Interaction of flap endonuclease-1 and replication protein A with photoreactive intermediates of DNA repair

A new method for enzymatic synthesis of radioactive DNA flapped structures containing a photoreactive dCMP moiety at a branch point with 4-(4-azido-2,3,5,6-tetrafluorobenzylidene-hydrazinocarbonyl)butylcarbamoyl group attached at exo-N-position of cytosine was developed. The formation of complexes of flap endonuclease-1 (FEN-1) with flapped DNA was shown by photoaffinity modification and gel retardation assays. The substrate properties of the flapped structures with different flap lengths were studied in the reaction of endonuclease cleavage catalyzed by FEN-1. It was demonstrated that inhibition of FEN-1 activity by replication protein A (RPA) depends on the length of the single-stranded part of the flapped substrate. A significant inhibition of cleavage was observed when the flap length was sufficient for effective RPA binding, while for structures with short single-stranded part the efficiency of cleavage was independent of the presence of RPA. FEN-1 and RPA were modified by photoaffinity labeling using flap structures with single-stranded parts consisting of 8 and 21 nucleotides. Products of DNA photoattachment to FEN-1 were observed in both cases, while the covalent adducts with RPA were obtained only with the 21-nucleotide-long flap. Photoaffinity modification demonstrated that FEN-1 and RPA compete for the binding of the flapped substrates with long single-stranded parts.     

AdVEGF165 gene transfer increases survival in overdimensioned skin flaps

BACKGROUND: Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. VEGF A also plays an important role in wound healing of the skin by promoting angiogenesis and by stimulating blood vessel growth. Therefore we tested the hypothesis that flap survival could be increased by the preoperative injection of AdVEGF(165). METHODS: We studied the effect of AdVEGF(165) in an overdimensioned ischemic random-pattern-flap model in the rat (n = 50) with a length-to-width ratio of 4 : 1. VEGF cDNA was administered in two concentrations of 5 x 10(8) plaque-forming units (pfU) and 1 x 10(9) pfU using a recombinant adenoviral vector. Recombinant virus was injected subdermally 7, 3 or 0 days prior to flap harvest for the lower concentration and 7 days prior for the higher concentration. Flap survival and necrosis were observed at day 7, the day the animals were sacrificed. RESULTS: Adenoviral gene transfer with VEGF(165) 3 and 7 days before flap harvest showed a significantly increased flap survival of 50% together with a significantly reduced necrosis (p < 0.01). Injection using a titer of 1 x 10(9) pfU 7 days prior to surgery increased flap survival even more, though failing to reach statistical significance compared to the lower concentration. VEGF protein concentration in the injected skin was significantly higher than in controls (p < 0.01). Flap perfusion was increased as well, demonstrated by indocyanine green (ICG) fluoroscopy (p < 0.001). CONCLUSIONS: Our results confirm the important role of VEGF(165) on angiogenesis in ischemic flaps. Indeed by injecting VEGF(165) at 3 to 7 days preoperatively in a concentration of 1 x 10(9) pfU our data show that length-to-width ratio for random-pattern-flaps could be increased from 2 : 1 to 3 : 1 and therefore may allow a wider range of applications of this simple flap technique.     

The two DNA clamps Rad9/Rad1/Hus1 complex and proliferating cell nuclear antigen differentially regulate flap endonuclease 1 activity

DNA damage leads to activation of several mechanisms such as DNA repair and cell-cycle checkpoints. It is evident that these different cellular mechanisms have to be finely co-ordinated. Growing evidence suggests that the Rad9/Rad1/Hus1 cell-cycle checkpoint complex (9-1-1 complex), which is recruited to DNA lesion upon DNA damage, plays a major role in DNA repair. This complex has been shown to interact with and stimulate several proteins involved in long-patch base excision repair. On the other hand, the well-characterised DNA clamp-proliferating cell nuclear antigen (PCNA) also interacts with and stimulates several of these factors. In this work, we compared the effects of the 9-1-1 complex and PCNA on flap endonuclease 1 (Fen1). Our data suggest that PCNA and the 9-1-1 complex can independently bind to and activate Fen1. Finally, acetylation of Fen1 by p300-HAT abolished the stimulatory effect of the 9-1-1 complex but not that of PCNA, suggesting a possible mechanism of regulation of this important repair pathway.     

