DNA repair by the cryptic endonuclease activity of Mu transposase

Phage Mu transposes by two distinct pathways depending on the specific stage of its life cycle. A common θ strand transfer intermediate is resolved differentially in the two pathways. During lytic growth, the θ intermediate is resolved by replication of Mu initiated within the flanking target DNA; during integration of infecting Mu, it is resolved without replication, by removal and repair of DNA from a previous host that is still attached to the ends of the incoming Mu genome. We have discovered that the cryptic endonuclease activity reported for the isolated C-terminal domain of the transposase MuA [Wu Z, Chaconas G (1995) A novel DNA binding and nuclease activity in domain III of Mu transposase: Evidence for a catalytic region involved in donor cleavage. EMBO J 14:3835–3843], which is not observed in the full-length protein or in the assembled transpososome in vitro, is required in vivo for removal of the attached host DNA or “5′flap” after the infecting Mu genome has integrated into the E. coli chromosome. Efficient flap removal also requires the host protein ClpX, which is known to interact with the C-terminus of MuA to remodel the transpososome for replication. We hypothesize that ClpX constitutes part of a highly regulated mechanism that unmasks the cryptic nuclease activity of MuA specifically in the repair pathway.     

Switch in the transposition products of Mu DNA mediated by proteins: Cointegrates versus simple insertions.

Bacteriophage Mu is a self-contained mobile unit encoding functions that mediate its movement. There appear to be two alternate pathways for Mu DNA transposition that differ with respect to the end products they generate. During the lytic cycle of phage Mu growth the end products of transposition are predominantly cointegrates in an experimental system in which the induced Mu prophage is located on pSC101, a low-copy-number plasmid. On the other hand, Mu insertions into the host genome during lysogenization contain Mu DNA as simple insertions. Two Mu functions, encoded by the A and B genes, are required for Mu DNA transposition during its lytic growth. However, during lysogeny the product of gene B is not required for integration of Mu DNA. Evidence is presented here which shows that in the absence of the B gene product the majority of transposition events are simple insertions. This is in striking contrast to the situation in which the majority of the products are cointegrates in the presence of both A and B gene products. Additional evidence also suggests that these simple insertions do not arise through the resolution of cointegrate structures.     

Dissecting the roles of MuB in Mu transposition: ATP regulation of DNA binding is not essential for target delivery

Collaboration between MuA transposase and its activator protein, MuB, is essential for properly regulated transposition. MuB activates MuA catalytic activity, selects target DNA, and stimulates transposition into the selected target site. Selection of appropriate target DNA requires ATP hydrolysis by the MuB ATPase. By fusing MuB to a site-specific DNA-binding protein, the Arc repressor, we generated a MuB variant that could select target DNA independently of ATP. This Arc-MuB fusion protein allowed us to test whether ATP binding and hydrolysis by MuB are necessary for stimulation of transposition into selected DNA, a process termed target delivery. We find that with the fusion proteins, MuB-dependent target delivery occurs efficiently under conditions where ATP hydrolysis is prevented by mutation or use of ADP. In contrast, no delivery was detected in the absence of nucleotide. These data indicate that the ATP- and MuA-regulated DNA-binding activity of MuB is not essential for target delivery but that activation of MuA by MuB strictly requires nucleotide-bound MuB. Furthermore, we find that the fusion protein directs transposition to regions of the DNA within 40–750 bp of its own binding site. Taken together, these results suggest that target delivery by MuB occurs as a consequence of the ability of MuB to stimulate MuA while simultaneously tethering MuA to a selected target DNA. This tethered-activator model provides an attractive explanation for other examples of protein-stimulated control of target site selection.     

Long-term growth following neonatal anatomic repair of transposition of the great arteries

