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Recombinational analysis Genetic recombination Gene 39 Gene 61 Topoisomerase II Primase a b s t r a c t The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i × j) and three-factor (i k × j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1 × i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1–i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1 × i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1 × i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII+ recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombinationproficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss-branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced sitespecific recombination, the topoisomerase and primase mutants demonstrated about 3–8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants.

The actin-binding and bundling protein, plastin 3 (PLS3), was identified as a protective modifier of spinal muscular atrophy (SMA) in some patient populations and as a disease modifier in animal models of SMA. How it functions in this process, however, is not known. Because PLS3 is an actin-binding/bundling protein, we hypothesized it would likely act via modification of the actin cytoskeleton in axons and neuromuscular junctions to protect motoneurons in SMA. To test this, we examined the ability of other known actin cytoskeleton organizing proteins to modify motor axon outgrowth phenotypes in an smn morphant zebrafish model of SMA. While PLS3 can fully compensate for low levels of smn, cofilin 1, profilin 2 and α-actinin 1 did not affect smn morphant motor axon outgrowth. To determine how PLS3 functions in SMA, we generated deletion constructs of conserved PLS3 structural domains. The EF hands were essential for PLS3 rescue of smn morphant phenotypes, and mutation of the Ca(2+)-binding residues within the EF hands resulted in a complete loss of PLS3 rescue. These results indicate that Ca(2+) regulation is essential for the function of PLS3 in motor axons. Remarkably, PLS3 mutants lacking both actin-binding domains were still able to rescue motor axons in smn morphants, although not as well as full-length PLS3. Therefore, PLS3 function in this process may have an actin-independent component.

Limb-girdle muscular dystrophy (LGMD) 2H is a slowly progressive condition characterized by proximal weakness, atrophy, and mildly to moderately raised levels of creatine kinase. Facial weakness, scapular winging, hypertrophied calves, and Achilles tendon contractions are not uncommon and the age of onset ranges between the first and fourth decade. LGMD2H was originally described in the Hutterite population that resides in central Canada and the Dakotas of the USA. LGMD2H was mapped to a specific mutation in the TRIM32 gene and it has subsequently been shown that the same mutation also results in the "sarcotubular myopathy" syndrome, which was described histopathologically. TRIM32 appears to be an E3 ubiquitin ligase, containing the tripartite motif common to this family of proteins (RING finger, B-box, coiled-coil). A few substrates have been identified, including actin and dysbindin. Recent studies have identified additional mutations in the C-terminal region of TRIM32 that result in a dystrophic myopathy. Although TRIM32 appears to be expressed ubiquitously, it is still not clear why certain mutations of TRIM32 would result in a phenotype relatively restricted to skeletal muscle. A mutation in the B-box region of TRIM32 has also been shown to result in a more pleiotropic disorder, Bardet-Biedl Syndrome (BBS11). This disorder is associated with obesity, retinopathy, diabetes, polydactyly, renal abnormalities, learning disability, and hypogenitalism. It is likely that C-terminal mutations in TRIM32 affect the ability of muscle proteins to be degraded by the ubiquitin-proteasome pathway.

Limb-girdle muscular dystrophy (LGMD) 2H is a slowly progressive condition characterized by proximal weakness, atrophy, and mildly to moderately raised levels of creatine kinase. Facial weakness, scapular winging, hypertrophied calves, and Achilles tendon contractions are not uncommon and the age of onset ranges between the first and fourth decade. LGMD2H was originally described in the Hutterite population that resides in central Canada and the Dakotas of the USA. LGMD2H was mapped to a specific mutation in the TRIM32 gene and it has subsequently been shown that the same mutation also results in the "sarcotubular myopathy" syndrome, which was described histopathologically. TRIM32 appears to be an E3 ubiquitin ligase, containing the tripartite motif common to this family of proteins (RING finger, B-box, coiled-coil). A few substrates have been identified, including actin and dysbindin. Recent studies have identified additional mutations in the C-terminal region of TRIM32 that result in a dystrophic myopathy. Although TRIM32 appears to be expressed ubiquitously, it is still not clear why certain mutations of TRIM32 would result in a phenotype relatively restricted to skeletal muscle. A mutation in the B-box region of TRIM32 has also been shown to result in a more pleiotropic disorder, Bardet-Biedl Syndrome (BBS11). This disorder is associated with obesity, retinopathy, diabetes, polydactyly, renal abnormalities, learning disability, and hypogenitalism. It is likely that C-terminal mutations in TRIM32 affect the ability of muscle proteins to be degraded by the ubiquitin-proteasome pathway.

