FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability.

FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea-induced stalled forks. The RAD51 recombinase has also been implicated in regulation of resection at stalled replication forks. The mechanistic contributions of these proteins to fork protection are not well understood. Here, we used purified FANCD2 and RAD51 to study how each protein regulates DNA resection at stalled forks. We characterized three mechanisms of FANCD2-mediated fork protection: (1) The N-terminal domain of FANCD2 inhibits the essential DNA2 nuclease activity by directly binding to DNA2 accounting for over-resection in FANCD2 defective cells. (2) Independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit multiple nucleases, including DNA2, MRE11 and EXO1. (3) Unexpectedly, we uncovered a new FANCD2 function: by stabilizing RAD51 filaments, FANCD2 acts to stimulate the strand exchange activity of RAD51. Our work biochemically explains non-canonical mechanisms by which FANCD2 and RAD51 protect stalled forks. We propose a model in which the strand exchange activity of FANCD2 provides a simple molecular explanation for genetic interactions between FANCD2 and BRCA2 in the FA/BRCA fork protection pathway.

Levofloxacin prophylaxis for pediatric leukemia patients: Longitudinal follow-up for impact on health care-associated infections.

BACKGROUND: Bloodstream infections (BSIs) cause morbidity and mortality in pediatric patients with leukemia. Antibiotic prophylaxis during periods of chemotherapy-induced neutropenia may reduce the incidence of BSIs. PROCEDURE: A levofloxacin prophylaxis guideline was implemented for pediatric patients with acute myeloid leukemia and relapsed acute lymphoblastic leukemia. We conducted a retrospective cohort study over 4 years (2 years pre and 2 years post implementation) of the practice guideline to assess the impact on central line-associated bloodstream infections (CLABSI) and BSI events. Secondary outcomes included incidence of Clostridioides difficile-associated diarrhea, bacteremia due to multidrug-resistant organisms (MDRO), and bacteremia due to levofloxacin nonsusceptible organisms. STATA was used for data analysis. RESULTS: Sixty-three and 72 patients met inclusion criteria for the pre- and postimplementation cohorts, respectively. Demographics were similar between the groups. We observed 60 BSI events in the pre-group versus 49 events in the post-group (p = .1). Bacteremia due to Gram-negative rods (risk ratio [RR] 0.37 [0.21, 0.66], p < .001) and National Healthcare Safety Network (NHSN) CLABSIs (RR 0.62 [0.44, 0.89], p = .01) were significantly reduced in the postimplementation group. The incidences of C. difficile-associated diarrhea and MDRO bacteremia were similar between groups. However, we observed an increase in the incidence of BSI due to Gram-negative rods that were nonsusceptible to levofloxacin (RR 3.38 [0.72, 6.65], p < .001). CONCLUSION: Following implementation of a levofloxacin prophylaxis guideline, we observed a significant decrease in BSIs due to Gram-negative rods and NHSN CLABSIs. Vigilant monitoring of outcomes post guideline implementation is critical to track emergence of resistant organisms.

Glycemic outcomes of Advanced Hybrid Closed Loop system in children and adolescents with Type 1 Diabetes, previously treated with Multiple Daily Injections (MiniMed 780G system in T1D individuals, previously treated with MDI).

BACKGROUND: The objective of this study was to evaluate the glycemic outcomes in children and adolescents with Type 1 Diabetes (T1D) previously treated with Multiple Daily Injections (MDI) using a structured initiation protocol for the Advanced Hybrid Closed Loop (AHCL) Minimed 780G insulin pump system. METHODS: In this prospective open label single-arm, single-center, clinical investigation, we recruited children and adolescents (aged 7-17 years) with T1D on MDI therapy and HbA1c below 12.5%. All participants followed a 10-day structured initiation protocol which included 4 steps: step 1: AHCL system assessment; step 2: AHCL system training; step 3: Sensor augmented pump therapy (SAP) for 3 days; step 4: AHCL system use for 12 weeks, successfully completing the training from MDI to AHCL in 10 days. The primary outcome of the study was the change in the time spent in the target in range (TIR) of 70-180 mg/dl and HbA1c from baseline (MDI + CGM, 1 week) to study phase (AHCL, 12 weeks). The paired student t-test was used for statistical analysis and a value < 0.05 was considered statistically significant. RESULTS: Thirty-four participants were recruited and all completed the 12 weeks study. TIR increased from 42.1 ± 18.7% at baseline to 78.8 ± 6.1% in the study phase (p < 0.001). HbA1c decreased from 8.6 ± 1.7% (70 ± 18.6 mmol/mol) at baseline, to 6.5 ± 0.7% (48 ± 7.7 mmol/mol) at the end of the study (p = 0.001). No episodes of severe hypoglycemia or DKA were reported. CONCLUSION: Children and adolescents with T1D on MDI therapy who initiated the AHCL system following a 10-days structured protocol achieved the internationally recommended goals of glycemic control with TIR > 70% and a HbA1c of < 7%.

