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DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5’ DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5’ terminus stimulates resection by EXO1. Ribonucleotides within a 5’ flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases., DNA double-strand break repair by homologous recombination initiates with nucleolytic resection of the 5’ DNA strand at the break ends. Here, the authors reveal that the lesion context influences the action and efficiency of the long range resection factors EXO1 and BLM-DNA2.

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70–80 base pairs per second) unwinding extensive tracts (∼8–10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

Nontraumatic cardiac arrest (NTCA) is a significant public health problem, affecting between 166,000 and 310,000 people annually in the United States. The probability of survival to hospital discharge remains low. Despite significant advances in care, the proportion of people surviving to hospital discharge has only minimally improved. In cardiac arrest patients, balloon occlusion of the aorta can be used in the critically ill as a method of temporizing the shock state and acting as a bridge to more definitive therapy such as the cardiac catheterization laboratory. During cardiac arrest, preclinical evidence demonstrates that the occlusion blood flow to the distal aorta effectively redirects the patient’s blood supply toward the heart and the brain, helping to preserve their function. Increased flow to these vital organs improves coronary and cerebral perfusion and oxygenation. A significant body of preclinical evidence dating back to the early 1980s supports the balloon occlusion of the aorta during NTCA as an adjunct to improve traditional advanced cardiac life support (ACLS); however, human trials have been limited to case series. Recent advances in percutaneous balloon technology have broadened this technique’s feasibility and set the stage for promising research into its role as an adjunct to ACLS.

The glomerulus exercises its filtration barrier function by establishing a complex filtration apparatus consisting of podocyte foot processes, glomerular basement membrane and endothelial cells. Disruption of any component of the glomerular filtration barrier leads to glomerular dysfunction, frequently manifested as proteinuria. Ultrastructural studies of the glomerulus by transmission electron microscopy (TEM) and conventional scanning electron microscopy (SEM) have been routinely used to identify and classify various glomerular diseases. Here we report the application of newly developed helium ion scanning microscopy (HIM) to examine the glomerulopathy in a Col4a3 mutant/Alport syndrome mouse model. Our study revealed unprecedented details of glomerular abnormalities in Col4a3 mutants including distorted podocyte cell bodies and disorganized primary processes. Strikingly, we observed abundant filamentous microprojections arising from podocyte cell bodies and processes, and presence of unique bridging processes that connect the primary processes and foot processes in Alport mice. Furthermore, we detected an altered glomerular endothelium with disrupted sub-endothelial integrity. More importantly, we were able to clearly visualize the complex, three-dimensional podocyte and endothelial interface by HIM. Our study demonstrates that HIM provides nanometer resolution to uncover and rediscover critical ultrastructural characteristics of the glomerulopathy in Col4a3 mutant mice.

Non-traumatic cardiac arrest is a major public health problem that carries an extremely high mortality rate. If we hope to increase the survivability of this condition, it is imperative that alternative methods of treatment are given due consideration. Balloon occlusion of the aorta can be used as a method of circulatory support in the critically ill patient. Intra-aortic balloon pumps have been used to temporize patients in cardiogenic shock for decades. More recently, resuscitative endovascular balloon occlusion of the aorta (REBOA) has been utilized in the patient in hemorrhagic shock or cardiac arrest secondary to trauma. Aortic occlusion in non-traumatic cardiac arrest has the effect of reducing the vascular volume that the generated cardiac output is distributed across. This augments myocardial and cerebral perfusion, increasing the probability of a return to a good quality of life for the patient. This phenomenon has been the subject of numerous animal studies dating back to the early 1980s; however, the human evidence is limited to several small case series. Animal research has demonstrated improvements in cerebral and coronary perfusion pressure during ACLS that lead to statistically significant differences in mortality. Several case series in humans have replicated these findings, suggesting the efficacy of this procedure. The objectives of this review are to: 1) introduce the reader to REBOA 2) review the physiology of NTCA and examine the current limitations of traditional ACLS 3) summarize the literature regarding the efficacy and feasibility of aortic balloon occlusion to support traditional ACLS.

DNA double-strand break repair by homologous recombination entails nucleolytic resection of the 5′ strand at break ends. Dna2, a flap endonuclease with 5′–3′ helicase activity, is involved in the resection process. The Dna2 helicase activity has been implicated in Okazaki fragment processing during DNA replication but is thought to be dispensable for DNA end resection. Unexpectedly, we found a requirement for the helicase function of Dna2 in end resection in budding yeast cells lacking exonuclease 1. Biochemical analysis reveals that ATP hydrolysis-fueled translocation of Dna2 on ssDNA facilitates 5′ flap cleavage near a single-strand–double strand junction while attenuating 3′ flap incision. Accordingly, the ATP hydrolysis-defective dna2-K1080E mutant is less able to generate long products in a reconstituted resection system. Our study thus reveals a previously unrecognized role of the Dna2 translocase activity in DNA break end resection and in the imposition of the 5′ strand specificity of end resection.

