a b s t r a c tGenomes are subject to constant threat by damaging agents that generate DNA double-strand breaks (DSBs). The ends of linear chromosomes need to be protected from DNA damage recognition and end-joining, and this is achieved through protein-DNA complexes known as telomeres. The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in detection and signaling of DSBs, as well as the repair pathways of homologous recombination (HR) and non-homologous end-joining (NHEJ). In addition, MRN associates with telomeres and contributes to their maintenance. Here, we provide an overview of MRN functions at DSBs, and examine its roles in telomere maintenance and dysfunction.

The kinetic mechanism and the structural bases of the fidelity of DNA polymerases are still highly controversial. Here we report the use of three probes in the stopped-flow studies of Pol beta to obtain new, direct evidence for our previous interpretations: (a) Increasing the viscosity of the reaction buffer by sucrose or glycerol is expected to slow down the conformational change differentially, and it was shown to slow down the first (fast) fluorescence transition selectively. (b) Use of dNTPalphaS in place of dNTP is expected to slow down the chemical step preferentially, and it was shown to slow down the second (slow) fluorescence transition selectively. (c) The substitution-inert Rh(III)dNTP was used to show for the first time that the slow fluorescence change occurs after mixing of Pol beta.DNA.Rh(III)dNTP with Mg(II). These results, along with crystal structures, suggest that the subdomain-closing conformational change occurs before binding of the catalytic Mg(II) while the rate-limiting step occurs after binding of the catalytic Mg(II). These results provide new evidence to the mechanism we suggested previously, but do not support the results of three recent papers of computational studies. The results were further supported by a "sequential mixing" stopped-flow experiment that used no analogues, and thus ruled out the possibility that the discrepancy between experimental and computational results is due to the use of analogues. The methodologies can be used to examine other DNA polymerases to answer whether the properties of Pol beta are exceptional or general.

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Our recent demonstration that DNA polymerase X (Pol X), the DNA repair polymerase encoded by the African swine fever virus (ASFV), is extremely error prone during single-nucleotide gap filling led us to hypothesize that it might contribute to genetic variability in ASFV. For the infidelity of Pol X to be relevant, however, the DNA ligase working downstream of it would need to be capable of sealing nicks containing 3'-OH mismatches. We therefore examined the nick ligation capabilities of the ASFV-encoded DNA ligase and here report the first complete 3' fidelity analysis, employing catalytic parameters, for any DNA ligase. The catalytic efficiency of nick sealing by both ASFV DNA ligase and bacteriophage T4 DNA ligase was determined in the steady state for substrates containing all 16 possible matched and mismatched base pair combinations at the 3' side of a nick. Our results indicate that ASFV DNA ligase is the lowest-fidelity DNA ligase ever reported, capable of ligating a 3' C:T mismatched nick (where C and T are the templating and nascent nucleotides, respectively) more efficiently than nicks containing Watson-Crick base pairs. Comparison of the mismatch specificity of Pol X with that of ASFV DNA ligase suggests that the latter may have evolved toward low fidelity for the purpose of generating the broadest possible spectrum of sealed mismatches. These findings are discussed in light of the genetic and antigenic variability observed among some ASFV isolates. Two novel assays for determining the concentration of active DNA ligase are also reported.

A growing understanding of the molecular interactions between immune effector cells and target tumor cells, coupled with refined gene therapy approaches, are giving rise to novel cancer immunotherapeutics with remarkable efficacy in the clinic against both solid and liquid tumors. While immunotherapy holds tremendous promise for treatment of certain cancers, significant challenges remain in the clinical translation to many other types of cancers and also in minimizing adverse effects. Therefore, there is an urgent need for functional potency assays, in vitro and in vivo, that could model the complex interaction of immune cells with tumor cells and can be used to rapidly test the efficacy of different immunotherapy approaches, whether it is small molecule, biologics, cell therapies or combinations thereof. Herein we report the development of an xCELLigence real-time cytolytic in vitro potency assay that uses cellular impedance to continuously monitor the viability of target tumor cells while they are being subjected to different types of treatments. Specialized microtiter plates containing integrated gold microelectrodes enable the number, size, and surface attachment strength of adherent target tumor cells to be selectively monitored within a heterogeneous mixture that includes effector cells, antibodies, small molecules, etc. Through surface-tethering approach, the killing of liquid cancers can also be monitored. Using NK92 effector cells as example, results from RTCA potency assay are very well correlated with end point data from image-based assays as well as flow cytometry. Several effector cells, i.e., PBMC, NK, CAR-T were tested and validated as well as biological molecules such as Bi-specific T cell Engagers (BiTEs) targeting the EpCAM protein expressed on tumor cells and blocking antibodies against the immune checkpoint inhibitor PD-1. Using the specifically designed xCELLigence immunotherapy software, quantitative parameters such as KT50 (the amount of time it takes to kill 50% of the target tumor cells) and % cytolysis are calculated and used for comparing the relative efficacy of different reagents. In summary, our results demonstrate the xCELLigence platform to be well suited for potency assays, providing quantitative assessment with high reproducibility and a greatly simplified work flow.

