Double-stranded DNA Ends Research Articles

Replication-transcription conflicts trigger extensive DNA degradation in Escherichia coli cells lacking RecBCD

Bacterial chromosome duplication is initiated at a single origin (oriC). Two forks are assembled and proceed in opposite directions with high speed and processivity until they fuse and terminate in a specialised area opposite to oriC. Proceeding forks are often blocked by tightly-bound protein-DNA complexes, topological strain or various DNA lesions. In Escherichia coli the RecBCD protein complex is a key player in the processing of double-stranded DNA (dsDNA) ends. It has important roles in the repair of dsDNA breaks and the restart of forks stalled at sites of replication-transcription conflicts. In addition, ΔrecB cells show substantial amounts of DNA degradation in the termination area. In this study we show that head-on encounters of replication and transcription at a highly-transcribed rrn operon expose fork structures to degradation by nucleases such as SbcCD. SbcCD is also mostly responsible for the degradation in the termination area of ΔrecB cells. However, additional processes exacerbate degradation specifically in this location. Replication profiles from ΔrecB cells in which the chromosome is linearized at two different locations highlight that the location of replication termination can have some impact on the degradation observed. Our data improve our understanding of the role of RecBCD at sites of replication-transcription conflicts as well as the final stages of chromosome duplication. However, they also highlight that current models are insufficient and cannot explain all the molecular details in cells lacking RecBCD.

Abstract 2628: Synthetic lethal targeting of ATR in alternative lengthening of telomeres-dependent rhabdomyosarcoma

Abstract Synthetic lethal dependencies have recently emerged as tumor-specific vulnerabilities which provide broad therapeutic windows and have successfully been used for rational therapeutic target discovery. Despite intense international efforts over many decades, cure rates for patients with high-risk rhabdomyosarcoma remain dismal. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. Telomere maintenance is a hallmark of cancer and requires either activation of telomerase (TERT) or alternative lengthening of telomeres (ALT). ALT is active in a large subset of rhabdomyosarcomas and not active in healthy tissues suggesting it is a unique, tumor-specific process. ALT has recently been shown to depend on DNA damage response activation through the ATR kinase, and studies in osteosarcoma have revealed that ALT renders cancer cells hypersensitive to ATR inhibition. This defines ALT as a tumor-specific synthetic lethal determinant for susceptibility to ATR inhibition. Consistent with previous reports, we observed that ALT-dependent rhabdomyosarcoma cells are hypersensitive to ATR inhibition via AZD6738, a small molecule ATR inhibitor currently being tested in clinical trials. Intriguingly, we found that treatment with ATR inhibitors not only induced DNA damage but also resulted in aneuploidy and subsequent apoptosis in ALT-dependent rhabdomyosarcoma cells, suggesting loss of telomere protection and inadequate chromosomal segregation occur during mitosis upon ATR inhibition. To test this, we measured unrepaired DNA double-strand break formation using TUNEL. ALT-dependent cells, but not TERT-dependent cells, showed increased TUNEL labeling, consistent with an increase in double-stranded DNA breaks and unprotected DNA ends. Consistent with reduced telomere maintenance, ATR inhibition significantly reduced the number of C-circles in rhabdomyosarcoma cells relying on ALT. Furthermore, AZD6738 treatment reduced phosphorylation of known ATR targets, and differentially phosphorylated peptides were enriched for proteins involved in alternative lengthening of telomeres as measured using SILAC labeling and LC-MS/MS phospho-proteomic analysis. Finally, treatment with AZD6738 reduced tumor burden in patient-derived primary rhabdomyosarcoma xenografts, which was potentiated by combined treatment with cisplatin. Our findings delineate a therapeutically actionable DNA repair dependency induced at least in part by ALT and may lead directly to clinical translation of ATR inhibitors for therapy of refractory rhabdomyosarcoma. Citation Format: Heathcliff Dorado García, Jennifer von Stebut, Ian MacArthur, Koshi Imami, Natalie Timme, Kerstin Schoenbeck, Annabell Szymansky, Georg Seifert, Patrick Hundsdoerfer, Andrej Lissat, Matthias Selbach, Angelika Eggert, Johannes Schulte, Anton Henssen. Synthetic lethal targeting of ATR in alternative lengthening of telomeres-dependent rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2628.