Background: Focused transthoracic echocardiography has been used to determine etiologies of cardiac arrest and evaluate utility of continuing resuscitation after cardiac arrest. Few guidelines exist advising ultrasound timing within the advanced cardiac life support algorithm. Natural timing of echocardiography occurs during the pulse check, when views are unencumbered by stabilization equipment or vigorous movements. However, recent studies suggest that ultrasound performance during pulse checks prolongs the pause duration of cardiopulmonary resuscitation. Transesophageal echocardiography studies have demonstrated benefits in this regard, but there have been no transthoracic echocardiography studies assessing the physical performance of compressions during cardiopulmonary resuscitation. Objective: The purpose of this study was to describe cases where echocardiography performed at the beginning of the cardiac arrest algorithm offers actionable information to cardiopulmonary resuscitation itself without delaying provision of compressions. Conclusion: Providers using focused echocardiography to evaluate cardiac arrest patients should consider initiating scans at the start of compressions to identify the optimal location for compression delivery and to detect inadequate compressions. Subsequent visualization of full left ventricular compression may be seen after a location change, and combined with end tidal carbon dioxide values, gives indication for improved forward circulatory flow. Although it is not possible in all patients, doing so hastens provision of quality compressions that affect hemodynamic parameters without causing prolongations to the pulse check pause. Further research is needed to determine patient outcomes from both out-of-hospital and in-hospital cardiac arrest when cardiopulmonary resuscitation is visually guided by focused echocardiography.

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.

The budding yeast Mre11–Rad50–Xrs2 (MRX) complex and Sae2 function together in DNA end resection during homologous recombination. Here we show that the Ku complex shields DNA ends from exonucleolytic digestion but facilitates endonucleolytic scission by MRX with a dependence on ATP and Sae2. The incision site is enlarged into a DNA gap via the exonuclease activity of MRX, which is stimulated by Sae2 without ATP being present. RPA renders a partially resected or palindromic DNA structure susceptible to MRX–Sae2, and internal protein blocks also trigger DNA cleavage. We present models for how MRX–Sae2 creates entry sites for the long-range resection machinery.

DNA double-strand breaks (DSBs) in cells can undergo nucleolytic degradation to generate long 3' single-stranded DNA tails. This process is termed DNA end resection, and its occurrence effectively commits to break repair via homologous recombination, which entails the acquisition of genetic information from an intact, homologous donor DNA sequence. Recent advances, prompted by the identification of the nucleases that catalyze resection, have revealed intricate layers of functional redundancy, interconnectedness, and regulation. Here, we review the current state of the field with an emphasis on the major questions that remain to be answered. Topics addressed will include how resection initiates via the introduction of an endonucleolytic incision close to the break end, the molecular mechanism of the conserved MRE11 complex in conjunction with Sae2/CtIP within such a model, the role of BRCA1 and 53BP1 in regulating resection initiation in mammalian cells, the influence of chromatin in the resection process, and potential roles of novel factors.

Summary: DNA double-strand break repair by homologous recombination entails the resection of DNA ends to reveal ssDNA tails, which are used to invade a homologous DNA template. CtIP and its yeast ortholog Sae2 regulate the nuclease activity of MRE11 in the initial stage of resection. Deletion of CtIP in the mouse or SAE2 in yeast engenders a more severe phenotype than MRE11 nuclease inactivation, indicative of a broader role of CtIP/Sae2. Here, we provide biochemical evidence that CtIP promotes long-range resection via the BLM-DNA2 pathway. Specifically, CtIP interacts with BLM and enhances its helicase activity, and it enhances DNA cleavage by DNA2. Thus, CtIP influences multiple aspects of end resection beyond MRE11 regulation. : Biochemical analysis by Daley et al. shows that CtIP not only functions as a cofactor for the MRN complex but also stimulates long-range resection by BLM-DNA2-RPA. CtIP interacts with BLM and enhances its helicase activity, and it upregulates the DNA flap cleavage activity of DNA2. Keywords: homologous recombination, double-strand break repair, end resection, Bloom syndrome, BLM, CtIP, DNA2

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. Our studies showed that DNA2 nuclease is an evolutionarily conserved essential component of HR repair machinery. Here we demonstrate that DNA2 is indeed overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90%-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anti-cancer drugs by targeting druggable nuclease DNA2. We 4, 16. In propose DNA2 inhibition as new strategy in cancer therapy by targeting replication stress, a molecular property of cancer cells that is acquired as a result of oncogene activation instead of targeting undruggable oncoprotein itself such as KRAS.

In homologous recombination (HR), DNA polymerase δ-mediated DNA synthesis occurs within the displacement loop (D-loop) that is made by the recombinase Rad51 in conjunction with accessory factors. We describe in this chapter the reconstitution of the D-loop and repair DNA synthesis reactions using purified Saccharomyces cerevisiae HR (Rad51, RPA, and Rad54) and DNA replication (PCNA, RFC, and DNA polymerase δ) proteins and document the role of the Pif1 helicase in DNA synthesis via a migrating DNA bubble intermediate. These reconstituted systems are particularly valuable for understanding the conserved mechanism of repair DNA synthesis dependent on DNA polymerase δ and its cognate helicase in eukaryotic organisms.