In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of DNA repair pathways via their tolerance/ intolerance of nicks containing mismatched base pairs. Although human DNA ligase I (Okazaki fragment processing) and the human DNA ligase III/XRCC1 complex (general DNA repair) have been shown to be relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex has not been studied in this regard. Ligase IV/XRCC4 is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway. During the repair of DSBs generated by chemical/physical damage as well as the repair of the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, a capacity for ligating nicks containing mismatched base pairs would appear to be advantageous. Herein we examine whether ligase IV/XRCC4 can contribute a mismatched nick ligation activity to NHEJ. Toward this end, we (i) describe an E. coli-based coexpression system that provides relatively high yields of the ligase IV/XRCC4 complex, (ii) describe a unique rate-limiting step, which has bearing on how the complex is assayed, (iii) specifically analyze how XRCC4 influences ligase IV catalysis and substrate specificity, and (iv) probe the mismatch tolerance/intolerance of DNA ligase IV/XRCC4 via quantitative in Vitro kinetic analyses. Analogous to most other DNA ligases, ligase IV/XRCC4 is shown to be fairly intolerant of nicks containing mismatched base pairs. These results are discussed in light of the biological roles of NHEJ.In addition to sealing broken DNA back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of the repair pathways they are involved in. This is well illustrated by the example of mammalian base excision repair (BER 1 ), where nicks containing 3′-OH mismatched base pairs, resulting from aberrant gap-filling by DNA polymerase (Pol ), are sealed inefficiently by the DNA ligase III/XRCC1 complex (1). This delay in mismatch ligation is expected to provide a greater window of opportunity for nick editing by the 3′ f 5′ exonuclease activity of APE1 (1, 2), the predominant mammalian AP endonuclease (APE).Though a large body of qualitative and quantitative data suggests that DNA ligases from a broad spectrum of organisms are intolerant of non-Watson-Crick base pairing schemes at the 3′-OH terminus of a nick (3 and references therein), our recent analysis of the African swine fever virus (ASFV)-encoded DNA ligase demonstrated that analogous to the recently discovered error-prone DNA polymerases, there are exceptions to this rule (3). The ASFV DNA ligase is not only tolerant of numerous 3′ mismatched base pairing schemes but also actually displays higher catalytic efficiency for sealing a 3′ C:T 2 mismatch than it does for sealing nicks containing Watson-Crick base pairs. Whether the errortolerance of t...