Replication Fork Breakage and Restart in Escherichia coli

In all organisms, replication impairments are an important source of genome rearrangements, mainly because of the formation of double-stranded DNA (dsDNA) ends at inactivated replication forks. Three reactions for the formation of dsDNA ends at replication forks were originally described for Escherichia coli and became seminal models for all organisms: the encounter of replication forks with preexisting single-stranded DNA (ssDNA) interruptions, replication fork reversal, and head-to-tail collisions of successive replication rounds. Here, we first review the experimental evidence that now allows us to know when, where, and how these three different reactions occur in E. coli. Next, we recall our recent studies showing that in wild-type E. coli, spontaneous replication fork breakage occurs in 18% of cells at each generation. We propose that it results from the replication of preexisting nicks or gaps, since it does not involve replication fork reversal or head-to-tail fork collisions. In the recB mutant, deficient for double-strand break (DSB) repair, fork breakage triggers DSBs in the chromosome terminus during cell division, a reaction that is heritable for several generations. Finally, we recapitulate several observations suggesting that restart from intact inactivated replication forks and restart from recombination intermediates require different sets of enzymatic activities. The finding that 18% of cells suffer replication fork breakage suggests that DNA remains intact at most inactivated forks. Similarly, only 18% of cells need the helicase loader for replication restart, which leads us to speculate that the replicative helicase remains on DNA at intact inactivated replication forks and is reactivated by the replication restart proteins.

DNA-bound ubiquitin coordinates repair

DNA Repair Ubiquitylation is a posttranslational modification that reversibly alters various protein properties. Liu et al. discovered that Lys63-linked polyubiquitin chains bound to the free ends of double-stranded DNA, bridged the broken ends of DNA, and recruited repair proteins. Ubiquitins with DNA-binding motif mutations were found in several types of tumors. When expressed in cultured cells, these mutant ubiquitins impaired the cellular response to DNA-damaging agents, suggesting that it might be possible to therapeutically exploit these mutations in some cancer patients. Sci. Signal. 11 , eaar8133 (2018).

MacroD1 Is a Promiscuous ADP-Ribosyl Hydrolase Localized to Mitochondria.

MacroD1 is a macrodomain containing protein that has mono-ADP-ribose hydrolase enzymatic activity toward several ADP-ribose adducts. Dysregulation of MacroD1 expression has been shown to be associated with the pathogenesis of several forms of cancer. To date, the physiological functions and sub-cellular localization of MacroD1 are unclear. Previous studies have described nuclear and cytosolic functions of MacroD1. However, in this study we show that endogenous MacroD1 protein is highly enriched within mitochondria. We also show that MacroD1 is highly expressed in human and mouse skeletal muscle. Furthermore, we show that MacroD1 can efficiently remove ADP-ribose from 5′ and 3′-phosphorylated double stranded DNA adducts in vitro. Overall, we have shown that MacroD1 is a mitochondrial protein with promiscuous enzymatic activity that can target the ester bonds of ADP-ribosylated phosphorylated double-stranded DNA ends. These findings have exciting implications for MacroD1 and ADP-ribosylation within the regulation of mitochondrial function and DNA-damage in vivo.

Sgs1 Binding to Rad51 Stimulates Homology-Directed DNA Repair in Saccharomyces cerevisiae.

Accurate repair of DNA breaks is essential to maintain genome integrity and cellular fitness. Sgs1, the sole member of the RecQ family of DNA helicases in Saccharomyces cerevisiae, is important for both early and late stages of homology-dependent repair. Its large number of physical and genetic interactions with DNA recombination, repair, and replication factors has established Sgs1 as a key player in the maintenance of genome integrity. To determine the significance of Sgs1 binding to the strand-exchange factor Rad51, we have identified a single amino acid change at the C-terminal of the helicase core of Sgs1 that disrupts Rad51 binding. In contrast to an SGS1 deletion or a helicase-defective sgs1 allele, this new separation-of-function allele, sgs1-FD, does not cause DNA damage hypersensitivity or genome instability, but exhibits negative and positive genetic interactions with sae2Δ, mre11Δ, exo1Δ, srs2Δ, rrm3Δ, and pol32Δ that are distinct from those of known sgs1 mutants. Our findings suggest that the Sgs1-Rad51 interaction stimulates homologous recombination (HR). However, unlike sgs1 mutations, which impair the resection of DNA double-strand ends, negative genetic interactions of the sgs1-FD allele are not suppressed by YKU70 deletion. We propose that the Sgs1-Rad51 interaction stimulates HR by facilitating the formation of the presynaptic Rad51 filament, possibly by Sgs1 competing with single-stranded DNA for replication protein A binding during resection.