BLM, a RecQ family DNA helicase mutated in Bloom's Syndrome, participates in homologous recombination at two stages: 5′ DNA end resection and double Holliday junction dissolution. BLM exists in a complex with Topo IIIα, RMI1 and RMI2. Herein, we address the role of Topo IIIα and RMI1-RMI2 in resection using a reconstituted system with purified human proteins. We show that Topo IIIα stimulates DNA unwinding by BLM in a manner that is potentiated by RMI1-RMI2, and that the processivity of resection is reliant on the Topo IIIα–RMI1-RMI2 complex. Topo IIIα localizes to the ends of double-strand breaks, thus implicating it in the recruitment of resection factors. While the single-stranded DNA binding protein RPA plays a major role in imposing the 5′ to 3′ polarity of resection, Topo IIIα also makes a contribution in this regard. Moreover, we show that DNA2 stimulates the helicase activity of BLM. Our results thus uncover a multifaceted role of the Topo IIIα–RMI1-RMI2 ensemble and of DNA2 in the DNA resection reaction.

SummaryThe G-rich single-stranded DNA at the 3′ end of human telomeres can self-fold into G-quaduplex (GQ). However, telomere lengthening by telomerase or the recombination-based alternative lengthening of telomere (ALT) mechanism requires protein loading on the overhang. Using single-molecule fluorescence spectroscopy, we discovered that lengthening the telomeric overhang also increased the rate of dynamic exchanges between structural conformations. Overhangs with five to seven TTAGGG repeats, compared with four repeats, showed much greater dynamics and accessibility to telomerase binding and activity and loading of the ALT-associated proteins RAD51, WRN, and BLM. Although the eight repeats are highly dynamic, they can fold into two GQs, which limited protein accessibility. In contrast, the telomere-specific protein POT1 is unique in that it binds independently of repeat number. Our results suggest that the telomeric overhang length and dynamics may contribute to the regulation of telomere extension via telomerase action and the ALT mechanism.

When DNA double-strand breaks occur, the cell cycle stage has a major influence on the choice of the repair pathway employed. Specifically, nonhomologous end joining is the predominant mechanism used in the G1 phase of the cell cycle, while homologous recombination becomes fully activated in S phase. Studies over the past 2 decades have revealed that the aberrant joining of replication-associated breaks leads to catastrophic genome rearrangements, revealing an important role of DNA break repair pathway choice in the preservation of genome integrity. 53BP1, first identified as a DNA damage checkpoint protein, and BRCA1, a well-known breast cancer tumor suppressor, are at the center of this choice. Research on how these proteins function at the DNA break site has advanced rapidly in the recent past. Here, we review what is known regarding how the repair pathway choice is made, including the mechanisms that govern the recruitment of each critical factor, and how the cell transitions from end joining in G1 to homologous recombination in S/G2.

Objectives: The primary objectives were to describe the diagnostic characteristics tricuspid annular plane systolic excursion (TAPSE) for pulmonary embolism (PE) and to optimize the measurement cutoff of TAPSE for the diagnosis of PE. Secondary objectives included assessment of interrater reliability and the quantitative visual estimation of TAPSE. Methods: This is a prospective observational cohort study involving a convenience sample of patients at an urban academic emergency department. Patients underwent focused right heart echocardiogram (FOCUS) before computed tomographic angiography (CTA) for suspected PE. Results: A total of 150 patients were enrolled, 32 of whom (21.3%) were diagnosed as having a PE. A receiver operating characteristic curve analysis yielded 2.0 cm as the optimal cutoff for TAPSE in the diagnosis of PE, with a sensitivity of 72% (95% confidence interval [CI], 53-86), a specificity of 66% (95% CI, 57-75), and an area under the curve of 0.73 (95% CI, 0.65-0.83). In patients with tachycardia or hypotension, post hoc analysis demonstrated that FOCUS is 100% (95% CI, 80-100) sensitive for PE, whereas TAPSE is 94% (95% CI, 71-99) sensitive for PE. The intraclass correlation coefficient was 0.87 (95% CI, 0.79-0.93). Emergency physicians with training in echocardiography accurately visually estimated TAPSE, with a κ statistic of 0.94 (95% CI, 0.87-0.98). Conclusions: Emergency physicians with training in echocardiography can reliably measure TAPSE and are able to accurately visually estimate TAPSE as either normal or abnormal. When using an abnormal cutoff of less than 2.0 cm, TAPSE has moderate diagnostic value in patients with suspected PE. On post hoc analysis, TAPSE and FOCUS appear to be highly sensitive for PE in patients with tachycardia or hypotension.

Nucleolytic resection of DNA double-strand breaks is the crucial first step in their repair via homologous recombination. New findings by Tkac et al. (2016) published in this issue of Molecular Cell identify HELB as a novel, cell-cycle-specific negative regulator of DNA end resection.

Apurinic/apyrimidinic (AP) endonucleases are versatile DNA repair enzymes that possess a variety of nucleolytic activities, including endonuclease activity at AP sites, 3′ phosphodiesterase activity that can remove a variety of ligation-blocking lesions from the 3′ end of DNA, endonuclease activity on oxidative DNA lesions, and 3′ to 5′ exonuclease activity. There are two families of AP endonucleases, named for the bacterial counterparts endonuclease IV (EndoIV) and exonuclease III (ExoIII). While ExoIII family members are present in all kingdoms of life, EndoIV members exist in lower organisms but are curiously absent in plants, mammals and some other vertebrates. Here, we review recent research on these enzymes, focusing primarily on the EndoIV family. We address the role(s) of EndoIV members in DNA repair and discuss recent findings from each model organism in which the enzymes have been studied to date.