We previously demonstrated that the DNA repair system encoded by the African swine fever virus (ASFV) is both extremely error-prone during the single-nucleotide gap-filling step (catalyzed by ASFV DNA Polymerase X) and extremely error-tolerant during the nick-sealing step (catalyzed by ASFV DNA ligase). On the basis of these findings we have suggested that at least some of the diversity known to exist among ASFV isolates may be a consequence of mutagenic DNA repairwherein damaged nucleotides are replaced with undamaged but incorrect nucleotides by Pol X and the resultant mismatched nicks are sealed by ASFV DNA ligase. Recently, this hypothesis appeared to be discredited by Salas and coworkers [J. Mol. Biol. 2003, 326, 1403-1412 who reported the fidelity of Pol X to be, on average, two orders of magnitude higher than what we previously published. In an effort to address this discrepancy and provide a definitive conclusion about the fidelity of Pol X, herein we examine the fidelity of Pol X-catalyzed single-nucleotide gap-filling in both the steady state and the pre-steady state under a diverse array of assay conditions (varying pH and ionic strength) and within different DNA sequence contexts. These studies corroborate our previously published data (demonstrating the low-fidelity of Pol X to be independent of assay condition/sequence context), do not reproduce the data of Salas et al., and therefore confirm Pol X to be one of the most errorprone polymerases known. These results are discussed in light of ASFV biology and the mutagenic DNA repair hypothesis described above.ASFV 1 causes a disease of varying mortality [depending on the particular isolate (1)] in domestic pigs in Africa, the Iberian Peninsula, and the Caribbean (2). Possessing a large [168-189 kb (3)] double-stranded DNA genome encoding 151 proteins (4), ASFV is one of the most complex viruses known. In its target cells, marcrophages and monocytes (5), ASFV utilizes the host cell nucleus during an early phase of viral DNA synthesis but appears to complete the replication/assembly of its genome in cytoplasmic/perinuclear viral factories (6 -8). Consistent with the latter intracellular location, ASFV encodes its own replicative polymerase in addition to a minimalist DNA repair system consisting of an AP endonuclease (APE), a repair polymerase (Pol X), and an ATP-dependent DNA ligase (4). While this tripartite repair system would appear to have been retained for the purpose of processing spontaneously 2 generated apurinic/apyrimidinic (AP) sites and/or reactive oxygen species (ROS)-induced singlestrand † This work was supported by NIH Grant GM43268. B.J.L. was supported in part by a predoctoral NIH CBIP fellowship (2T32 GM08512).*To whom correspondence should be addressed at the Department of Chemistry [phone: (614) breaks in the viral genome (9), the unique substrate specificities of both Pol X and ASFV DNA ligase have led us to hypothesize that this "repair" system may have subsequently evolved a secondary function, that of viral genome muta...

: We recently demonstrated that African swine fever virus DNA polymerase X (Pol X) is extremely error-prone during single-nucleotide gap-filling and that the downstream ASFV DNA ligase seals 3′ mismatched nicks with high efficiency. To further assess the credence of our hypothesis that these proteins may promote viral diversification by functioning within the context of an aberrant DNA repair pathway, herein we characterize the third protein expected to function in this system, a putative AP endonuclease (APE). Assays of the purified protein using oligonucleotide substrates unequivocally establish canonical APE activity, 3′-phosphatase and 3′-phosphodiesterase activities (in the context of a singlenucleotide gap), 3′ f 5′ exonuclease activity (in the context of a nick), and nucleotide incision repair activity against 5,6-dihydrothymine. The 3′ f 5′ exonuclease activity is shown to be highly dependent upon the identity of the nascent 3′ base pair and to be inhibited when 2-deoxyribose-5-phosphate, rather than phosphate, constitutes the 5′ moiety of the nick. ASFV APE retains activity when assayed in the presence of EDTA but is inactivated by incubation with 1,10-phenanthroline in the absence of a substrate, suggesting that it is an endonuclease IV homologue possessing intrinsic metal cofactors. The activities of ASFV APE, when considered alongside those of Pol X and ASFV DNA ligase, provide an enhanced understanding of (i) the types of damage that are likely to be sustained by the viral genome and (ii) the mechanisms by which the minimalist ASFV DNA repair pathway, consisting of just these three proteins, contributes to the fitness of the virus.Apurinic/apyrimidinic (AP) 1 sites, generated either by DNA glycosylase-mediated or spontaneous base loss, can be both mutagenic (1, 2) and cytotoxic (3) and are among the most abundant types of damage found in DNA (4). Although they can be processed by AP lyases, which cleave 3′ to the lesion by a -elimination mechanism to generate a single-nucleotide gap flanked by 5′-phosphate and a 3′-polymerase-blocking R, -unsaturated aldehyde (5-7), AP sites are likely to be processed predominantly by AP endonucleases (8), which catalyze hydrolysis of the sugarphosphate backbone 5′ of the lesion to generate a singlenucleotide gap flanked by a polymerase-usable 3′-hydroxyl and 5′-2-deoxyribose-5-phosphate (5′-dRP) (7). Two families of APEs have been identified to date; these consist of proteins with homology to Escherichia coli exonuclease III 2 and proteins with homology to E. coli endonuclease IV. While the reaction that these proteins catalyze is identical, they differ dramatically in both tertiary structure and mechanisms of catalysis (9). Whereas exonuclease III displays a fourlayered R, -sandwich fold and employs a single readily dissociatable magnesium ion (9, 10), endonuclease IV is an R 8 8 TIM barrel that utilizes three tightly bound zinc ions (9,11).In addition to their APE activities, both exonuclease III and endonuclease IV (and their respective homologues) pos...