Mutations in the non-lysosomal cysteine protease calpain-3 cause autosomal recessive limb girdle muscular dystrophy. Pathological mechanisms occurring in this disease have not yet been elucidated. Here, we report both morphological and biochemical evidence of mitochondrial abnormalities in calpain-3 knockout (C3KO) muscles, including irregular ultrastructure and distribution of mitochondria. The morphological abnormalities in C3KO muscles are associated with reduced in vivo mitochondrial ATP production as measured by (31)P magnetic resonance spectroscopy. Mitochondrial abnormalities in C3KO muscles also correlate with the presence of oxidative stress; increased protein modification by oxygen free radicals and an elevated concentration of the anti-oxidative enzyme Mn-superoxide dismutase were observed in C3KO muscles. Previously we identified a number of mitochondrial proteins involved in beta-oxidation of fatty acids as potential substrates for calpain-3. In order to determine if the mitochondrial abnormalities resulted from the loss of direct regulation of mitochondrial proteins by calpain-3, we validated the potential substrates that were identified in previous proteomic studies. This analysis showed that the beta-oxidation enzyme, VLCAD, is cleaved by calpain-3 in vitro, but we were not able to confirm that VLCAD is an in vivo substrate for calpain-3. However, the activity of VLCAD was decreased in C3KO mitochondrial fractions compared with wild type, a finding that likely reflects a general mitochondrial dysfunction. Taken together, these data suggest that mitochondrial abnormalities leading to oxidative stress and energy deficit are important pathological features of calpainopathy and possibly represent secondary effects of the absence of calpain-3.

Calpain-3 (CAPN3) is a non-lysosomal cysteine protease that is necessary for normal muscle function, as mutations in CAPN3 result in an autosomal recessive form of limb girdle muscular dystrophy type 2A. To elucidate the biological roles of CAPN3 in skeletal muscle, we performed a search for potential substrates and interacting partners. By yeast-two-hybrid analysis we identified the glycolytic enzyme aldolase A (AldoA) as a binding partner of CAPN3. In co-expression studies CAPN3 degraded AldoA; however, no accumulation of AldoA was observed in total extracts from CAPN3-deficient muscles suggesting that AldoA is not an in vivo substrate of CAPN3. Instead, we found CAPN3 to be necessary for recruitment of AldoA to one specific location, namely the triads, which are structural components of muscle responsible for calcium transport and excitation-contraction coupling. Both aldolase and CAPN3 are present in the triad-enriched fraction and are able to interact with ryanodine receptors (RyR) that form major calcium release channels. Levels of triad-associated AldoA and RyR were decreased in CAPN3-deficient muscles compared with wild-type. Consistent with these observations we found calcium release to be significantly reduced in fibers from CAPN3-deficient muscles. Together, these data suggest that CAPN3 is necessary for the structural integrity of the triad-associated protein complex and that impairment of calcium transport is a phenotypic feature of CAPN3-deficient muscle.

Calpain 3 (CAPN3) is a calcium-dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To explore the physiological function of CAPN3, we compared the proteomes of transgenic mice that overexpress CAPN3 (CAPN3 Tg) and their nontransgenic (non-Tg) counterparts. We first examined known muscular dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known muscular dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to compare and identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of ten possible substrates for CAPN3, classified into two major functional categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyze MLC1 in vitro. These studies are the first to identify possible substrates for CAPN3 in an in vivo system and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.

The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca(2+) overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.