Mutations in Aprataxin cause the neurodegenerative syndrome ataxia oculomotor apraxia type 1. Aprataxin catalyzes removal of adenosine monophosphate (AMP) from the 5' end of a DNA strand, which results from an aborted attempt to ligate a strand break containing a damaged end. To gain insight into which DNA lesions are substrates for Aprataxin action in vivo, we deleted the Saccharomyces cerevisiae HNT3 gene, which encodes the Aprataxin homolog, in combination with known DNA repair genes. While hnt3Delta single mutants were not sensitive to DNA damaging agents, loss of HNT3 caused synergistic sensitivity to H(2)O(2) in backgrounds that accumulate strand breaks with blocked termini, including apn1Delta apn2Delta tpp1Delta and ntg1Delta ntg2Delta ogg1Delta. Loss of HNT3 in rad27Delta cells, which are deficient in long-patch base excision repair (LP-BER), resulted in synergistic sensitivity to H(2)O(2) and MMS, indicating that Hnt3 and LP-BER provide parallel pathways for processing 5' AMPs. Loss of HNT3 also increased the sister chromatid exchange frequency. Surprisingly, HNT3 deletion partially rescued H(2)O(2) sensitivity in recombination-deficient rad51Delta and rad52Delta cells, suggesting that Hnt3 promotes formation of a repair intermediate that is resolved by recombination.

Nonhomologous end joining (NHEJ) directly rejoins DNA double-strand breaks (DSBs) when recombination is not possible. In Saccharomyces cerevisiae , the DNA polymerase Pol4 is required for gap filling when a short 3′ overhang must prime DNA synthesis. Here, we examined further end variations to test specific hypotheses regarding Pol4 usage in NHEJ in vivo . Surprisingly, Pol4 dependence at 3′ overhangs was reduced when a nonhomologous 5′ flap nucleotide was present across from the gap, even though the mismatched nucleotide was corrected, not incorporated. In contrast, a gap with a 5′ deoxyribosephosphate (dRP) was as Pol4-dependent as a gap with a 5′ phosphate, demonstrating the importance of the downstream base in relaxing the Pol4 requirement. Combined with prior observations of Pol4-independent NHEJ of nicks with 5′ hydroxyls, we suggest that base stacking interactions across the broken strands can stabilize a joint, allowing another polymerase to substitute for Pol4. This model predicts that a unique function of Pol4 is to actively stabilize template strands that lack stacking continuity. We also explored whether NHEJ end processing can occur via short- and long-patch pathways analogous to base excision repair. Results demonstrated that 5′ dRPs could be removed in the absence of Pol4 lyase activity. The 5′ flap endonuclease Rad27 was not required for repair in this or any situation tested, indicating that still other NHEJ 5′ nucleases must exist.

DNA double-strand breaks can be eliminated via non-homologous end joining or homologous recombination. Non-homologous end joining is initiated by the association of Ku with DNA ends. In contrast, homologous recombination entails nucleolytic resection of the 5′-strands, forming 3′-ssDNA tails that become coated with replication protein A (RPA). Ku restricts end access by the resection nuclease Exo1. It is unclear how partial resection might affect Ku engagement and Exo1 restriction. Here, we addressed these questions in a reconstituted system with yeast proteins. With blunt-ended DNA, Ku protected against Exo1 in a manner that required its DNA end-binding activity. Despite binding poorly to ssDNA, Ku could nonetheless engage a 5′-recessed DNA end with a 40-nucleotide (nt) ssDNA overhang, where it localized to the ssDNA-dsDNA junction and efficiently blocked resection by Exo1. Interestingly, RPA could exclude Ku from a partially resected structure with a 22-nt ssDNA tail and thus restored processing by Exo1. However, at a 40-nt tail, Ku remained stably associated at the ssDNA-dsDNA junction, and RPA simultaneously engaged the ssDNA region. We discuss a model in which the dynamic equilibrium between Ku and RPA binding to a partially resected DNA end influences the timing and efficiency of the resection process.

Customer service is identified in this paper, as the fundamental difference between the traditional department store and the "off-price" retailer. The off-price trend is first described and then, the customer service components of off-pricers and department store retailers are evaluated. The paper concludes with suggested customer service strategies for the off-price retailer.

Ku-dependent nonhomologous end joining (NHEJ) is a double-strand break repair process conserved in all branches of cellular life but has not previously been implicated in the DNA metabolic processes of viruses. We identified Ku homologs in Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis. These proteins formed homodimers and bound DNA ends in a manner identical to other Ku's and stimulated joining of ends by the host NHEJ DNA ligase (LigD). Omega and Corndog are unusual in having short 4 base cos ends that would not be expected to self-anneal and would therefore require NHEJ during phage genome circularization. Consistently, M. smegmatis LigD null strains are entirely and selectively unable to support infection by Corndog or Omega, with concomitant failure of genome circularization. These results establish a new paradigm for sequestration of the host cell NHEJ process by bacteriophage and provide a framework for understanding similar transactions in eukaryotic viral infections.

E-commerce and China are two widely discussed business topics. Despite the political, legal and infrastructure obstacles, the potential of e-commerce in China (PRC) is too great to ignore. This paper addresses the perceptions of Chinese concerning the obstacles and benefits of conducting e-commerce. Analysis identifies the rank-order of benefits and obstacles to conducting e-commerce in China (PRC).

