Role of mammalian Rad54 in telomere length maintenance

The homologous recombination (HR) DNA repair pathway participates in telomere length maintenance in yeast but its putative role at mammalian telomeres is unknown. Mammalian Rad54 is part of the HR machinery, and Rad54-deficient mice show a reduced HR capability. Here, we show that Rad54-deficient mice also show significantly shorter telomeres than wild-type controls, indicating that Rad54 activity plays an essential role in telomere length maintenance in mammals. Rad54 deficiency also resulted in an increased frequency of end-to-end chromosome fusions involving telomeres compared to the controls, suggesting a putative role of Rad54 in telomere capping. Finally, the study of mice doubly deficient for Rad54 and DNA-PKcs showed that telomere fusions due to DNA-PKcs deficiency were not rescued in the absence of Rad54, suggesting that they are not mediated by Rad54 activity.     

AUF1/HnRNP D RNA binding protein functions in telomere maintenance

In the current issue of Molecular Cell, Yu et al. (2012) establish H3K56 monomethylation (H3K56me1) as a new mammalian chromatin mark, imposed by the G9a methyltransferase and recognized by the replication clamp PCNA.     

In vitro reconstitution of hnRNP particles

The assembly of hnRNP-like particles was studied by in vitro reconstitution, UV-crosslinking and CsCl-equilibrium centrifugation. Using total nuclear protein and RNA extracts from HeLa cells for RNP reconstitution, RNP particles sedimenting with the same buoyant density of p = 1.4 g/cm3 as 'native' 40 S core hnRNPs were obtained. Under the stringent reconstitution conditions used, hnRNP complexes containing only the Cl-core hnRNP protein could be identified.     

Unwinding protein complexes in ALTernative telomere maintenance

Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA‐binding proteins and a number of three dimensional nucleic acid structures including G‐quartets and D‐loops. A number of studies suggest that the BLM and WRN recQ‐like helicases play important roles in recombination‐mediated mechanisms of telomere elongation or A lternative L engthening of T elomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT‐associated nuclear bodies in telomerase‐negative immortalized cell lines and interact with the telomere‐specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double‐strand break repair processes such as non‐homologous end joining, homologous recombination‐mediated repair, resolution of stalled replication forks and synthesis‐dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ‐like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec‐Q‐like helicases and their role in telomere elongation during ALT processes. J. Cell. Biochem. 109: 7–15, 2010. © 2009 Wiley‐Liss, Inc.     

In vitro assembly of feline immunodeficiency virus capsid protein: biological role of conserved cysteines

Nitric oxide (NO) can modulate numerous genes through several pathways, yet some genes may be modulated only in the presence of the inflammatory stimuli that upregulate the inducible nitric oxide synthase (iNOS) rather than by NO alone. Furthermore, the role of prior expression of iNOS in the modulation of genes by NO is unknown. We addressed these issues in hepatocytes harvested from iNOS-null (iNOS(-/-)) mice exposed to NO by treatment with NO donors or by infection with an adenovirus-expressing human iNOS (Ad-iNOS), rather than by stimulation with inflammatory cytokines. Differential display and gene array analyses performed on mRNA derived from iNOS(-/-) hepatocytes demonstrated that infection with Ad-iNOS, but not infection with a control adenovirus expressing the beta-galactosidase gene (Ad-LacZ), induced a gene fragment identical to cytochrome P450 2E1 (CYP2E1). Northern analysis performed with this fragment demonstrated that treatment of iNOS(-/-) hepatocytes with Ad-iNOS or with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), but not control treatment or infection with Ad-LacZ, resulted in increased expression of CYP2E1. Inhibition of soluble guanylyl cyclase partially blocked the induction of CYP2E1 mRNA by Ad-iNOS. Rat hepatocytes treated with SNAP also exhibited increased expression of CYP2E1 mRNA. Preliminary studies, however, suggest that the induction of CYP2E1 in the rat hepatocytes treated with cytokines was not reduced in the presence of a NOS inhibitor. Our results suggest that CYP2E1 can be induced solely by NO derived from iNOS, at least partly in a cyclic GMP-dependent manner and independently of inflammatory stimuli or of prior exposure to NO.     

In vitro characterization of native mammalian smooth-muscle protein synaptopodin 2

An analysis of the primary structure of the actin-binding protein fesselin revealed it to be the avian homologue of mammalian synaptopodin 2 [Schroeter, Beall, Heid, and Chalovich (2008) Biochem. Biophys. Res. Commun. 371, 582-586]. We isolated two synaptopodin 2 isoforms from rabbit stomach that corresponded to known types of human synaptopodin 2. The purification scheme used was that developed for avian fesselin. These synaptopodin 2 forms shared several key functions with fesselin. Both avian fesselin and mammalian synaptopodin 2 bound to Ca(2+)-calmodulin, alpha-actinin and smooth-muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca(2+)-calmodulin-dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of alpha-actinin, to polymerize actin in a Ca(2+)-calmodulin-dependent manner, or to bind to Ca(2+)-calmodulin or myosin. These properties are consistent with the proposed function of synaptopodin 2 in organizing the cytoskeleton.     

