum test was performed to determine whether EXO1 expression was upregulated in LUAD. The correlation between EXO1 expression and clinicopathological parameters was analyzed by Chi-square test, and Kaplan-Meier survival analysis and COX regression models were adopted to analyze and verify the correlation of EXO1 expression with OS of LUAD patients for the exploration of prognostic value of EXO1 in LUAD patients. The signaling pathway related to EXO1 was predicted by gene set enrichment analysis (GSEA). In addition, sera from LUAD patients and healthy subjects were collected, and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was conducted to detect EXO1 expression. EXO1 expression was upregulated in LUAD patients with respect to normal individuals. EXO1 expression was negatively correlated with the prognosis and thus could independently predict the prognosis of LUAD patients. EXO1 gene was involved in 128 signal pathways, of which 9 pathways may be closely related. EXO1 was highly expressed in the blood of LUAD patients. High EXO1 expression can serve as an independent risk factor for poor prognosis, and the expression of serum EXO1 has certain diagnostic value for LUAD.

EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.

Biophysical Characterization and Computational Modeling of Histone mRNA Degradation Intermediates

Histone mRNA is the only known metazoan mRNA that is not polyadenylated at its 3′ end; instead, it ends in a highly conserved stem-loop formed by Watson-Crick base pairing. In histone mRNA degradation, the stem-loop is trimmed by 3′ human exonuclease (3′hExo) and uridylated by terminal uridylyl transferase 7. This process creates uridylated intermediates—some of which are more common in the cell than expected. It is unknown how this trimming and uridylation affects the binding interactions between the stem-loop and its protein binding partners, the stem-loop binding protein (SLBP) and 3′hExo. In this work, we characterized the binding interactions of a wild-type stem-loop, a uridylated intermediate, and a truncated stem-loop using a combination of experimental and computational biophysical techniques. The uridylated intermediate represents one of the most prominent degradation intermediates identified in the cell, and the truncated stem-loop represents the stem-loop post-trimming and pre-uridylation. Through native electrophoretic mobility shift assays, we show that SLBP and 3′hExo are able to bind both the uridylated intermediate and the truncated stem-loop despite 3′ end modification. We also performed molecular dynamics simulations (1 µs) to evaluate structural changes induced by stem-loop modification and to understand how these changes impact SLBP and 3′hExo binding. RNA-protein systems were generated by applying AMBER force fields to modified versions of the histone mRNA ternary complex crystal structures (PDB 4QOZ and 4L8R). This work shows that modification of the stem-loop through trimming and uridylation does not interfere with the tertiary structure of the ternary complex.

Recruiting Mechanism and Functional Role of a Third Metal Ion in the Enzymatic Activity of 5' Structure-Specific Nucleases.

Enzymes of the 5′ structure-specific nuclease family are crucial for DNA repair, replication, and recombination. One such enzyme is the human exonuclease 1 (hExo1) metalloenzyme, which cleaves DNA strands, acting primarily as a processive 5′-3′ exonuclease and secondarily as a 5′-flap endonuclease. Recently, in crystallo reaction intermediates have elucidated how hExo1 exerts hydrolysis of DNA phosphodiester bonds. These hExo1 structures show a third metal ion intermittently bound close to the two-metal-ion active site, to which recessed ends or 5′-flap substrates bind. Evidence of this third ion has been observed in several nucleic-acid-processing metalloenzymes. However, there is still debate over what triggers the (un)binding of this transient third ion during catalysis and whether this ion has a catalytic function. Using extended molecular dynamics and enhanced sampling free-energy simulations, we observed that the carboxyl side chain of Glu89 (located along the arch motif in hExo1) flips frequently from the reactant state to the product state. The conformational flipping of Glu89 allows one metal ion to be recruited from the bulk and promptly positioned near the catalytic center. This is in line with the structural evidence. Additionally, our simulations show that the third metal ion assists the departure, through the mobile arch, of the nucleotide monophosphate product from the catalytic site. Structural comparisons of nuclease enzymes suggest that this Glu(Asp)-mediated mechanism for third ion recruitment and nucleic acid hydrolysis may be shared by other 5′ structure-specific nucleases.

4Fe–4S] cluster-containing human exonuclease V acts as a novel replication fork restart factor

4Fe–4S] cluster-containing human exonuclease V acts as a novel replication fork restart factor

Human Exonuclease 1 (EXO1) Regulatory Functions in DNA Replication with Putative Roles in Cancer.

Human exonuclease 1 (EXO1), a 5′→3′ exonuclease, contributes to the regulation of the cell cycle checkpoints, replication fork maintenance, and post replicative DNA repair pathways. These processes are required for the resolution of stalled or blocked DNA replication that can lead to replication stress and potential collapse of the replication fork. Failure to restart the DNA replication process can result in double-strand breaks, cell-cycle arrest, cell death, or cellular transformation. In this review, we summarize the involvement of EXO1 in the replication, DNA repair pathways, cell cycle checkpoints, and the link between EXO1 and cancer.