Efficient N-tailing of blunt DNA ends by Moloney murine leukemia virus reverse transcriptase

Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is a widely used enzyme for cDNA synthesis. Here we show that MMLV-RT has a strong template-independent polymerase activity using blunt DNA ends as substrate that generates 3′ overhangs of A, C, G, or T. Nucleotides were appended efficiently in the order A > G > T > C, and tail lengths varied from 4 to 5, 2 to 7, 2 to 4, and 2 to 3 for A, C, G, and T, respectively. The activity was so strong that nearly all our test DNA ends were appended with at least one A, C, G, or T. The N-tailing activity of MMLV-RT was enhanced in the presence of Mn2+, and the G-, C-, and T-tailing activities were further enhanced by dCMP, dGMP, and dAMP, respectively. This is the first report of an enzymatic activity that almost thoroughly appends two or more As, or one or more Cs, Gs, or Ts to the 3′ end of double-stranded DNA, which would enable exhaustive analysis of DNA samples. The N-tailing activity of MMLV-RT is potentially useful in many biotechnological applications.

DNA flap creation by the RarA/MgsA protein of Escherichia coli.

We identify a novel activity of the RarA (also MgsA) protein of Escherichia coli, demonstrating that this protein functions at DNA ends to generate flaps. A AAA+ ATPase in the clamp loader clade, RarA protein is part of a highly conserved family of DNA metabolism proteins. We demonstrate that RarA binds to double-stranded DNA in its ATP-bound state and single-stranded DNA in its apo state. RarA ATPase activity is stimulated by single-stranded DNA gaps and double-stranded DNA ends. At these double-stranded DNA ends, RarA couples the energy of ATP binding and hydrolysis to separating the strands of duplex DNA, creating flaps. We hypothesize that the creation of a flap at the site of a leading strand discontinuity could, in principle, allow DnaB and the associated replisome to continue DNA synthesis without impediment, with leading strand re-priming by DnaG. Replication forks could thus be rescued in a manner that does not involve replisome disassembly or reassembly, albeit with loss of one of the two chromosomal products of a replication cycle.

DNA-PK and P38 MAPK: A Kinase Collusion in Alzheimer's Disease?

The pathogenesis of Alzheimer's disease (AD), characterized by prevalent neuronal death and extracellular deposit of amyloid plaques, is poorly understood. DNA lesions downstream of reduced DNA repair ability have been reported in AD brains. Neurons predominantly use a mechanism to repair double-strand DNA breaks (DSB), which is non-homologous end joining (NHEJ). NHEJ requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK is a holoenzyme comprising the p460 kD catalytic subunit (DNA-PKcs) and its activator Ku, a heterodimer of p86 and p70 subunits. Ku first binds and then recruits DNA-PKcs to double-stranded DNA ends before NHEJ process begins. Studies have shown reduced NHEJ activity as well as DNA-PKcs and Ku protein levels in AD brains suggesting possible contribution of unrepaired DSB to AD development. However, normal aging brains also show reduced DNA-PKcs and Ku levels thus challenging the notion of any direct link between NHEJ and AD. Another kinase, p38 MAPK is induced by various DNA damaging agents and DSB itself. Increased DNA damage with aging could induce p38 MAPK and its induction may be sustained when DNA repair is compromised in the brain with reduced DNA-PK activity. Combined, these two events may potentially set the stage for an awry nervous system approaching AD.

λ Recombination and Recombineering.

The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.

DNA-PK Deficiency in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by neuronal death with an accumulaton of intra-cellular neurofibrillary tangles (NFT) and extracellular amyloid plaques. Reduced DNA repair ability has been reported in AD brains. In neurons, the predominant mechanism to repair double-strand DNA breaks (DSB) is non-homologous end joining (NHEJ) that requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK is a holoenzyme comprising the p460 kD DNA-PK catalytic subunit (DNA-PKcs) and its activator Ku, a heterodimer of p86 (Ku80) and p70 (Ku70) subunits. Upon binding to double-stranded DNA ends, Ku recruits DNA-PKcs to process NHEJ. In AD brains, reduced NHEJ activity as well as DNA-PKcs and Ku protein levels have been shown. Normal aging brains also show a reduction in both DNA-PKcs and Ku levels questioning a direct link between NHEJ ability and AD, and suggesting additional players/events in AD pathogenesis. Deficiency of Ku80, a somatostatin receptor, can disrupt somatostatin signaling thus inducing amyloid beta (Aβ) generation, which in turn can potentiate DNA-PKcs degradation and consequently loss of NHEJ activity, an additional step negatively affecting DSB repair. Trigger of these two different pathways culminating in genome instability may differentiate the outcomes between AD and normal aging.