Interaction of Replication Protein A and Flap Endonuclease 1 with DNA Duplexes Containing a Nick or a Flap

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase (Pol) interaction with DNA. Using Pol, a photoreactive dTTP analog was added to the 3" end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5" end of a nick or a flap. Photoaffinity labeling of Pol substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol during long-patch base excision repair.     

带血管髂骨移植动物模型的建立与评估

目的:探索一种相对简单稳定的大鼠髂骨皮瓣移植手术方式,为髂骨移植诱导免疫嵌合状态的研究建立可靠的动物模型。方法:实验分为3组:空白对照组,不做任何处理;旧术式组,将供者髂骨肌皮瓣移植入受者腹股沟区,吻合供者髂腰动静脉和受者股动静脉;实验组,剥离供者髂骨臀大肌,取供者的髂骨连带肌肉及皮瓣移植于受者的腹股沟区域,将供者的髂总动脉和髂腰静脉分别与受者的股动静脉吻合,随后缝合皮肤。术后所有大鼠给予环孢素A 28天,始剂量16 mg/kg/d,第三周始每周剂量减半,第28天处死大鼠并选取髂骨做病理学检测,观测骨髓细胞量,骨陷窝是否有骨细胞等,以此判断髂骨是否成活。结果:实验组共进行手术15例,皮瓣成活11例,成功率73%;病理检测皮下髂骨成活7例,成功率47%;旧术式组共进行手术8例,皮瓣成活4例,成功率50%,病理检测皮下髂骨成活3例,成功率37.5%,其中实验组动脉吻合时间(24.7±2.3)min,明显短于旧术式组(36.7±1.5)min(均P0.05),而静脉吻合时间、缺血时间,供受体准备时间并无差异(均P0.05)。结论:通过剥离供者臀大肌,改变动脉吻合对象,可以建立相对简单、稳定的髂骨皮瓣移植模型,为探索带血管骨髓移植诱导嵌合状态提供了新的动物模型。     

Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease

Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures. The FEN-catalysed reaction properties of nicked DNA and flap structures possessing an extrahelical 3'-nucleotide (nt) were also characterised. The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated. Only larger DNAs containing two duplex regions are effective substrates for the archaeal enzyme and undergo reaction at multiple sites when they lack a 3'-extrahelical nucleotide. However, a single product corresponding to reaction 1 nt into the double-stranded region occurred with A. fulgidus FEN when substrates possessed a 3'-extrahelical nt. Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested. Single-turnover maximal rates of reaction are similar with most substrates. In contrast, turnover numbers for T5FEN decrease as the size of the DNA substrate is increased. Comparison of the catalytic parameters of the A. fulgidus FEN employing flap and double-flap substrates indicates that binding interactions with the 3'-extrahelical nucleotide stabilise the ground state FEN-DNA interaction, leading to stimulation of comparative reactions at DNA concentrations below saturation with the single flap substrate. Maximal multiple turnover rates of the archaeal enzyme with flap and double flap substrates are similar. A model is proposed to account for the varying specificities of the two enzymes with regard to cleavage patterns and substrate preferences.     

Molecular dynamics insights for PI3K-δ inhibition & structure guided identification of novel PI3K-δ inhibitors