A nuclease specific to lepidopteran insects suppresses RNAi

More than 70% of all agricultural pests are insects in the order Lepidoptera, which, unlike other related insect orders, are not very sensitive to RNAi, limiting genetic studies of this insect group. However, the reason for this distinct lepidopteran characteristic is unknown. Previously, using transcriptome analysis of the Asian corn borer Ostrinia furnacalis, we identified a gene, termed up56, that is up-regulated in response to dsRNA. Here we report that this Lepidoptera-specific gene encodes a nuclease that contributes to RNAi insensitivity in this insect order. Its identity was experimentally validated, and sequence analysis indicated that up56 encodes a previously uncharacterized protein with homologous sequences in seven other lepidopteran species. Its computationally predicted three-dimensional structure revealed a high structural similarity to human exonuclease I. Exposure to dsRNA in O. furnacalis strongly up-regulated this gene's expression, and the protein could digest single-stranded RNA (ssRNA), dsRNA, and dsDNA both in vitro and in vivo Of note, we found that this up-regulation of up56 expression is faster than that of the gene encoding the key RNAi-associated nuclease Dicer. up56 knockdown in O. furnacalis significantly enhanced RNAi efficiency. Moreover, up56 overexpression in Drosophila melanogaster suppressed RNAi efficiency. Finally, up56 knockdown significantly increased the amount and diversity of small RNAs. Therefore, we renamed this protein RNAi efficiency-related nuclease (REase). In conclusion, we propose that REase may explain why lepidopterans are refractory to RNAi and that it represents a target for further research of RNAi efficiency in this insect order.

Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro.

DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display a mild mutator phenotype, while nuclear extracts of these cells exhibit reduced MMR activity in vitro, and these defects are complemented by overexpression or addition of exogenous human Exonuclease 1 (EXO1). By contrast, another proofreading-deficient mutant, PolδD515V, which has a weaker strand displacement activity, does not decrease the MMR activity as significantly as PolδD316A;E318A. In addition, PolδD515V does not increase the mutation frequency in MMR-proficient cells. Based on our findings, we propose that the proofreading activity restricts the strand displacement activity of Polδ in MMR. This contributes to maintain the nicks required for EXO1 entry, and in this manner ensures the dominance of the EXO1-dependent MMR pathway.

Human Exonuclease 1 Threads 5'-Flap Substrates through Its Helical Arch.

Human exonuclease 1 (hEXO1) is a member of the 5'-nuclease superfamily and plays important roles in DNA repair. Along with acting as a 5'-exonuclease on blunt, gapped, nicked, and 3'-overhang DNAs, hEXO1 can also act as an endonuclease removing protruding 5'-single-stranded flaps from duplex ends. How hEXO1 and related 5'-nuclease human flap endonuclease 1 (hFEN1) are specific for discontinuous DNA substrates like 5'-flaps has been controversial. Here we report the first functional data that imply that hEXO1 threads the 5'-flap through a hole in the protein known as the helical arch, thereby excluding reactions of continuous single strands. Conjugation of bulky 5'-streptavidin that would "block" threading through the arch drastically slowed the hEXO1 reaction. In contrast, addition of streptavidin to a preformed hEXO1 5'-biotin flap DNA complex trapped a portion of the substrate in a highly reactive threaded conformation. However, another fraction behaves as if it were "blocked" and decayed very slowly, implying there were both threaded and unthreaded forms of the substrate present. The reaction of an unmodified hEXO1-flap DNA complex did not exhibit marked biphasic kinetics, suggesting a fast re-equilibration occurs that produces more threaded substrate when some decays. The finding that a threading mechanism like that used by hFEN1 is also used by hEXO1 unifies the mode of operation for members of the 5'-nuclease superfamily that act on discontinuous substrates. As with hFEN1, intrinsic disorder of the arch region of the protein may explain how flaps can be threaded without a need for a coupled energy source.

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.

Associations between single-nucleotide polymorphisms of human exonuclease 1 and the risk of hepatocellular carcinoma.

Human exonuclease 1 (hEXO1) is an important nuclease involved in mismatch repair system that contributes to maintain genomic stability and modulate DNA recombination. This study is aimed to explore the associations between single-nucleotide polymorphisms (SNPs) of hEXO1 and the hereditary susceptibility of hepatocellular carcinoma (HCC). SNPs rs1047840, rs1776148, rs3754093, rs4149867, rs4149963, and rs1776181 of hEXO1 were examined from a hospital-based case-control study including 1,196 cases (HCC patients) and 1,199 controls (non-HCC patients) in Guangxi, China. We found the rs3754093 AG genotype decreased the risk of HCC (OR=0.714, 95% CI: 0.539∼0.946). According to the results of stratification analysis, rs3754093 mutant genotype AG/GG decreased the risk of HCC with some HCC protective factors such as non-smoking, non-alcohol consumption and non-HCC family history, but also decreased the risk of HCC with HBV infection. Moreover, it was correlated to non-tumor metastasis and increased the survival of HCC patients. The results from gene-environment interaction assay indicated all hEXO1 SNPs interacted with smoking, alcohol consumption, HBV infection in pathogenesis of HCC. However, gene-gene interaction assay suggested the interaction between rs3754093 and other 5 SNPs were associated with reducing the HCC risk. These results suggest rs3754093 exhibits a protective activity to decrease the incidence risk of HCC in Guangxi, China. In addition, all SNPs in this study interacted with environment risk factors in pathogenesis of HCC.