Multiple roles of DNA2 nuclease/helicase in DNA metabolism, genome stability and human diseases.

DNA2 nuclease/helicase is a structure-specific nuclease, 5'-to-3' helicase, and DNA-dependent ATPase. It is involved in multiple DNA metabolic pathways, including Okazaki fragment maturation, replication of 'difficult-to-replicate' DNA regions, end resection, stalled replication fork processing, and mitochondrial genome maintenance. The participation of DNA2 in these different pathways is regulated by its interactions with distinct groups of DNA replication and repair proteins and by post-translational modifications. These regulatory mechanisms induce its recruitment to specific DNA replication or repair complexes, such as DNA replication and end resection machinery, and stimulate its efficient cleavage of various structures, for example, to remove RNA primers or to produce 3' overhangs at telomeres or double-strand breaks. Through these versatile activities at replication forks and DNA damage sites, DNA2 functions as both a tumor suppressor and promoter. In normal cells, it suppresses tumorigenesis by maintaining the genomic integrity. Thus, DNA2 mutations or functional deficiency may lead to cancer initiation. However, DNA2 may also function as a tumor promoter, supporting cancer cell survival by counteracting replication stress. Therefore, it may serve as an ideal target to sensitize advanced DNA2-overexpressing cancers to current chemo- and radiotherapy regimens.

Characterization of the dimeric CMG/pre-initiation complex and its transition into DNA replication forks.

The pre-initiation complex (pre-IC) has been proposed for two decades as an intermediate right before the maturation of the eukaryotic DNA replication fork. However, its existence and biochemical nature remain enigmatic. Here, through combining several enrichment strategies, we are able to isolate an endogenous dimeric CMG-containing complex (designated as d-CMG) distinct from traditional single CMG (s-CMG) and in vitro reconstituted dimeric CMG. D-CMG is assembled upon entry into the S phase and shortly matures into s-CMG/replisome, leading to the fact that only ~ 5% of the total CMG-containing complexes can be detected as d-CMG in vivo. Mass spectra reveal that RPA and DNA Pol α/primase co-purify with s-CMG, but not with d-CMG. Consistently, the former fraction is able to catalyze DNA unwinding and de novo synthesis, while the latter catalyzes neither. The two CMGs in d-CMG display flexibly orientated conformations under an electronic microscope. When DNA Pol α-primase is inactivated, d-CMG % rose up to 29%, indicating an incomplete pre-IC/fork transition. These findings reveal biochemical properties of the d-CMG/pre-IC and provide in vivo evidence to support the pre-IC/fork transition as a bona fide step in replication initiation.

RAD51 and mitotic function of mus81 are essential for recovery from low-dose of camptothecin in the absence of the WRN exonuclease.

Stabilization of stalled replication forks prevents excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined. Recently, the WRN exonuclease has been linked to protection of stalled forks from degradation. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells. Thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon treatment with clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Ser87, which is essential to prevent excessive mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.

TRAF6 mediates human DNA2 polyubiquitination and nuclear localization to maintain nuclear genome integrity.

The multifunctional human DNA2 (hDNA2) nuclease/helicase is required to process DNA ends for homology-directed recombination repair (HDR) and to counteract replication stress. To participate in these processes, hDNA2 must localize to the nucleus and be recruited to the replication or repair sites. However, because hDNA2 lacks the nuclear localization signal that is found in its yeast homolog, it is unclear how its migration into the nucleus is regulated during replication or in response to DNA damage. Here, we report that the E3 ligase TRAF6 binds to and mediates the K63-linked polyubiquitination of hDNA2, increasing the stability of hDNA2 and promoting its nuclear localization. Inhibiting TRAF6-mediated polyubiquitination abolishes the nuclear localization of hDNA2, consequently impairing DNA end resection and HDR. Thus, the current study reveals a mechanism for the regulation of hDNA2 localization and establishes that TRAF6-mediated hDNA2 ubiquitination activates DNA repair pathways to maintain nuclear genome integrity.

Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint.