It is coincidence, in the strictest meaning of the word, that the demand for electrical energy in a manufacturing facility maximizes when the cost of that energy maximizes. Maintaining the competitive edge in business means controlling energy costs among others. Given the cost of on-peak energy and the potential for its increase under the utility deregulation scenario, businesses will be looking for ways to optimize return on equity invested in on-site generation equipment. A likely opportunity is the adaptation of installed emergency and standby power generation for peaking scenarios. This article discusses the path followed by a manufacturer in the varied use of on-site generation for peaking power production. The variations were dictated by evolving conditions, not the least of which was environmental, over 18 years of peaking. The goal has been cost control in each of the variations. This experience is shared as objective evidence that existing on-site generation can be cost effectively deployed in a variety of operating modes to meet changing business and environmental conditions.

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90–95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting replication stress, a molecular property of cancer cells that is acquired as a result of oncogene activation instead of targeting currently undruggable oncoprotein itself such as KRAS.

Provides results from a recent empirical study of US‐based international freight forwarders (IFFs). Compares IFF demographics with those from previous empirical research and finds that the IFF industry is still dominated by small companies and that IFFs continue to diversify their revenue bases. Presents information concerning contemporary issues affecting the forwarding industry and finds that IFFs generally view the Internet as a complement to, rather than a substitute for, EDI.

The DNA double-strand break (DSB), arising from exposure to ionizing radiation or various chemotherapeutic agents or from replication fork collapse, is among the most dangerous of chromosomal lesions. DSBs are highly cytotoxic and can lead to translocations, deletions, duplications, or mutations if mishandled. DSBs are eliminated by either homologous recombination (HR), which uses a homologous template to guide accurate repair, or by nonhomologous end joining (NHEJ), which simply rejoins the two broken ends after damaged nucleotides have been removed. HR generates error-free repair products and is also required for generating chromosome arm crossovers between homologous chromosomes in meiotic cells. The HR reaction includes several distinct steps: resection of DNA ends, homologous DNA pairing, DNA synthesis, and processing of HR intermediates. Each occurs in a highly regulated fashion utilizing multiple protein factors. These steps are being elucidated using a combination of genetic tools, cell-based assays, and in vitro reconstitution with highly purified HR proteins. In this review, we summarize contributions from our laboratory at Yale University in understanding HR mechanisms in eukaryotic cells.

Summarizes previous logistics research into the benefits of, and barriers to, electronic data interchange (EDI). Argues that information management in general, and EDI in particular, is vitally important for today’s international freight forwarder. Presents the findings from empirical studies of international freight forwarders and customers of international freight forwarders with respect to EDI benefits and barriers. Suggests that numerous opportunities exist for further research into EDI benefits and barriers across various logistics constituencies.

As the level of cross-border trade continues to expand, so does the prominence and importance of efficient logistics management. Indeed, there is little question that international logistics is more costly and more challenging than domestic logistics. With respect to the former, Rise (1995) has estimated that between 10% and 30% of the costs of international orders are logistics-related. Challenges associated with cross-border logistics include, but are not limited to, longer lead times, increased inventory levels, and unfamiliar and/or inadequate transportation systems. While it has been suggested (Czinkota and Ronkainen, 1998) "... that logistics may well become the key dimension by which firms distinguish themselves internationally ...", logistical considerations may not assume high priority when companies are making decisions about 1) countries to do business in and 2) the appropriate organizational strategy (e.g., exporting, direct investment) for entering these countries. Previous research by the current authors has suggested that logistical considerations can be incorporated into the country of choice and method of entry decisions by evaluating a country's logistical "friendliness" or "unfriendliness." Briefly, logistical "friendliness" ("unfriendliness") refers (Murphy and Daley, 1994) to the ease (difficulty) of arranging international freight operations to/from a particular country. Previous empirical research involving both international freight forwarders (IFFs) and smaller businesses revealed that participants could clearly articulate logistically friendly and unfriendly countries. IFFs, for instance, listed (Murphy, Daley, and Dalenberg, 1993a) Great Britain, Germany, Japan, and Holland as particularly friendly countries; China, Saudi Arabia, and Brazil emerged as particularly unfriendly. According to small business managers, Canada, Great Britain, and Hong Kong (Murphy, Daley, and Dalenberg, 1993b) were viewed as the most logistically friendly countries, while Japan, Brazil, and China were the most logistically unfriendly. Unfortunately, neither the IFF study nor the small business study identified features or attributes of logistical friendliness (unfriendliness). A subsequent research project (Murphy and Daley, 1994) identified a number of overriding themes associated with logistical friendliness, and suggested that many of these themes were non-logistical in nature. Prominent non-logistical themes included "trade relationships", "economic conditions", and "cultural issues." THE PRESENT STUDY Our previous research on logistical friendliness, while valuable, is lacking in several respects. First, as pointed out above, the research on the features or attributes of logistical friendliness was conducted separately from that involving the delineation of logistically friendly (unfriendly) countries. In short, the features or attributes of logistical friendliness cannot be linked directly/explicitly with individual countries. Second, the research on the features/attributes of logistical friendliness only investigated logistical friendliness, and not logistical unfriendliness. Is it possible that certain features/attributes are associated with logistical friendliness, while different features/ attributes are associated with logistical unfriendliness? In an attempt to address these shortcomings, the present paper reports the results of a study involving international freight forwarders (IFFs) designed to learn 1) about logistically friendly and logistically unfriendly countries and 2) the reasons why these countries are viewed as logistically friendly (unfriendly). IFF's appear to be an excellent sampling frame for investigating logistical friendliness (unfriendliness) because they are widely used logistical intermediaries (Lambert, Stock, and Ellram, 1998) that provide numerous functions (e.g., preparing export declarations, determining shipment routings) to facilitate cross-border trade. …