哺乳动物精子体外发生

精子的发生是一个高度复杂而有序的过程 ,涉及到细胞的增殖和分化。体外培养生精细胞在近年来取得了较大进展 ,建立了睾丸组织培养、曲精细管小段培养、支持细胞 生精细胞共培养及藻酸钙胶囊包裹培养等方法。建立生精细胞体外培养模型有助于 :(1)研究精子发生的调控机制 ;(2 )直接对雄性生殖细胞进行遗传修饰 ;(3)用于辅助生育技术 ,治疗精子发生阻滞的患者。如何改善培养条件 ,进一步提高生殖细胞的存活、分化、增殖效率 ,是使哺乳动物体外精子发生发展成为一项适用性较强的技术所必须解决的问题。     

Structure of hnRNP D complexed with single-stranded telomere DNA and unfolding of the quadruplex by heterogeneous nuclear ribonucleoprotein D

Heterogeneous nuclear ribonucleoprotein D, also known as AUF1, has two DNA/RNA-binding domains, each of which can specifically bind to single-stranded d(TTAGGG)n, the human telomeric repeat. Here, the structure of the C-terminal-binding domain (BD2) complexed with single-stranded d(TTAGGG) determined by NMR is presented. The structure has revealed that each residue of the d(TAG) segment is recognized by BD2 in a base-specific manner. The interactions deduced from the structure have been confirmed by gel retardation experiments with mutant BD2 and DNA. It is known that single-stranded DNA with the telomeric repeat tends to form a quadruplex and that the quadruplex has an inhibitory effect on telomere elongation by telomerase. This time it is revealed that BD2 unfolds the quadruplex of such DNA upon binding. Moreover, the effect of BD2 on the elongation by telomerase was examined in vitro. These results suggest the possible involvement of heterogeneous nuclear ribonucleoprotein D in maintenance of the telomere 3'-overhang either through protection of a single-stranded DNA or destabilization of the potentially deleterious quadruplex structure for the elongation by telomerase.     

Studies of the strand-annealing activity of mammalian hnRNP complex protein A1.

A1 is a major core protein of the mammalian hnRNP complex, and as a purified protein of approximately 34 kDa, A1 is a strong single-stranded nucleic acid binding protein. Several lines of evidence suggest that the protein is organized in discrete domains consisting of an N-terminal segment of approximately 22 kDa and a C-terminal segment of approximately 12 kDa. Each of these domains as a purified fragment is capable of binding to both ssDNA and RNA. We report here that A1 and its C-terminal domain fragment are capable of potent strand-annealing activity for base-pair complementary single-stranded polynucleotides of both RNA and DNA. This effect is not stimulated by ATP. Compared with A1 and the C-terminal fragment, the N-terminal domain fragment has negligible annealing activity. These results indicate that A1 has biochemical activity consistent with a strand-annealing role in relevant reactions, such as pre-mRNA splicing.     

Initiation of mammalian protein synthesis: Dynamic properties of the assembly process in vitro

Binding of the Met-tRNA^Met_f·eIF-2 GTP complex to the 40 S ribosomal subunit is the first step in initiation of eukaryotic protein synthesis. The extent of binding and the stability of the complex are enhanced by initiation factors eIF-3 and eIF-4C, AUG and elevated magnesium concentration. The reversibility of reaction steps occurring during the assembly of the initiation complex is measured as the rate of Met-tRNA^Met_f exchange in the initiation complex and its intermediates. This rate progressively decreases and Met-tRNA^Met_f binding becomes irreversible upon binding of mRNA. The association of the 40 S Met-tRNA^Met_f mRNA initiation complex with the 60 S ribosomal subunit is again reversible as long as elongation does not occur.     

Initiation of mammalian protein synthesis: dynamic properties of the assembly process in vitro

Binding of the Met-tRNAMetf . eIf-2 GTP complex to the 40 S ribosomal subunit is the first step in initiation of eukaryotic protein synthesis. The extent of binding and the stability of the complex are enhanced by initiation factors eIF-3 and eIF-4C, AUG and elevated magnesium concentration. The reversibility of reaction steps occurring during the assembly of the initiation complex is measured as the rate of Met-tRNAMetf exchange in the initiation complex and its intermediates. This rate progressively decreases and Met-tRNAMetf binding becomes irreversible upon binding of mRNA. The association of the 40 S Met-tRNAMetf mRNA initiation complex with the 60 S ribosomal subunit is again reversible as long as elongation does not occur.     