Despite generally normal prenatal growth, surviving infants with transposition of the great arteries (TGA) frequently develop severe and progressive growth impairment which is not always fully reversed by elective atrial repair within the first year of life. This study was undertaken to determine the effect of neonatal anatomic repair of TGA on long-term growth. Twenty-three children with uncomplicated TGA were followed for a mean of 60 (12–90) months after anatomic repair at a mean age of 11 (1–40) days. Standardized measurements of weight, height, and head circumference for both patients and normal siblings were expressed as percentiles as well as in Z scores (in standard deviations from the mean for age and sex) based on internationally recognized standards. At latest follow-up, 22 (96%) of the patients were above the 3rd percentile for weight and 21 (91%) for both height and head circumference, with 13 (57%), 11 (48%), and 13 (57%) above the 50th percentile for each respective parameter. The mean Z scores (± SD) for weight, height, and head circumference for the patient group were – 0.1 ± 1.2, – 0.2 ± 1.3, and – 0.1 ± 1.1, respectively, and did not differ significantly from those of the reference population (p > 0.05 for each comparison). Paired comparisons of mean Z scores for each growth parameter with those of 35 normal siblings demonstrated no significant difference for weight or height and a small but significant difference for head circumference. Age at surgical repair (within the first 6 weeks of life), duration of follow-up and the development of moderate supravalvar pulmonary stenosis were not statistically related to long-term growth. These results indicate that in patients without extracardiac abnormalities, neonatal anatomic repair of uncomplicated TGA results in normal long-term growth.     

Pregnancy after atrial repair for transposition of the great arteries

OBJECTIVE: To investigate the risk of pregnancy in patients with transposition of the great arteries (TGA) who have undergone atrial repair. DESIGN: Retrospective analysis (1962-94) of 342 TGA patients who underwent atrial repair. Of 231 known late survivors, 48 were women over 18 years old who were interviewed about possible reproductive plans and previous pregnancies. As a control, comparison was made with data of 57 500 women (mean age 26 years) obtained from the Swiss Statistical Bank in Bern. RESULTS: Mean follow up was 13.7 years; 66% remained asymptomatic, 29% had mild to moderate cardiac symptoms, and 5% suffered from severe cardiac symptoms (New York Heart Association grade III-IV). Thirty six of the 48 women wished to bear children and, to date, there have been 10 live births, two spontaneous first trimester abortions, and one induced abortion at 16 weeks. During pregnancy there was one case of cardiac deterioration and two cases of pneumonia. There was no evidence of congenital heart disease in the children. CONCLUSIONS: In this relatively small series the completion of pregnancy in women with TGA who had undergone atrial repair and who had normal functional cardiac status was uncomplicated     

The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

The RecBCD enzyme is required for homologous recombination and DNA repair in Escherichia coli. The structure and function of RecBCD enzyme is altered on its interaction with the recombination hotspot Chi (5'-GCTGGTGG-3'). It has been hypothesized that the RecD subunit plays a role in Chi-dependent regulation of enzyme activity [Thaler, D. S., Sampson, E., Siddiqi, I., Rosenberg, S. M., Stahl, F. W. & Stahl, M. (1988) in Mechanisms and Consequences of DNA Damage Processing, eds. Friedberg, E. & Hanawalt, P. (Liss, New York), pp. 413-422; Churchill, J. J., Anderson, D. G. & Kowalczykowski, S. C. (1999) Genes Dev. 13, 901-911]. We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease- and recombination-deficient mutant recB(D1080A)CD. We report here that the resulting strain, recB(D1080A)C, was proficient for recombination and DNA repair. Recombination proficiency was accompanied by a change in enzyme activity: RecB(D1080A)C enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecB(D1080A)CD enzyme did not. Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E. coli cells and suggest that RecD interferes with the enzyme domain required for its loading. A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi.     