Advances in the study of Lynch syndrome in China.

Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer, is an autosomal dominant genetic condition that has a high risk of colon cancer as well as other cancers due to inherited mutations in mismatch repair (MMR) genes. During the last decades, there have been great advances in research on Chinese Lynch syndrome. This review mainly focuses on the genetic basis, clinicopathologic features, diagnosis, intervention, chemoprevention, and surveillance of Lynch syndrome in China. In addition to frequently altered MMR genes, such as MLH1, MSH2, MSH6, and MLH3, other MMR-associated genes, such as those encoding human exonuclease 1, transforming growth factor β receptor 2, and alanine aminopeptidase, metastasis-associated protein 2, adenomatosis polyposis coli down-regulated 1, and hepatic and glial cell adhesion molecule have also been implicated in Chinese Lynch syndrome. Most Chinese researchers focused on the clinicopathologic features of Lynch syndrome, and it is noticeable that the most frequent extracolonic tumor in northeast China is lung cancer, which is different from other areas in China. The Chinese diagnostic criteria for Lynch syndrome have been established to identify gene mutation or methylation. With regard to chemoprevention, celecoxib may be effective to prevent polyps relapse in Lynch syndrome carriers. Additionally, a colonoscopy-based surveillance strategy for the prevention and early detection of neoplasms in Lynch-syndrome carriers has been proposed.

Human exonuclease 1 (EXO1) activity characterization and its function on flap structures.

Human exonuclease 1 (EXO1) is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of EXO1 have been described in yeast. Here, we report a biochemical characterization of human full-length EXO1. Prior to assay EXO1 on different DNA flap structures, we determined factors essential for the thermodynamic stability of EXO1. We show that enzymatic activity and stability of EXO1 on DNA is modulated by temperature. By characterization of EXO1 flap activity using various DNA flap substrates, we show that EXO1 has a strong capacity for degrading double stranded DNA and has a modest endonuclease or 5' flap activity. Furthermore, we report novel mechanistic insights into the processing of flap structures, showing that EXO1 preferentially cleaves one nucleotide inwards in a double stranded region of a forked and nicked DNA flap substrates, suggesting a possible role of EXO1in strand displacement.

Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1.

Mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1 and PMS2 are associated with Lynch Syndrome (LS), a familial predisposition to early-onset cancer of the colon and other organs. Because not all LS families carry mutations in these four genes, the search for cancer-associated mutations was extended to genes encoding other members of the mismatch repairosome. This effort identified mutations in EXO1, which encodes the sole exonuclease implicated in MMR. One of these mutations, E109K, was reported to abrogate the catalytic activity of the enzyme, yet, in the crystal structure of the EXO1/DNA complex, this glutamate is far away from both DNA and the catalytic site of the enzyme. In an attempt to elucidate the reason underlying the putative loss of function of this variant, we expressed it in Escherichia coli, and tested its activity in a series of biochemical assays. We now report that, contrary to earlier reports, and unlike the catalytic site mutant D173A, the EXO1 E109K variant resembled the wild-type (wt) enzyme on all tested substrates. In the light of our findings, we attempt here to reinterpret the results of the phenotypic characterization of a knock-in mouse carrying the E109K mutation and cells derived from it.

Human Exonuclease 5 Is a Novel Sliding Exonuclease Required for Genome Stability

Previously, we characterized Saccharomyces cerevisiae exonuclease 5 (EXO5), which is required for mitochondrial genome maintenance. Here, we identify the human homolog (C1orf176; EXO5) that functions in the repair of nuclear DNA damage. Human EXO5 (hEXO5) contains an iron-sulfur cluster. It is a single-stranded DNA (ssDNA)-specific bidirectional exonuclease with a strong preference for 5'-ends. After loading at an ssDNA end, hEXO5 slides extensively along the ssDNA prior to cutting, hence the designation sliding exonuclease. However, the single-stranded binding protein human replication protein A (hRPA) restricts sliding and enforces a unique, species-specific 5'-directionality onto hEXO5. This specificity is lost with a mutant form of hRPA (hRPA-t11) that fails to interact with hEXO5. hEXO5 localizes to nuclear repair foci in response to DNA damage, and its depletion in human cells leads to an increased sensitivity to DNA-damaging agents, in particular interstrand cross-linking-inducing agents. Depletion of hEXO5 also results in an increase in spontaneous and damage-induced chromosome abnormalities including the frequency of triradial chromosomes, suggesting an additional defect in the resolution of stalled DNA replication forks in hEXO5-depleted cells.

14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.