The chaperone-like properties of mammalian inhibitor-2 are conserved in a Drosophila homologue

Phosphatase inhibitor-2 (I-2) is a mammalian phosphoprotein that binds to the catalytic subunit of type 1 serine/threonine phosphoprotein phosphatase (PP1c) and inhibits its activity in vitro. Recombinant PP1c differs from native PP1c in several biochemical criteria, including the requirement for Mn(2+), sensitivity to vanadate, and p-nitrophenyl phosphate (pNPP) phosphatase activity. I-2 can convert recombinant PP1c into a native-like activity in vitro. It has therefore been suggested that I-2 may act as a molecular chaperone for PP1 in vivo. We have identified a Drosophila homologue (I-2Dm) in a two-hybrid screen for PP1c-binding proteins. The sequence of I-2Dm is 35% identical with that of I-2, whereas the catalytic subunits themselves are >85% identical in flies and humans; however, we show that many biochemical properties of I-2 are conserved. Like I-2, I-2Dm can convert recombinant PP1c to a native-like activity. This strongly suggests that this ability is an essential, conserved role of I-2 and I-2Dm.     

In vitro characterization of a gamma-secretase radiotracer in mammalian brain

Inhibition of gamma-secretase is a potential therapeutic target for Alzheimer's disease (AD). The present studies have characterized the in vitro properties of a radiolabeled small molecule gamma-secretase inhibitor, [3H]compound D (Yan et al., 2004, J. Neurosci.24, 2942-2952) in mammalian brain. [3H]Compound D was shown to bind with nanomolar affinity (Kd = 0.32-1.5 nM) to a single population of saturable sites in rat, rhesus and human brain cortex homogenates, the density of binding sites ranging from 4 to 7 nM across the species. Competition studies with a structurally diverse group of gamma-secretase inhibitors with a wide range of binding affinities showed that the binding affinities of these compounds correlated well with their ability to inhibit gamma-secretase in vitro. Autoradiographic studies showed that the specific binding of [3H]compound D was widely distributed throughout adult rat, rhesus and normal human brain. There did not appear to be any difference in distribution of [3H]compound D specific binding sites in AD cortex compared with control human cortex as measured using tissue section autoradiography, nor any correlation between gamma-secretase binding and plaque burden as measured immunohistochemically. [3H]compound D is a useful tool to probe the expression and pharmacology of gamma-secretase in mammalian brain.     

The role of SMARCAL1 in replication fork stability and telomere maintenance

SMARCAL1 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A-Like 1), also known as HARP, is an ATP-dependent annealing helicase that stabilizes replication forks during DNA damage. Mutations in this gene are the cause of Schimke immune-osseous dysplasia (SIOD), an autosomal recessive disorder characterized by T-cell immunodeficiency and growth dysfunctions. In this review, we summarize the main roles of SMARCAL1 in DNA repair, telomere maintenance and replication fork stability in response to DNA replication stress.     

Telomere protection without a telomerase; the role of ATM and Mre11 in Drosophila telomere maintenance

The conserved ATM checkpoint kinase and the Mre11 DNA repair complex play essential and overlapping roles in maintaining genomic integrity. We conducted genetic and cytological studies on Drosophila atm and mre11 knockout mutants and discovered a telomere defect that was more severe than in any of the non-Drosophila systems studied. In mutant mitotic cells, an average of 30% of the chromosome ends engaged in telomere fusions. These fusions led to the formation and sometimes breakage of dicentric chromosomes, thus starting a devastating breakage-fusion-bridge cycle. Some of the fusions depended on DNA ligase IV, which suggested that they occurred by a nonhomologous end-joining (NHEJ) mechanism. Epistasis analyses results suggest that ATM and Mre11 might also act in the same telomere maintenance pathway in metazoans. Since Drosophila telomeres are not added by a telomerase, our findings support an additional role for both ATM and Mre11 in telomere maintenance that is independent of telomerase regulation.     

Posttranslational control of telomere maintenance and the telomere damage response

Telomeres help maintain genome integrity by protecting natural chromosome ends from being recognized as damaged DNA. When telomeres become dysfunctional, they limit replicative lifespan and prevent outgrowth of potentially cancerous cells by activating a DNA damage response that forces cells into senescence or apoptosis. On the other hand, chromosome ends devoid of proper telomere protection are subject to DNA repair activities that cause end-to-end fusions and, when cells divide, extensive genomic instability that can promote cancer. While telomeres represent unique chromatin structures with important roles in cancer and aging, we have limited understanding of the way telomeres and the response to their malfunction are controlled at the level of chromatin. Accumulating evidence indicates that different types of posttranslational modifications act in both telomere maintenance and the response to telomere uncapping. Here, we discuss the latest insights on posttranslational control of telomeric chromatin, with emphasis on ubiquitylation and SUMOylation events.