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.DNA double-strand break repair (DSBR) is essential for cell survival and repair-deficient cells are highly sensitive to chromosome breakage. In Escherichia coli, a single unrepaired DNA DSB per replication cycle is lethal, illustrating the critical nature of the repair reaction (1). DSBR in E. coli is mediated by homologous recombination, which relies on the RecA protein to efficiently recognize DNA sequence identity between two molecules. RecA homologs are widely conserved from bacteriophages to mammals, where they are known as the Rad51 proteins (2). The RecA protein plays its central role by binding single-stranded DNA (ssDNA) to form a presynaptic filament that searches for a homologous double-stranded DNA (dsDNA) donor from which to repair. It then catalyzes a strand-exchange reaction to form a joint molecule (3), which is stabilized by the branch migration activities of the RecG and RuvAB proteins (4). The joint molecule is then resolved by cleavage at its four-way Holliday junction by the nuclease activity of RuvABC (5, 6).RecA binding at the site of a DSB is dependent on the activity of the RecBCD enzyme (Fig. 1A). RecBCD is a helicase-nuclease that binds to dsDNA ends, then separates and unwinds the two DNA strands using the helicase activities of the RecB and RecD subunits (see refs. 7 and 8 for recent reviews). RecD is the faster motor of the two and this consequently results in the formation of a ssDNA loop ahead of RecB (Loop 1 in Fig. 1A) (9). As the enzyme translocates along dsDNA, the 3′-terminated strand is continually passed through the Chi-scanning site thought to be located in the RecC protein (10). When a Chi sequence (the octamer 5′-GCTGGTGG-3′) enters this recognition domain, the RecD motor is disengaged and the 3′ strand continues to be unwound by RecB. Under in vitro conditions, where the concentration of magnesium exceeds that of ATP, the 3′ end (unwound by RecB) is rapidly digested before Chi recognition, whereas the 5′ end (unwound by RecD) is intermittently cleaved (11, 12). After Chi recognition the 3′ end is no longer cleaved but the nuclease domain of RecB continues to degrade the 5′ end as it exits the enzyme (11, 12). Under in vitro conditions where the concentration of ATP exceeds that of magnesium, unwinding takes place but the only site of cleavage detected is ∼5 nucleotides 3′ of the Chi sequence (13, 14). Because the RecB motor continues to operate while the RecD motor is disengaged, Loop 1 is converted to a second loop located between the RecB and RecC subunits or to a tail upon release of the Chi sequence from its recognition site. We therefore describe this single-stranded region as Loop/Tail 2 in Fig. 1A. After the whole of Loop 1 is converted to Loop/Tail 2, this second single-stranded region continues to grow as long as the RecB subunit unwinds the dsDNA. The RecBCD enzyme enables RecA protein to load on to Loop/Tail 2 to generate the presynaptic filament necessary to search for homology and initiate strand-exchange (15). Finally, the RecBCD enzyme stops translocation and disassembles as it dissociates from the DNA, releasing a DNA-free RecC subunit (16).Open in a separate windowFig. 1.DSBR in E. coli. (A and B) Schematic representation of DSB processing by the RecBCD complex. (A) Before Chi recognition, both the RecB and RecD motors progress along the DNA. RecD is the faster motor and as a result a loop of ssDNA (Loop 1) is formed ahead of the slower RecB motor. The 3′ ssDNA strand is scanned for the Chi sequence by the RecC protein. (B) After Chi recognition, RecBCD likely undergoes a conformational change so that only the RecB motor is engaged. The RecA protein is recruited by the RecB nuclease domain and loaded onto the ssDNA loop generated by RecB unwinding to promote RecA nucleoprotein filament formation. In this schematic representation, the Chi site is shown held in its recognition site. However, the Chi site will be released either by disassembly of the RecBCD complex or at some point before this and the second single-stranded region on the 3′ terminating strand will be converted from a loop to a tail. Therefore, this region is denoted Loop/Tail 2. The mathematical model described in SI Appendix does not depend on the ATP/magnesium dependent differential cleavage of DNA strands (7, 8), nor does it depend on the precise time that the 3′ end is released from the complex following Chi recognition. (C) The hairpin endonuclease SbcCD is used to cleave a 246-bp interrupted palindrome inserted in the lacZ gene of the E. coli chromosome. Cleavage of this DNA hairpin results in the generation of a site-specific DSB on only one of a pair of replicating sister chromosomes, thus leaving an intact sister chromosome to serve as a template for repair by homologous recombination.Our understanding of the action of RecBCD and RecA has been the result of more than 40 years of genetic analysis and biochemical investigation of these purified proteins in vitro. However, relatively little is known about their activities on the genomic scale. To investigate these reactions in vivo, we have used RecA chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) in an experimental system that allows us to introduce a single and fully repairable DSB into the chromosome of E. coli (1). Because DSBR by homologous recombination normally involves the repair of a broken chromosome by copying the information on an unbroken sister chromosome, our laboratory has previously developed a procedure for the cleavage of only one copy of two genetically identical sister chromosomes (1). We have made use of the observation that the hairpin nuclease SbcCD specifically cleaves only one of the two sister chromosomes following DNA replication through a 246-bp interrupted palindrome to generate a two-ended DSB (1). As shown in Fig. 1B, this break is fully repairable and we have shown that recombination-proficient cells suffer very little loss of fitness in repairing such breaks (17).Here we investigate in vivo and in a quantitative manner the first steps of DSBR: because the outcome of RecBCD action is understood to be the loading of RecA on DNA in a Chi-dependent manner, we use RecA-ChIP to reveal the consequences of RecBCD action on a genomic scale during DSBR. Analyses of most ChIP-Seq datasets focus on the identification of regions of significant enrichment of a given protein but do not take into account the underlying mechanisms giving rise to the binding (18). We reasoned that given the detailed mechanistic understanding of RecBCD in vitro, we could gain a deeper insight into its in vivo functions by developing a mathematical model of RecBCD action that would enable us to estimate the mechanistic parameters of the complex in live cells. Our ChIP data indicate that RecA is indeed loaded on to DNA in a Chi-dependent manner and we have used our mathematical model to infer the parameters of RecBCD action in vivo on a genomic scale. Furthermore, our analysis reveals that DSBR at lacZ induces DSBR in the terminus region of the chromosome, an unanticipated observation illuminated by the genomic scale of our data.     