To preserve genome integrity, the S-phase checkpoint senses damaged DNA or nucleotide depletion and when necessary, arrests replication progression and delays cell division. Previous studies, based on two pol2 mutants have suggested the involvement of DNA polymerase epsilon (Pol ε) in sensing DNA replication accuracy in Saccharomyces cerevisiae. Here we have studied the involvement of Pol ε in sensing proper progression of DNA replication, using a mutant in DPB2, the gene coding for a non-catalytic subunit of Pol ε. Under genotoxic conditions, the dpb2-103 cells progress through S phase faster than wild-type cells. Moreover, the Nrm1-dependent branch of the checkpoint, which regulates the expression of many replication checkpoint genes, is impaired in dpb2-103 cells. Finally, deletion of DDC1 in the dpb2-103 mutant is lethal supporting a model of strand-specific activation of the replication checkpoint. This lethality is suppressed by NRM1 deletion. We postulate that improper activation of the Nrm1-branch may explain inefficient replication checkpoint activation in Pol ε mutants.

BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair.

Repair of dsDNA breaks requires processing to produce 3'-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 5' → 3' resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells.

Brucellosis in Adults and Children: A 10-Year Case Series at Two Large Academic Hospitals in Houston, Texas.

OBJECTIVES: Brucellosis is one of the most common zoonoses worldwide. Most cases in the United States occur among travelers or immigrants from endemic regions, mostly Central America. In this study, we aimed at describing and comparing the epidemiology and clinical presentation of brucellosis in pediatric and adult patients at two large tertiary care centers in Houston, Texas. METHODS: We identified patients diagnosed as having brucellosis between January 2000 and December 2009 by searching electronic medical records and reviewing microbiology records for positive cultures. Cases were defined as those with a positive blood culture for Brucella sp, a serum agglutination titer ≥1:80 (or both positive blood culture and serum agglutination titer ≥1:80), along with an epidemiologic risk factor and clinical presentation that is consistent with brucellosis. RESULTS: Six adult and 12 pediatric cases were identified; 13 of 18 (72%) cases were immigrants, mostly from Central America. The median ages for adult and pediatric patients were 53 and 3 years old, respectively. Ingestion of unpasteurized milk products was frequently reported. Common clinical features included fever (83%), arthralgias or arthritis (67%), and hepatosplenomegaly (61%). Positive blood cultures were more frequently reported among children than adults (83% vs 33%, P = 0.03). The most common laboratory finding was mildly elevated transaminases. Three adults (50%) but no children developed thrombocytopenia (P = 0.02). Relapsed infection was a frequent occurrence. CONCLUSIONS: In the southern United States, brucellosis is an important consideration in the differential diagnosis of immigrants presenting with undifferentiated fever and joint complaints. A careful history often reveals an epidemiologic risk factor such as ingestion of unpasteurized dairy products.

The SOSS1 single-stranded DNA binding complex promotes DNA end resection in concert with Exo1.

The human SSB homologue 1 (hSSB1) has been shown to facilitate homologous recombination and double-strand break signalling in human cells. Here, we compare the DNA-binding properties of the SOSS1 complex, containing SSB1, with Replication Protein A (RPA), the primary single-strand DNA (ssDNA) binding complex in eukaryotes. Ensemble and single-molecule approaches show that SOSS1 binds ssDNA with lower affinity compared to RPA, and exhibits less stable interactions with DNA substrates. Nevertheless, the SOSS1 complex is uniquely capable of promoting interaction of human Exo1 with double-strand DNA ends and stimulates its activity independently of the MRN complex in vitro. Both MRN and SOSS1 also act to mitigate the inhibitory action of the Ku70/80 heterodimer on Exo1 activity in vitro. These results may explain why SOSS complexes do not localize with RPA to replication sites in human cells, yet have a strong effect on double-strand break resection and homologous recombination.

Mammalian DNA2 helicase/nuclease cleaves G-quadruplex DNA and is required for telomere integrity.

Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G-quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2's role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2-deficient mice develop aneuploidy-associated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4.

Angiostrongylus cantonensis Infection: A Cause of Fever of Unknown Origin in Pediatric Patients.

Fever of unknown origin (FUO) in children is frequently caused by infectious diseases. Angiostrongylus cantonensis, while a primary cause of eosinophilic meningitis, is rarely a cause of FUO. We present 2 pediatric cases of FUO caused by Angiostrongylus cantonensis acquired in Houston, Texas, outside its usual geographic distribution.

Clinical and Molecular Features of Decreased Chlorhexidine Susceptibility among Nosocomial Staphylococcus aureus Isolates at Texas Children's Hospital.