Regulation of genetic flux between bacteria by restriction–modification systems

Restriction-modification (R-M) systems are often regarded as bacteria's innate immune systems, protecting cells from infection by mobile genetic elements (MGEs). Their diversification has been recently associated with the emergence of particularly virulent lineages. However, we have previously found more R-M systems in genomes carrying more MGEs. Furthermore, it has been suggested that R-M systems might favor genetic transfer by producing recombinogenic double-stranded DNA ends. To test whether R-M systems favor or disfavor genetic exchanges, we analyzed their frequency with respect to the inferred events of homologous recombination and horizontal gene transfer within 79 bacterial species. Genetic exchanges were more frequent in bacteria with larger genomes and in those encoding more R-M systems. We created a recognition target motif predictor for Type II R-M systems that identifies genomes encoding systems with similar restriction sites. We found more genetic exchanges between these genomes, independently of their evolutionary distance. Our results reconcile previous studies by showing that R-M systems are more abundant in promiscuous species, wherein they establish preferential paths of genetic exchange within and between lineages with cognate R-M systems. Because the repertoire and/or specificity of R-M systems in bacterial lineages vary quickly, the preferential fluxes of genetic transfer within species are expected to constantly change, producing time-dependent networks of gene transfer.

A novel fluorescent aptasensor based on silica nanoparticles, PicoGreen and exonuclease III as a signal amplification method for ultrasensitive detection of myoglobin

Measurement of myoglobin (Mb) in human blood serum is of great interest for quick diagnosis of acute myocardial infarction (AMI). In this study, a novel fluorescent aptasensor was designed for ultrasensitive and selective detection of Mb, based on target-induced high fluorescence intensity, complementary strand of aptamer (CS), PicoGreen (PG) dye, exonuclease III (Exo III) and silica nanoparticles coated with streptavidin (SNPs-Streptavidin). The developed aptasensor obtains characteristics of SNPs as enhancers of fluorescence intensity, Exo III as an enzyme which selectively digests the 3'-end of double-stranded DNA (dsDNA), PG as a fluorescent dye which could selectively bind to dsDNA and high selectivity and sensitivity of aptamer (Apt) toward its target. In the absence of Mb, no free CS remains in the environment of SNPs-Streptavidin, resulting in a weak fluorescence emission. In the present of Mb, dsDNA-modified SNPs-Streptavidin complex forms, leading to a very strong fluorescence emission. The developed fluorescent aptasensor exhibited high specificity toward Mb with a limit of detection (LOD) as low as 52 pM. In addition, the designed fluorescent aptasensor was efficiently used to detect Mb in human serum.

Sequence-Dependent Fluorescence of Cy3- and Cy5-Labeled Double-Stranded DNA.

The fluorescent intensity of Cy3 and Cy5 dyes is strongly dependent on the nucleobase sequence of the labeled oligonucleotides. Sequence-dependent fluorescence may significantly influence the data obtained from many common experimental methods based on fluorescence detection of nucleic acids, such as sequencing, PCR, FRET, and FISH. To quantify sequence dependent fluorescence, we have measured the fluorescence intensity of Cy3 and Cy5 bound to the 5′ end of all 1024 possible double-stranded DNA 5mers. The fluorescence intensity was also determined for these dyes bound to the 5′ end of fixed-sequence double-stranded DNA with a variable sequence 3′ overhang adjacent to the dye. The labeled DNA oligonucleotides were made using light-directed, in situ microarray synthesis. The results indicate that the fluorescence intensity of both dyes is sensitive to all five bases or base pairs, that the sequence dependence is stronger for double- (vs single-) stranded DNA, and that the dyes are sensitive to both the adjacent dsDNA sequence and the 3′-ssDNA overhang. Purine-rich sequences result in higher fluorescence. The results can be used to estimate measurement error in experiments with fluorescent-labeled DNA, as well as to optimize the fluorescent signal by considering the nucleobase environment of the labeling cyanine dye.