Intermodal transportation may be one of the most misunderstood concepts (Jennings and Holcomb, 1996) in the logistics discipline. In some instances, intermodal is not even defined, resulting in an assumption that there is an implicit knowledge about what is meant by intermodal. Alternatively, there are myriad definitions of intermodal, such as (Coyle, Bardi, and Novack 1994) "... the use of two or more modes of transportation in moving a shipment from origin to destination." Indeed, there are so many definitions of intermodal (Jennings and Holcomb, 1996) "... that researchers, government bodies, and practitioners may wind up spending more time arguing over its definition than implementing its ideas." For purposes of this paper, intermodal transportation will refer to (Jennings and Holcomb, 1996) "... a container or other device which can be transferred from one vehicle or mode to another without the contents of said device being reloaded or disturbed." While intermodal transportation has registered impressive growth during the past two decades, there has been relatively little academic research dealing with intermodalism. In fact, a review of two key logistics journals, Transportation Journal and the Journal of Business Logistics, reveals a total of three empirical studies on intermodalism in the five year time period from 1993 to 1997. These articles are summarized below. Jennings and Holcomb (1996) used interview type case studies to learn about noncontainerized intermodal (transload) movements by mode and by commodity. Transload activities tend to involve large volume or large-sized commodities; shippers, rather than carriers, are the initiating party for transload movements. Transload shippers cited a variety of reasons (e.g., service abandonment, location) for being involved in transloading activities. Johnston and Marshall (1993) looked at shipper perceptions about intermodal equipment in six categories such as cubic capacity, ease of loading and unloading, and cleanliness. They found that various types of intermodal equipment have different strengths and weaknesses. For example, TOFC (trailer-on-flatcars) trailers are perceived to be strong in cubic capacity, but weak in cleanliness; RoadRailers are strong in cleanliness, but weak in capacity. Harper and Evers (1993) investigated competitive issues in intermodal rail-truck (IRT) service among manufacturers in the state of Minnesota. Their research suggested that IRT service was not available to many potential customers, that larger firms tend to use IRT, and that shippers do not have a very good perception of IRT. In particular, shippers emphasized the seriousness of poor IRT transit times. THE PRESENT STUDY The Harper and Evers research is particularly valuable because a portion of it looked at the perspectives of both users and nonusers of IRT services with respect to select intermodal issues. Their findings involving the users and nonusers serve as an excellent source for the development of a series of propositions concerning rail-truck intermodal. The present paper will evaluate the various propositions using data compiled from a recent study of business organizations located in a major metropolitan area. These propositions will be developed below. One portion of the Harper and Evers research involved a mail survey of manufacturers located in the state of Minnesota. Their findings (1993) suggested that larger firms were more likely than smaller firms to be users of IRT services. The Harper and Evers research also investigated the modal splits of users and nonusers of IRT services. Their findings suggested different modal split patterns between users and nonusers for their outbound, shipments. More specifically, IRT users tend to rely more heavily than nonusers on truckload (TL) motor carriage service, while less-than-truckload (LTL) service is the preferred form for IRT nonusers. Furthermore, based on aggregate figures, IRT tends to be a secondary mode of outbound transportation among IRT users. …

INTRODUCTION International freight forwarders (IFFs) are key specialists in cross-border trade. They can provide a variety of services, and are used by the great majority of companies engaged in international commerce (Johnson and Wood, 1996). Despite the important role of IFFs in efficient cross-border trade, there is relatively limited empirical information about them. The literature has suggested (Pope and Thomchick, 1985; Murphy, Daley, and Dalenberg, 1992a) that IFFs are small companies, often employing fewer than 10 people. IFFs are becoming more diversified in their customer offerings; many contemporary IFFs provide forwarding services for both air and water shipments, and a number also provide such multiple intermediary services as non-vessel operating common carrier service and customshouse brokerage (Murphy and Daley, 1995). Moreover, the rapidly changing global business environment has had important implications for the forwarding industry. More specifically, the forwarding industry has been characterized by tremendous volatility over the past decade (Ozsomer, Mitri and Cavusgil, 1993), as manifested in various acquisitions, consolidations, and bankruptcies. This volatility has led some to question the continued viability of smaller forwarders. Consider the following statement from the president of a smaller IFF (Gillis, 1996): Tm a firm believer that the smaller forwarder and broker will be extinct by 2000." One of the key aspects in the future viability of individual IFFs is how well they can meet the needs and wants of current and future customers. While this philosophy essentially represents the marketing concept, providers of logistics services have not always embraced the notion of satisfying customer needs and wants, in part because logistics service providers have sometimes used a very narrow definition of "customer". As an example, international water ports (Murphy, Daley, and Dalenberg, 1992b) have appeared to understand the requirements of water carriers--traditionally considered to be the ports' primary customers--with respect to key factors in water port selection, but are not so well aligned with other customer groups such as shippers and international freight forwarders. Furthermore, although the marketing concept stresses that service providers should satisfy customer needs and wants, the service quality research has identified a variety of gaps (Parasuraman, Zeithaml, and Berry 1985) between expected and perceived service quality, and that service quality is a key determinant of customer satisfaction. With this in mind, the present paper will examine IFFs and current users of IFFs with respect to various services which might be provided by IFFs. In so doing, the paper seeks to identify possible gaps between the services actually provided by IFFs and the services IFF users would like provided. In addition, because customer satisfaction is a desired output of service quality, the paper will report on IFF users perceived satisfaction with the general performance of the IFFs uBed by their respective companies. METHODOLOGY AND PARTICIPANT PROFILES The IFF information comes from a mail survey sent to IFFs identified in The Official Directory of Transportation Middlemen (now, The Official Intermodal Guide). Of 336 eligible IFFs, usable responses were received from 98, for an effective response rate of 29.2%. Nearly two thirds of the responding IFFs reported annual revenues of less than $10 million, a finding consistent with previous IFF research (Pope and Thomchick, 1985; Murphy, Daley, and Dalenberg, 1992a). Approximately 75%of the respondents classified themselves as a Vice President, President, CEO, or Owner. These senior-level managers should be quite familiar with the services provided by their companies. The user information is drawn from a mail survey of 370 randomly selected members of the Council of Logistics Management (CLM). …