Conjugated structure based and ligand based drug design techinques have been used previously to unearth putative binding ligands for kinase inhibition. PI3K-δ is a lipid kinase and it has been found abberant in diseases such as cancer,inflammation etc. Preliminarily, protein crystal structure analysis suggest avaibility of two crystal structures with varying degree of root mean square de throughtion in protein back bone and root mean square fluctuation in side chain geometry. Therefore, PI3K-δ crystal structure was selected based on charactristic reciever operating characterstic curve and % enrichment of actives analysis. Active site analysis through molecular dynamics simulations provided insights about four residues Ile910, Asp911, Met752, Lys755, which act as flap. These residues fecilitate ligand binding in a unique manner.Thereafter, a validated designed protocol has been used to screen asinex ligand database using molecular docking and binding energy calculations. Based on binding affinity & energy scores and interaction pattern analysis total top 50 ligands were selected for PI3K-δ inhibition studies. Moreover, two molecules ethyl 2-(2-((4-chloro-1-methyl-1H-pyrazole-3-carbonyl) oxy)acetamido) benzo[1]thiazole-6-carboxylate and 1,6,7-trimethyl-8-((tetrahydrofuran-2-yl) methyl)-1H-imidazo [1',2':1,5] pyrrolo[3,2-d]pyrimidine-2,4(3H,8H)-dione have been identified, which could be potential hits for PI3K-δ using insights provided by molecular modelling studies. The identified compunds were subjected to pan assay interference compound filter and were found to be compliant. Quantum mechanical calculations were perfromed for identified hits. The above strategy could be implemented as a strategy for rational drug design.

Communicated by Ramaswamy H. Sarma     

CTA联合CEUS在锁骨上皮瓣术前设计中的应用

目的:探讨计算机断层扫描血管造影(CTA)联合对比增强超声(CEUS)在锁骨上皮瓣术前设计中的应用价值。方法:将2016年1月2018年12月本院收治的15例锁骨上皮瓣术前患者作为研究对象。所有患者术前进行CTA和CEUS联合检查以观察锁骨上动脉穿支解剖变异及走行,应用于锁骨上皮瓣术前的辅助设计,评估该方法的成功率和并发症的发生情况。结果:15例患者的锁骨上动脉来源于颈横动脉,其中5例(33.33%)来自甲状颈干,10例(66.67%)来自锁骨下动脉。CTA检查中,10例识别出右锁骨上动脉,血管平均长度为(38.25±11.08)mm,血管平均直径(1.52±0.45)mm;13例确定了左锁骨上动脉,血管平均长度为(38.14±11.05)mm,血管平均直径(1.52±0.51)mm。CEUS检查的15个皮瓣中,发现27个胸锁骨上动脉的胸廓分支(TBSA),平均口径为(0.8±0.2)mm,平均收缩期峰值流速(PSV)为(11.95±2.08)cm/s。所有病例(100%)术中观察皮瓣血管数量、走形等情况与术前影像学相一致的手术结果。与造影剂有关的并发症发生率为6.67%。所有患者均随访1年以上,无进一步并发症,手术效果满意。结论:将CTA和CEUS相结合用于锁骨上皮瓣术前的辅助设计,可互为补充,尤其适用于锁骨上皮瓣(SCF)存在血管解剖变异而且管径细小的皮瓣术前设计。     

Mechanism of drug resistance revealed by the crystal structure of the unliganded HIV-1 protease with F53L mutation

Mutations in HIV-1 protease (PR) that produce resistance to antiviral PR inhibitors are a major problem in AIDS therapy. The mutation F53L arising from antiretroviral therapy was introduced into the flexible flap region of the wild-type PR to study its effect and potential role in developing drug resistance. Compared to wild-type PR, PR(F53L) showed lower (15%) catalytic efficiency, 20-fold weaker inhibition by the clinical drug indinavir, and reduced dimer stability, while the inhibition constants of two peptide analog inhibitors were slightly lower than those for PR. The crystal structure of PR(F53L) was determined in the unliganded form at 1.35 Angstrom resolution in space group P4(1)2(1)2. The tips of the flaps in PR(F53L) had a wider separation than in unliganded wild-type PR, probably due to the absence of hydrophobic interactions of the side-chains of Phe53 and Ile50'. The changes in interactions between the flaps agreed with the reduced stability of PR(F53L) relative to wild-type PR. The altered flap interactions in the unliganded form of PR(F53L) suggest a distinct mechanism for drug resistance, which has not been observed in other common drug-resistant mutants.