Calpain 3 (CAPN3) belongs to a family of Ca-dependent, nonlysosomal, cysteine proteases that includes both ubiquitously expressed and tissue-specific members (19). CAPN3 (with a molecular mass of 94 kDa) is the major calpain isoform expressed in adult skeletal muscles. Mutations in CAPN3 cause limb girdle muscular dystrophy 2A (LGMD2A), an autosomal recessive muscle disease characterized by progressive atrophy and weakness of the proximal limb muscles (40). We generated CAPN3 knockout (C3KO) mice and showed that these mice develop moderate muscle atrophy and growth deficiencies which are consistent with the human phenotype of LGMD2A. Examination of muscles from adult C3KO mice revealed abnormal sarcomere structure. Furthermore, studies on primary myoblast cultures revealed severe abnormalities during the terminal steps of myogenesis. C3KO myoblasts were able to fuse to form myotubes, but myofibrillogenesis was inhibited (or delayed) as was shown by ultrastructural and biochemical analyses (30). Mechanisms occurring during the final steps of myogenic differentiation (i.e., myoblast fusion and sarcomere assembly) are not well understood in mammals. However, several classes of transmembrane molecules, including members of the cadherin and immunoglobulin families, have been shown to play a role in the fusion process (26). It is likely that the mammalian fusion complex comprises many components with redundant functions, a feature that makes its analysis very difficult. Numerous observations indicate that M-cadherin is involved in the fusion of myogenic cells. For example, synthetic peptides that bind to the extracellular domain of M-cadherin and block homophilic interactions were able to block myoblast fusion in a dose-dependent manner (48). Recently, the importance of M-cadherin for fusion of cultured myoblasts was confirmed by the RNA interference method (11). Moreover, changes in M-cadherin levels led to changes in fusion: downregulation of M-cadherin caused inhibition, while upregulation of M-cadherin caused enhancement of the fusion process (11, 46). Although these data show that M-cadherin is involved in myoblast fusion, mice lacking M-cadherin develop normal skeletal musculature and M-cadherin knockout myogenic cells can fuse normally, suggesting that other molecules can largely compensate for the lack of M-cadherin in vivo (23). Thus, these observations indicate that, even though M-cadherin loss of function does not prevent fusion, perturbation of M-cadherin levels modulates fusion of myogenic cells in vitro, suggesting that M-cadherin plays a role in mediating the fusion process. One of the intracellular components of the cadherin complex is β-catenin, a protein that has been shown to play a dual role in myogenesis. First, a cytoplasmic pool of β-catenin plays a role in Wnt signaling that regulates myogenic fate specification during both embryonic development (14) and adult muscle regeneration (38, 41). β-Catenin also plays a role in later stages of myogenic differentiation as a component of the cadherin cell adhesion complex (32). Shortly after myoblasts are transferred to differentiation medium, β-catenin levels increase and β-catenin is recruited to the cadherin complex. This process has been shown to be crucial for expression of myogenin, a transcription factor that regulates progression towards the terminal stages of myogenic differentiation (18). However, perplexingly, overexpression of β-catenin had an inhibitory effect on myogenesis (18), suggesting that there is a mechanism for regulating the β-catenin pool not only in the cytoplasm, which is a well-known phenomenon in Wnt signaling, but also at the membrane. Recently, a muscle-specific ubiquitin ligase, called Ozz-E3 that regulates specific degradation of β-catenin associated with the cell membrane, was identified (36). Interestingly, accumulation of β-catenin due to null mutation of Ozz-E3 or due to overexpression of a β-catenin fragment that competes for binding to Ozz leads to severe defects in myofibrillogenesis, resulting in the absence of visible sarcomere formation in mutant myotubes. In spite of this in vitro phenotype, Ozz null mice form well-developed muscles, albeit with sarcomere abnormalities. These in vitro and in vivo characteristics are very similar to those which we observed in C3KO mice (30). In this study we further examined the biochemical and morphological features of C3KO myotubes to elucidate a role for CAPN3 in myogenesis. In particular, we investigated the relationship between CAPN3 and the cadherin complex. This study revealed that in addition to defects in myofibrillogenesis, some C3KO myotubes were morphologically abnormal and contained big clusters of myonuclei. Overall, the myonuclear indices of C3KO myotubes and adult myofibers were higher than those in wild-type (WT) myotubes and myofibers. We found that both β-catenin and M-cadherin are substrates for CAPN3 protease and that CAPN3 specifically controls the level of membrane-associated β-catenin and M-cadherin during myogenesis. We hypothesize that persistence of the M-cadherin-β-catenin complex at the membranes of C3KO myotubes causes excessive fusion and delayed myofibrillogenesis in the absence of CAPN3.