The Cohesin Complex Prevents the End Joining of Distant DNA Double-Strand Ends

The end joining of distant DNA double-strand ends (DSEs) can produce potentially deleterious rearrangements. We show that depletion of cohesion complex proteins specifically stimulates the end joining (both C-NHEJ and A-EJ) of distant, but not close, I-SceI-induced DSEs in S/G2 phases. At the genome level, whole-exome sequencing showed that ablation of RAD21 or Sororin produces large chromosomal rearrangements (translocation, duplication, deletion). Moreover, cytogenetic analysis showed that RAD21 silencing leads to the formation of chromosome fusions synergistically with replication stress, which generates distant single-ended DSEs. These data reveal a role for the cohesin complex in protecting against genome rearrangements arising from the ligation of distant DSEs in S/G2 phases (both long-range DSEs and those that are only a few kilobases apart), while keeping end joining fully active for close DSEs. Therefore, this role likely involves limitation of DSE motility specifically in S phase, rather than inhibition of the end-joining machinery itself.

The linear plastid chromosomes of maize: terminal sequences, structures, and implications for DNA replication.

The structure of a chromosomal DNA molecule may influence the way in which it is replicated and inherited. For decades plastid DNA (ptDNA) was believed to be circular, with breakage invoked to explain linear forms found upon extraction from the cell. Recent evidence indicates that ptDNA in vivo consists of linear molecules with discrete termini, although these ends were not characterized. We report the sequences of two terminal regions, End1 and End2, for maize (Zea mays L.) ptDNA. We describe structural features of these terminal regions and similarities found in other plant ptDNAs. The terminal sequences are within inverted repeat regions (leading to four genomic isomers) and adjacent to origins of replication. Conceptually, stem-loop structures may be formed following melting of the double-stranded DNA ends. Exonuclease digestion indicates that the ends in maize are unobstructed, but tobacco (Nicotiana tabacum L.) ends may have a 5'-protein. If the terminal structure of ptDNA molecules influences the retention of ptDNA, the unprotected molecular ends in mature leaves of maize may be more susceptible to degradation in vivo than the protected ends in tobacco. The terminal sequences and cumulative GC skew profiles are nearly identical for maize, wheat (Triticum aestivum L.) and rice (Oryza sativa L.), with less similarity among other plants. The linear structure is now confirmed for maize ptDNA and inferred for other plants and suggests a virus-like recombination-dependent replication mechanism for ptDNA. Plastid transformation vectors containing the terminal sequences may increase the chances of success in generating transplastomic cereals.

A Structure-Activity Analysis for Probing the Mechanism of Processive Double-Stranded DNA Digestion by λ Exonuclease Trimers.

λ exonuclease (λexo) is an ATP-independent 5'-to-3' exonuclease that binds to double-stranded DNA (dsDNA) ends and processively digests the 5'-strand into mononucleotides. The crystal structure of λexo revealed that the enzyme forms a ring-shaped homotrimer with a central funnel-shaped channel for tracking along the DNA. On the basis of this structure, it was proposed that dsDNA enters the open end of the channel, the 5'-strand is digested at one of the three active sites, and the 3'-strand passes through the narrow end of the channel to emerge out the back. This model was largely confirmed by the structure of the λexo-DNA complex, which further revealed that the enzyme unwinds the DNA by 2 bp prior to cleavage, to thread the 5'-end of the DNA into the active site. On the basis of this structure, an "electrostatic ratchet" model was proposed, in which the enzyme uses a hydrophobic wedge to insert into the base pairs to unwind the DNA, a two-metal mechanism for nucleotide hydrolysis, a positively charged pocket to bind to the terminal 5'-phosphate generated after each round of cleavage, and an arginine residue (Arg-45) to bind to the minor groove of the downstream end of the DNA. To test this model, in this study we have determined the effects of 11 structure-based mutations in λexo on DNA binding and exonuclease activities in vitro, and on DNA recombination in vivo. The results are largely consistent with the model for the mechanism that was proposed on the basis of the structure and provide new insights into the roles of particular residues of the protein in promoting the reaction. In particular, a key role for Arg-45 in DNA binding is revealed.

Mutant poisoning demonstrates a nonsequential mechanism for digestion of double-stranded DNA by λ exonuclease trimers.