From tricuspid to double orifice Morphology: Percutaneous tricuspid regurgitation repair with the MitraClip device in congenitally corrected‐transposition of great arteries

Edge to edge transcatheter mitral valve repair with MitraClip (Abbott Vascular, Menlo Park, CA) is increasing for high‐risk surgical patients with significant mitral regurgitation. Patients with congenitally corrected transposition of the great arteries (CCTGA) presenting with tricuspid valve regurgitation of a systemic right ventricle may represent particularly challenging candidates for MitraClip given their anatomy. We report the case of a 67‐year‐old gentleman with CCTGA and severe tricuspid regurgitation who was referred for MitraClip implantation after heart team consensus. Successful implantation of one clip was performed, achieving a significant reduction of the regurgitation. Similarly, favorable findings were confirmed at 6 months, 1 and 2 years follow‐up and the patient had no recurrent heart failure admissions after 2‐year follow‐up. We describe the technical considerations and the importance of 3D‐transoesophageal echocardiography for performing the MitraClip of a trileaflet systemic atrioventricular valve. © 2016 Wiley Periodicals, Inc.     

Natural insertions in rice commonly form tandem duplications indicative of patch-mediated double-strand break induction and repair

The insertion of DNA into a genome can result in the duplication and dispersal of functional sequences through the genome. In addition, a deeper understanding of insertion mechanisms will inform methods of genetic engineering and plant transformation. Exploiting structural variations in numerous rice accessions, we have inferred and analyzed intermediate length (10–1,000 bp) insertions in plants. Insertions in this size class were found to be approximately equal in frequency to deletions, and compound insertion–deletions comprised only 0.1% of all events. Our findings indicate that, as observed in humans, tandem or partially tandem duplications are the dominant form of insertion (48%), although short duplications from ectopic donors account for a sizable fraction of insertions in rice (38%). Many nontandem duplications contain insertions from nearby DNA (within 200 bp) and can contain multiple donor sources—some distant—in single events. Although replication slippage is a plausible explanation for tandem duplications, the end homology required in such a model is most often absent and rarely is >5 bp. However, end homology is commonly longer than expected by chance. Such findings lead us to favor a model of patch-mediated double-strand-break creation followed by nonhomologous end-joining. Additionally, a striking bias toward 31-bp partially tandem duplications suggests that errors in nucleotide excision repair may be resolved via a similar, but distinct, pathway. In summary, the analysis of recent insertions in rice suggests multiple underappreciated causes of structural variation in eukaryotes.Genomic DNA insertion causes genome expansion and, potentially, the rearrangement and diffusion of protein domains and regulatory elements throughout the genome (1, 2). Additionally, genetic engineers generally aim to integrate specific DNA into the nuclear genome, so the natural mechanisms by which this integration occurs may serve as a starting point to elaborate and improve genome modification (3, 4). Common causes of gene-sized insertions are unequal recombination (5), transposable element replication (1), and ectopic recombination stimulated by double-strand breaks (DSBs) in the genome (2, 6). Shorter events are less well characterized, but it appears that these can be created by similar processes (7). Still, high-throughput sequencing of DSB repair events in humans (8) and plants (9) suggests that insertions related to induced breaks are very rare and very short.Although the processes described above can produce duplications at distant genetic loci, the most common form of non-microsatellite-associated insertions in humans is tandem duplications (10). Once created, tandem duplications can be dramatically expanded by unequal recombination or replication slippage. Such duplications may be deleterious, or they may be promoted by selection for a novel or expanded function (11, 12).Although tandem repeats are ubiquitous in eukaryotic genomes, the mechanisms for their origin are still in question. Early analysis of human indel mutations indicated that replication slippage was the most effective model to explain the origin of assorted repeats (13). In other studies, longer, de novo tandem duplications were also hypothesized to be caused by slippage because, out of 85 insertions producing such duplications, 50 were associated with flanking repeats >2 bp (14). Replication slippage would presumably require a preexisting short repeat because priming must occur between the end of the loop that will become the duplication and the position to where replication slips. Authors of more recent work investigating insertions across the human genome suggest alternatives to replication slippage on the grounds that homology is often either nonexistent or very short, whereas the length of homology and the length of insertion are not correlated (10). These researchers favor a model based on DSBs being repaired by nonhomologous end-joining (NHEJ). However, conventional models of DSB repair are strained to predict tandem duplications >10 bp, much less >100 bp. Such models require extensive single-stranded, complementary ends to be preserved during the break. Moreover, DSBs produced by Tal-effector nucleases in humans do not yield insertions that form tandem repeats, despite the fact that the breaks generate a 5′ overhang (15). Thus, this common class of mutations currently lacks a firm molecular explanation.Similar to tandem duplications, short duplications are commonly found within 100 bp of one another, but with unique intervening DNA (16). By comparing human polymorphisms with chimp sequence, Thomas et al. (16) inferred that the repeats were recent insertions. As discussed by the authors and herein, a mechanism for such duplications is even less forthcoming than for tandem duplications.In this study, we used extensive population-scale rice resequencing data to confirm that tandem duplications are also abundant natural polymorphisms in the plant kingdom. Additionally, we found that many insertions in rice, although not perfectly tandem, are from a ∼50-bp window around the insertion site. We rarely found the end homology in tandem repeats that is expected for replication slippage, although we did note a bias toward short microhomology between insertion ends and insertion site. These data led us to elaborate on the DSB model of tandem duplication, proposing that long patch base excision repair (BER) on complementary strands commonly leads to such patterns (17). Additionally, we characterized common forms of nontandem, but local, duplication.     