Coordination of DNA ends during double-strand break (DSB) repair was studied in crosses of bacteriophage T4 in which DSBs were induced site-specifically by SegC endonuclease in the DNA of only one of the parents. Coupling of the genetic exchanges to the left and to the right of the DSB was measured in the wild-type genetic background as well as in T4 strains bearing mutations in several recombination genes: 47, uvsX, uvsW, 59, 39 and 61. The observed quantitative correlation between the degree of coupling and position of the recombining markers in relation to the DSB point implies that the two variants of the splice/patch-coupling (SPC) pathway, the “sequential SPC” and the “SPC with fork collision”, operate during DSB repair. In the 47 mutant with or without a das suppressor, coupling of the exchanges was greatly reduced, indicating a crucial role of the 47/46 complex in coupling of the genetic exchanges on the two sides of the DSB. From the observed dependence of the apparent coupling on the intracellular ratio of breakable and unbreakable chromosomes in different genetic backgrounds it is inferred that linking of the DNA ends by 47/46 protein is the mechanism that accounts for their concerted action during DSB repair. A mechanism of replicative resolution of D-loop intermediate (RR pathway) is suggested to explain the phenomenology of DSB repair in DNA arrest and uvsW mutants. A “left”–“right” bias in the recombinogenic action of two DNA ends of the broken chromosome was observed which was particularly prominent in the 59 (41-helicase loader) and 39 (topoisomerase) mutants. Phage topoisomerase II (gp39-52-60) is indispensable for growth in the DNA arrest mutants: the doubles 47− 39−, uvsX 39− and 59− 39− are lethal.

Human tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J) are caused by mutations in the giant sarcomeric protein titin (TTN) adjacent to a binding site for the muscle-specific protease calpain 3 (CAPN3). Muscular dystrophy with myositis (mdm) is a recessive mouse mutation with severe and progressive muscular degeneration caused by a deletion in the N2A domain of titin (TTN-N2AΔ83), disrupting a putative binding site for CAPN3. To determine whether the muscular dystrophy in mutant mdm mice is caused by misregulation of CAPN3 activity, genetic crosses with CAPN3 overexpressing transgenic (C3Tg) and CAPN3 knockout (C3KO) mice were generated. Here we report that overexpression of CAPN3 exacerbates the mdm disease, leading to a shorter lifespan and more severe muscular dystrophy. However, in a direct genetic test of CAPN3’s role as a mediator of mdm pathology, C3KO;mdm double mutant mice showed no change in the progression or severity of disease indicating that aberrant CAPN3 activity is not a primary mechanism in this disease. To determine whether we could detect a functional deficit in titin in a non-disease state, we examined the treadmill locomotion of heterozygous +/mdm mice and detected a significant increase in stride time with a concomitant increase in stance time. Interestingly, these altered gait parameters were completely corrected by CAPN3 overexpression in transgenic C3Tg;+/mdm mice, supporting a CAPN3-dependent role for the N2A domain of TTN in the dynamics of muscle contraction.

Trim32 belongs to the tripartite motif (TRIM) protein family, which is characterized by a common domain structure composed of a RING-finger, a B-box, and a coiled-coil motif. In addition to these motifs, Trim32 possesses six C-terminal NHL-domains. A point mutation in one NHL domain (D487N) has been linked to two forms of muscular dystrophy called limb girdle muscular dystrophy type 2H and sarcotubular myopathy. In the present study we demonstrate that Trim32 is an E3 ubiquitin ligase that acts in conjunction with ubiquitin-conjugating enzymes UbcH5a, UbcH5c, and UbcH6. Western blot analysis showed that Trim32 is expressed primarily in skeletal muscle, and revealed its differential expression from one muscle to another. The level of Trim32 expression was elevated significantly in muscle undergoing remodeling due to changes in weight bearing. Furthermore, expression of Trim32 was induced in myogenic differentiation. Thus, variability in Trim32 expression in different skeletal muscles could be due to induction of Trim32 expression upon changes in physiological conditions. We show that Trim32 associates with skeletal muscle thick filaments, interacting directly with the head and neck region of myosin. Our data indicate that myosin is not a substrate of Trim32; however, Trim32 was found to ubiquitinate actin in vitro and to cause a decrease in the level of endogenous actin when transfected into HEK293 cells. In conclusion, our results demonstrate that Trim32 is a ubiquitin ligase that is expressed in skeletal muscle, can be induced upon muscle unloading and reloading, associates with myofibrils and is able to ubiquitinate actin, suggesting its likely participation in myofibrillar protein turnover, especially during muscle adaptation.