λ Exonuclease (λexo) is a highly processive 5'-3' exonuclease that binds double-stranded DNA (dsDNA) ends and digests the 5'-strand into mononucleotides. The enzyme forms a toroidal homotrimer with a central tapered channel for tracking along the DNA. During catalysis, dsDNA enters the open end of the channel, and the 5'-strand is digested at one of the three active sites. It is currently not known if λexo uses a sequential mechanism, in which the DNA moves from one active site to the next around the trimer for each round of catalysis or a nonsequential mechanism, in which the DNA locks onto a single active site for multiple rounds. To understand how λexo uses its three active sites, we used a mutant poisoning approach, in which a 6xHis-tagged K131A inactive mutant of λexo was mixed with untagged wild type (WT) to form hybrid trimers. Nickel-spin pull-down analysis confirmed complete subunit exchange after 1 h at 37 °C. Exonuclease assays revealed an approximately linear decrease in activity with increasing fraction of mutant, as expected for a nonsequential mechanism. By fitting the observed rates of digestion to a simple mathematical model, the individual rates of the two hybrid species of trimer were determined. This analysis showed that trimers containing only one or two WT subunits contribute significantly to the observed activity, in further agreement with a nonsequential mechanism. Finally, purification of hybrid trimer mixtures by Ni-spin chromatography, to remove the contribution from fully WT trimers, also resulted in significant levels of activity, again consistent with a nonsequential mechanism.

Crystal structure of λ exonuclease in complex with DNA and Ca(2+).

Bacteriophage λ exonuclease (λexo) is a ring-shaped homotrimer that resects double-stranded DNA ends in the 5'-3' direction to generate a long 3'-overhang that is a substrate for recombination. λexo is a member of the type II restriction endonuclease-like superfamily of proteins that use a Mg(2+)-dependent mechanism for nucleotide cleavage. A previous structure of λexo in complex with DNA and Mg(2+) was determined using a nuclease defective K131A variant to trap a stable complex. This structure revealed the detailed coordination of the two active site Mg(2+) ions but did not show the interactions involving the side chain of the conserved active site Lys-131 residue. Here, we have determined the crystal structure of wild-type (WT) λexo in complex with the same DNA substrate, but in the presence of Ca(2+) instead of Mg(2+). Surprisingly, there is only one Ca(2+) bound in the active site, near the position of Mg(A) in the structure with Mg(2+). The scissile phosphate is displaced by 2.2 Å relative to its position in the structure with Mg(2+), and the network of interactions involving the attacking water molecule is broken. Thus, the structure does not represent a catalytic configuration. However, the crystal structure does show clear electron density for the side chain of Lys-131, which is held in place by interactions with Gln-157 and Glu-129. By combining the K131A-Mg(2+) and WT-Ca(2+) structures, we constructed a composite model to show the likely interactions of Lys-131 during catalysis. The implications with regard to the catalytic mechanism are discussed.

The Ku heterodimer: function in DNA repair and beyond.

Ku is an abundant, highly conserved DNA binding protein found in both prokaryotes and eukaryotes that plays essential roles in the maintenance of genome integrity. In eukaryotes, Ku is a heterodimer comprised of two subunits, Ku70 and Ku80, that is best characterized for its central role as the initial DNA end binding factor in the "classical" non-homologous end joining (C-NHEJ) pathway, the main DNA double-strand break (DSB) repair pathway in mammals. Ku binds double-stranded DNA ends with high affinity in a sequence-independent manner through a central ring formed by the intertwined strands of the Ku70 and Ku80 subunits. At the break, Ku directly and indirectly interacts with several C-NHEJ factors and processing enzymes, serving as the scaffold for the entire DNA repair complex. There is also evidence that Ku is involved in signaling to the DNA damage response (DDR) machinery to modulate the activation of cell cycle checkpoints and the activation of apoptosis. Interestingly, Ku is also associated with telomeres, where, paradoxically to its DNA end-joining functions, it protects the telomere ends from being recognized as DSBs, thereby preventing their recombination and degradation. Ku, together with the silent information regulator (Sir) complex is also required for transcriptional silencing through telomere position effect (TPE). How Ku associates with telomeres, whether it is through direct DNA binding, or through protein-protein interactions with other telomere bound factors remains to be determined. Ku is central to the protection of organisms through its participation in C-NHEJ to repair DSBs generated during V(D)J recombination, a process that is indispensable for the establishment of the immune response. Ku also functions to prevent tumorigenesis and senescence since Ku-deficient mice show increased cancer incidence and early onset of aging. Overall, Ku function is critical to the maintenance of genomic integrity and to proper cellular and organismal development.