Abnormalities of right ventricular long axis function after atrial repair of transposition of the great arteries

BACKGROUND—While volume derived global indices of right ventricle (RV) function are frequently abnormal after the Mustard procedure, the mechanism for these abnormalities is poorly understood. RV muscle fibres are predominantly arranged longitudinally and thus indices derived in the long axis may better describe RV function.
METHODS—20 survivors of the Mustard operation were studied (age 7.8-37.3 years, median 14.2 years). Long axis recordings from the apical four chamber view were obtained with the M mode cursor positioned through the lateral angle of the tricuspid valve annulus. M mode traces were recorded on paper and later digitised to derive total atrioventricular ring excursion, peak lengthening rate, and peak shortening rate. These data were averaged and compared with control data for the normal RV and left ventricle (LV).
RESULTS—RV total atrioventricular ring excursion was lower than that for the RV (p < 0.0001) or LV (p < 0.005) of controls. Peak lengthening rate was lower than the normal RV (p < 0.0001) and LV (p < 0.0001) rates. Furthermore, peak shortening rate was less than that of normal RV (p < 0.0001) and normal LV (p < 0.005) controls.
CONCLUSION—Systemic RV long axis function is notably reduced compared with that of either the normal subpulmonary RV or the systemic LV. This presumably reflects the response of the predominantly longitudinally arranged myocardial fibres to increased afterload. However, such measurements may provide a more sensitive marker for progressive changes in global function during long term follow up.


Keywords: Mustard procedure; right ventricular function; transposition of the great arteries     

A pilot study of total pelvic floor repair or gluteus maximus transposition for postobstetric neuropathic fecal incontinence