The giant protein titin serves a primary role as a scaffold for sarcomere assembly; however, proteins that mediate this remodeling have not been identified. One potential mediator of this process is the protease calpain 3 (C3), the protein mutated in limb girdle muscular dystrophy type 2A. To test the hypothesis that C3 mediates remodeling during myofibrillogenesis, C3 knockout (C3KO) mice were generated. The C3KO mice were atrophic containing small foci of muscular necrosis. Myogenic cells fused normally in vitro, but lacked well-organized sarcomeres, as visualized by electron microscopy (EM). Titin distribution was normal in longitudinal sections from the C3KO mice; however, EM of muscle fibers showed misaligned A-bands. In vitro studies revealed that C3 can bind and cleave titin and that some mutations that are pathogenic in human muscular dystrophy result in reduced affinity of C3 for titin. These studies suggest a role for C3 in myofibrillogenesis and sarcomere remodeling.

Calpain 3 (C3) is the only muscle-specific member of the calcium-dependent protease family. Although neither its physiological function nor its in vivo substrates are known, C3 must be an important protein for normal muscle function as mutations in the C3 gene result in limb-girdle muscular dystrophy type 2A. Previous reports have shown that the ubiquitous calpains (mu and m) proteolyze filamins in nonmuscle cells. This observation suggests that the muscle-specific filamin C (FLNC) is a good candidate substrate for C3. Binding studies using recombinant proteins establish that recombinant C3 and native FLNC can interact. When these two proteins are translated in vitro and incubated together, C3 cleaves the C-terminal portion of FLNC. Cleavage is specific as C3 fails to cleave FLNC lacking its C-terminal hinge and putative dimerization domains. Cotransfection experiments in COS-7 cells confirm that C3 can cleave the C-terminus of FLNC in live cells. The C-terminus of FLNC has been shown to bind the cytoplasmic domains of both delta- and gamma-sarcoglycan. Removal of the last 127 amino acids from FLNC, a protein that mimics FLNC after C3 cleavage, abolishes this interaction with the sarcoglycans. These studies confirm that C3 can cleave FLNC in vitro and suggest that FLNC may be an in vivo substrate for C3, functioning to regulate protein-protein interactions with the sarcoglycans. Thus, calpain-mediated remodeling of cytoskeletal-membrane interactions, such as those that occur during myoblast fusion and muscle repair, may involve regulation of FLNC-sarcoglycan interactions.

Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3'-->5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49)-->3'-->5' exonuclease (gp43)-->DNA polymerase (gp43)-->DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates.

In standard crosses, some rIIB mutants of T4 phage were found to be susceptible to an extra recombination mechanism to which the other mutants were much less susceptible. The following observations were interpreted as evidence for the mismatch-repair nature of the phenomenon: (1) Marker-dependent recombination generates exclusively double exchanges at both sides of the marker. (2) Marker-dependent recombination is highly sensitive to DNA base sequence at the site of the marker and to that at the corresponding site on the chromosome of the other parent. (3) Within certain limits, the contribution of the marker-dependent mechanism to the total recombination frequency is distance-independent and thus constitutes a constant component.

The contribution of mismatch repair to genetic recombination in T4 phage has been evaluated by three independent approaches: (1) testing for non-additivity of recombinant frequencies; (2) measurements of double exchange frequencies in three-factor crosses: (3) comparisons of recombination abilities of mutations occupying the same site. Quantitative agreement among the results of these approaches suggests that within distances much less than the mean length of hybrid regions, mismatch repair accounts perfectly for high negative interference as measured in three-factor crosses and as manifested by non-additivity in two-factor crosses. The mismatch repair mechanism readily recognizes only particular mismatches, the repair frequency being dependent on the base sequence in both strands of the mismatched region.