Previous carrier selection studies involving both shipper and carrier perspectives have discovered numerous differences between shippers and carriers, at least in terms of mean score comparisons. However, analysis of the relative importance of the selection factors (not reported in previous studies) uniformly shows a high degree of similarity between shippers and carriers. A new shipper-carrier study, investigating a more specific selection (of truckload general freight carriers), discovered a similar dichotomy between the mean score and relative comparisons. The paper concludes by developing several research propositions and by offering suggestions for additional research.

In mammalian cells, repair of DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ) is critical for genome stability. Although the end-bridging and ligation steps of NHEJ have been reconstituted in vitro, little is known about the end-processing reactions that occur before ligation. Recently, functionally homologous end-bridging and ligation activities have been identified in prokarya. Consistent with its homology to polymerases and nucleases, we demonstrate that DNA ligase D from Mycobacterium tuberculosis (Mt-Lig) possesses a unique variety of nucleotidyl transferase activities, including gap-filling polymerase, terminal transferase, and primase, and is also a 3′ to 5′ exonuclease. These enzyme activities allow the Mt-Ku and Mt-Lig proteins to join incompatible DSB ends in vitro, as well as to reconstitute NHEJ in vivo in yeast. These results demonstrate that prokaryotic Ku and ligase form a bona fide NHEJ system that encodes all the recognition, processing, and ligation activities required for DSB repair.

International freight forwarders (IFFs) are such important logistical intermediaries that almost every company engaged in international trade uses an IFF at one time or another.[1] In fact, a recent study of some of the largest U.S. companies found that more than 90 percent of them use IFFs to facilitate cross-border shipments.[2] While IFFs are frequently used because of the logistical expertise they bring to international shipments, there is some disagreement as to what is meant by an IFF as well as the functions provided by such firms. Common definitions portray IFFs as logistical specialists for export shipments.[3] Other views, however, indicate that IFFs provide both export and import services.[4] Recent research has identified approximately two dozen functions - ranging from the payment of freight charges to the provision of legal services - that are offered by IFFs.[5] What is the relevance of IFFs for purchasing managers? Because purchasing's primary responsibilities involve the acquisition Of goods and services for their firms, there is more of an inbound, vis-a-vis outbound, emphasis. With respect to international shipments, this inbound focus means that purchasing managers are generally importing products for their companies. As a result, according to "conventional wisdom," purchasing managers should be more likely to use customshouse brokers (CHBs), whose expertise lies in facilitating import shipments.[6] Several recent studies, however, suggest that dramatic changes are occurring in the functions and activities of IFFs. For example, one group of researchers point out that many IFFs are expanding their service offerings in an effort to provide customers and potential customers with one-stop shopping.[7] Another study suggests that some IFFs are choosing to provide one-stop shopping by offering services such as nonvessel operating common carriers (NVOCCs) and/or CHBs.[8] The relevance of these changes for the purchasing function is illustrated in a recent study by the authors.[9] Results from this study, which involved a regional sample of purchasing managers, indicated that "international freight forwarders - generally regarded as export specialists - are used slightly more than customshouse brokers - the import experts - in global sourcing activities." Although no explanations were offerred for the heavier utilization of IFFs by the purchasing professionals studied, the findings suggest the need for purchasing managers to be knowledgeable about the attributes, functions, and activities of both types of service firms. A review of the literature reveals only a limited number of empirical studies dealing with IFFs. The earliest such study investigated a regional (Northeast United States) sample of IFFs - and found most of them to be small, emerging one stop service companies.[10] A second empirical study, national in scope, likewise showed IFFs to be smaller enterprises, and also found that service was the primary reason shippers use IFFs.[11] WHY WAS THIS RESEARCH DONE? The previous studies, while providing valuable baseline information about IFFs, also generated important questions for further research. These include (1) the actual functions provided by forwarders; (2) issues associated with the provision of one-stop services; (3) issues associated with co-loading; and (4) the influence of intermodalism. Consequently, this study was undertaken to find answers to the following questions: 1. What are the various functions currently provided by IFFs? 2. To what degree have the growth of intermodalism and logistics outsourcing influenced the nature of forwarding operations? Has this influence been positive or negative? 3. To what degree is one-stop service being offered by IFFs? 4. Among those companies providing several intermediary services, how prevalent is conflict among the intermediaries' roles? What types of conflict exist between (among) these roles? …