PURPOSE: The aim of this study was to report pilot data comparing the morbidity and functional outcome of total pelvic floor repair with gluteus maximus transposition for women with postobstetric fecal incontinence. METHODS: This is a prospective, randomized trial of two surgical procedures in 24 women so far. Functional assessment was performed with use of a 20-point clinical incontinence score and patient questionnaire before and after operation. The physiologic parameters, before and after operation, included resting and squeeze anal pressures, length of the high pressure zone, anal and rectal mucosal sensitivity, and pudendal nerve latency. RESULTS: So far, 12 patients have been treated by total pelvic floor repair and 12 by gluteus maximus transposition. Of these, three patients developed wound complications after gluteus maximus transposition compared with none after total pelvic floor repair. Among these cases there was a significant overall improvement in functional score (given as mean ± standard deviation) after both total pelvic floor repair (13.1±2.7vs. 6.6±4.5;P<0.001) and gluteus maximus transposition (13.8±3.8vs. 7.7±6.1;P<0.01), although no difference existed between the groups. There was no change in any of the physiologic measurements after either operation, and preoperative measurements did not identify patients likely to do badly. CONCLUSIONS: We conclude from these preliminary data that both total pelvic floor repair and gluteus maximus transposition significantly improve continence in women with postobstetric neuropathic fecal incontinence. Gluteus maximus transposition gives equivalent results to total pelvic floor repair. Neither procedure has any influence on anorectal physiologic parameters.Preliminary results presented at the Association of Surgeons of Great Britain and Ireland, Glasgow, Scotland, April 9 to 11, 1997.     

Ultrasonography of entheseal insertions in the lower limb in spondyloarthropathy

OBJECTIVE: To compare ultrasonography (US) with clinical examination in the detection of entheseal abnormality of the lower limb in patients with spondyloarthropathy (SpA). METHODS: 35 patients with SpA (ankylosing spondylitis 27; psoriatic arthritis 7; reactive arthritis 1) underwent independent clinical and ultrasonographic examination of both lower limbs at five entheseal sites-superior pole and inferior pole of patella, tibial tuberosity, Achilles tendon, and plantar aponeurosis. US was performed using an ATL (Advanced Technology Laboratories, Bothell, Washington, USA) high definition imaging 3000 machine with linear 7-4 MHz and compact linear 10-5 MHz probes to detect bursitis, structure thickness, bony erosion, and enthesophyte (bony spur). An enthesitis score was formulated from these US findings giving a possible maximum total score of 36. RESULTS: On clinical examination 75/348 (22%) entheseal sites were abnormal and on US examination 195/348 (56%) sites were abnormal. In 19 entheseal sites with bursitis on US, only five were detected by clinical examination. Compared with US, clinical examination had a low sensitivity (22.6%) and moderate specificity (79.7%) for the detection of enthesitis of the lower limbs. There was no significant correlation between the US score of enthesitis and acute phase parameters such as erythrocyte sedimentation rate (ESR) or C reactive protein (CRP). The intraobserver kappa value for analysis of all sites was 0.9. CONCLUSIONS: Most entheseal abnormality in SpA is not detected at clinical examination. US is better than clinical examination in the detection of entheseal abnormality of the lower limbs in SpA. A quantitative US score of lower limb enthesitis is proposed but further studies are required to validate it in SpA.     

Predicted poxvirus FEN1-like nuclease required for homologous recombination,double-strand break repair and full-size genome formation

Poxviruses encode many if not all of the proteins required for viral genome replication in the cytoplasm of the host cell. In this context, we investigated the function of the vaccinia virus G5 protein because it belongs to the FEN1-like family of nucleases and is conserved in all poxviruses. A vaccinia virus G5 deletion mutant was severely impaired, as the yield of infectious virus was reduced by approximately two orders of magnitude. The mutant virions contained an apparently normal complement of proteins but appeared spherical rather than brick-shaped and contained no detectable DNA. The inability of G5 with substitutions of the predicted catalytic aspartates to complement the deletion mutant suggested that G5 functions as a nuclease during viral DNA replication. Although the amount of viral DNA produced in the absence of G5 was similar to that made by wild-type virus, the mean size was approximately one-fourth of the genome length. Experiments with transfected plasmids showed that G5 was required for double-strand break repair by homologous recombination, suggesting a similar role during vaccinia virus genome replication.     

A case of traumatic intramural hematoma of the duodenum effectively treated with ultrasonically guided aspiration drainage and endoscopic balloon catheter dilatation

A 52-year-old man was admitted on February 15, 1990, with hiccups and vomiting. He had been well until 13 days before admission when he stumbled and fell when intoxicated, striking his abdomen. A diagnosis of intramural hematoma was made with computerized tomography and sonography of the abdomen after admission, revealing a mass that was intimately related to the duodenum. Treatment of the intramural duodenal hematoma is controversial. However, this case illustrates the ideal situation where conservative management could be applied with total parenteral nutrition, percutaneous aspiration drainage, and endoscopic balloon catheter dilatation of the narrowed lumen of the duodenum. The patient’s subsequent course supports the concept of planned conservative management.