One of the strategies utilized to decrease infections in the hospital setting relies on topical antimicrobials and antiseptics. While their use is beneficial, concerns arise over the potential to develop resistance or tolerance to these agents. We examined nosocomial Staphylococcus aureus isolates from 2007 to 2013 for the presence of genes associated with tolerance to chlorhexidine. Isolates and patients were identified from an S. aureus surveillance study at Texas Children's Hospital. Nosocomial S. aureus isolates (those causing infection at ≥72 h of hospitalization) were identified and underwent PCR for the qacA or qacB (qacA/B) and smr genes associated with elevated minimum bactericidal concentrations of chlorhexidine. Molecular typing with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and agr typing and a review of the medical record were performed. Two hundred forty-seven nosocomial S. aureus infections were identified. Overall, 111 isolates carried one or both genes (44.9%); 33.1% were positive for smr, 22.7% were positive for qacA/B, and 10.9% of the isolates possessed both genes. The smr-positive isolates were more often resistant to methicillin, ciprofloxacin, and/or clindamycin. The isolates positive for qacA/B were more often associated with indwelling central venous catheters and a vancomycin MIC of ≥2 µg/ml. Isolates carrying either smr or qacA/B were associated with a diagnosis of bacteremia. The smr-positive isolates more often belonged to sequence type 8 (ST8) than the isolates that were positive for qacA/B. Mupirocin resistance was detected in 2.8% of the isolates. Antiseptic-tolerant S. aureus strains are common in our children's hospital and are associated with decreased susceptibility to other systemic antimicrobials and with bloodstream infections. Further work is needed to understand the implications that these organisms have on the hospital environment and antiseptic use in the future.

DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2.

The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3'-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases and topoisomerases. Here we biochemically reconstitute elements of the resection process and reveal that it requires the nuclease Dna2, the RecQ-family helicase Sgs1 and the ssDNA-binding protein replication protein-A (RPA). We establish that Dna2, Sgs1 and RPA constitute a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5'-terminated strand of the DNA break and to inhibit 3' to 5' degradation by Dna2, actions that generate and protect the 3'-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11-Rad50-Xrs2 complex (MRX) have important roles as stimulatory components. Stimulation of end resection by the Top3-Rmi1 heterodimer and the MRX proteins is by complex formation with Sgs1 (refs 5, 6), which unexpectedly stimulates DNA unwinding. We suggest that Top3-Rmi1 and MRX are important for recruitment of the Sgs1-Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding the initial steps of recombinational DNA repair in eukaryotes.

Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint.

Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA polymerase epsilon, essential for leading-strand DNA replication and for the checkpoint. In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C). Strikingly, phosphorylation of Mrc1 during the S phase checkpoint abolishes Pol2N binding, but not Pol2C interaction. Mrc1 is required to stabilize Pol2 at replication forks stalled in HU. The bimodal Mrc1/Pol2 interaction may be an additional step in regulating the S phase checkpoint response to DNA damage on the leading strand. We propose that Mrc1, which also interacts with the MCMs, may modulate coupling of polymerization and unwinding at the replication fork.

The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1.

Single-stranded DNA (ssDNA) at DNA ends is an important regulator of the DNA damage response. Resection, the generation of ssDNA, affects DNA damage checkpoint activation, DNA repair pathway choice, ssDNA-associated mutation and replication fork stability. In eukaryotes, extensive DNA resection requires the nuclease Exo1 and nuclease/helicase pair: Dna2 and Sgs1(BLM). How Exo1 and Dna2-Sgs1(BLM) coordinate during resection remains poorly understood. The DNA damage checkpoint clamp (the 9-1-1 complex) has been reported to play an important role in stimulating resection but the exact mechanism remains unclear. Here we show that the human 9-1-1 complex enhances the cleavage of DNA by both DNA2 and EXO1 in vitro, showing that the resection-stimulatory role of the 9-1-1 complex is direct. We also show that in Saccharomyces cerevisiae, the 9-1-1 complex promotes both Dna2-Sgs1 and Exo1-dependent resection in response to uncapped telomeres. Our results suggest that the 9-1-1 complex facilitates resection by recruiting both Dna2-Sgs1 and Exo1 to sites of resection. This activity of the 9-1-1 complex in supporting resection is strongly inhibited by the checkpoint adaptor Rad9(53BP1). Our results provide important mechanistic insights into how DNA resection is regulated by checkpoint proteins and have implications for genome stability in eukaryotes.

Replication stress by Py-Im polyamides induces a non-canonical ATR-dependent checkpoint response.

Pyrrole-imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition.

Dna2 is involved in CA strand resection and nascent lagging strand completion at native yeast telomeres.

Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3'-GT-overhangs that extend beyond the complementary 5'-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G2 phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5'-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5'-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.