Homologous recombination (HR) is an evolutionarily conserved process that eliminates DNA double-strand breaks from chromosomes, repairs injured DNA replication forks, and helps orchestrate meiotic chromosome segregation. Recent studies have shown that DNA helicases play multifaceted roles in HR mediation and regulation. In particular, the S. cerevisiae Sgs1 helicase and its human ortholog BLM helicase are involved in not only the resection of the primary lesion to generate single-stranded DNA to prompt the assembly of the HR machinery, but they also function in somatic cells to suppress the formation of chromosome arm crossovers during HR. On the other hand, the S. cerevisiae Mph1 and Srs2 helicases, and their respective functional equivalents in other eukaryotes, suppress spurious HR events and favor the formation of noncrossovers via distinct mechanisms. Thus, the functional integrity of the HR process and HR outcomes are dependent upon these helicase enzymes. Since mutations in some of these helicases lead to cancer predisposition in humans and mice, studies on them have clear relevance to human health and disease.

Most firms have traditionally offered one level of logistics service to all customers. This often results in some customer groups receiving more service than necessary, while other groups receive less service than necessary. The emerging concept of logistical segmentation suggests that companies can structure their logistical offerings to meet the needs and requirements of different customer groups. Uses the “nested” approach from industrial marketing to illustrate an application of logistical segmentation. More specifically, examines the importance of selection factors in modal choice for international sourcing according to geographic location, primary mode of transport, and job responsibilities. Uses empirical data from a group of regional purchasing managers to facilitate understanding of the nested approach.

Logistics problems have been identified as the biggest obstacle facing U.S. businesses in international sourcing. However, neither the purchasing nor the logistics literature contains much information on logistical considerations in global sourcing. This article presents the results of an exploratory study designed to learn about selected logistics issues in global sourcing. The article also identifies a number of opportunities for further research in the field.

A frequently overlooked component of succesful international trade involves the efficient distribution of products. The present paper looked at international freight forwarder perceptions of the easiest and most difficult countries for arranging international freight operations. The forwarders overwhelmingly indicated that Great Britain was the easiest country for arranging international freight operations, which China emerged as the most difficult. Study results could be helpful both as competitive benchmarks and in formulating public policy.

Responding to Randall and Gibson's (1990) call for more rigorous methodologies in empirically-based ethics research, this paper develops propositions — based on both previous ethics research as well as the larger organizational behavior literature — examining the impact of attitudes, leadership, presence/absence of ethical codes and organizational size on corporate ethical behavior. The results, which come from a mail survey of 149 companies in a major U.S. service industry, indicate that attitudes and organizational size are the best predictors of ethical behavior. Leadership and ethical codes contribute little to predicting ethical behavior. The paper concludes with an assessment of the relevant propositions, as well as a delineation of future research needs.

Presents a contemporary baseline study of US‐based international freight forwarders. Provides much needed information on industry characteristics, which should reduce the occurrences of managers making important distribution decisions based only on conjecture, subjective perceptions and commonly held assumptions. Addresses three research questions, focusing on the selected demographic characteristics of US‐based forwarders; the differences between “pure” forwarders (those concentrating primarily on consolidating shipments for international water transport) and “diversified” forwarders (those providing other intermediary services, those deriving a noticeable portion of revenues from air shipments); and the forwarders′ views as to the primary reason for shippers using their companies. Describes methodology and analyses, results and implications.

Nonhomologous end joining (NHEJ) in yeast depends on eight different proteins in at least three different functional complexes: Yku70–Yku80 (Ku), Dnl4–Lif1–Nej1 (DNA ligase IV), and Mre11–Rad50–Xrs2 (MRX). Interactions between these complexes at DNA double-strand breaks (DSBs) are poorly understood but critical for the completion of repair. We previously identified two such contacts that are redundantly required for NHEJ, one between Dnl4 and the C terminus of Yku80 and one between the forkhead-associated (FHA) domain of Xrs2 and the C terminus of Lif1. Here, we first show that mutation of the Yku80 C terminus did not impair Ku binding to DSBs, supporting specificity of the mutant defect to the ligase interaction. We next show that the Xrs2–Lif1 interaction depends on Xrs2 FHA residues (R32, S47, R48, and K75) analogous to those known in other proteins to contact phosphorylated threonines. Two potential target threonines in Lif1 (T417 and T387) were inferred by identifying regions similar to a site in the human Lif1 homolog, XRCC4, known to be bound by the FHA domain of polynucleotide kinase. Mutating these threonines, especially T417, abolished the Xrs2–Lif1 interaction and impaired NHEJ epistatically with Xrs2 FHA mutation. Combining mutations that selectively disable the Yku80–Dnl4 and Xrs2–Lif1 interactions abrogated both NHEJ and DNA ligase IV recruitment to a DSB. The collected results indicate that the Xrs–Lif1 and Yku80–Dnl4 interactions are important for formation of a productive ligase–DSB intermediate.