76 research outputs found
SUMO Modification Regulates BLM and RAD51 Interaction at Damaged Replication Forks
SUMO modification of BLM controls the switch between BLM's pro- and anti-recombinogenic roles in homologous recombination following DNA damage during replication
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SUMO Modification Regulates BLM and RAD51 Interaction at Damaged Replication Forks
The gene mutated in Bloom's syndrome, BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased Ξ³-H2AX foci. Because the increased Ξ³-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM's function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess Ξ³-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM's pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.</p
Proteomic analysis of the mammalian nuclear pore complex
As the sole site of nucleocytoplasmic transport, the nuclear pore complex (NPC) has a vital cellular role. Nonetheless, much remains to be learned about many fundamental aspects of NPC function. To further understand the structure and function of the mammalian NPC, we have completed a proteomic analysis to identify and classify all of its protein components. We used mass spectrometry to identify all proteins present in a biochemically purified NPC fraction. Based on previous characterization, sequence homology, and subcellular localization, 29 of these proteins were classified as nucleoporins, and a further 18 were classified as NPC-associated proteins. Among the 29 nucleoporins were six previously undiscovered nucleoporins and a novel family of WD repeat nucleoporins. One of these WD repeat nucleoporins is ALADIN, the gene mutated in triple-A (or Allgrove) syndrome. Our analysis defines the proteome of the mammalian NPC for the first time and paves the way for a more detailed characterization of NPC structure and function
Molecular Composition of Staufen2-Containing Ribonucleoproteins in Embryonic Rat Brain
Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Numerous mRNA-binding and regulatory proteins within mRNPs finely regulate the fate of bound-mRNAs. Their specific combination defines different types of mRNPs that in turn are related to specific synaptic functions. One of these mRNA-binding proteins, Staufen2 (Stau2), was shown to transport dendritic mRNAs along microtubules. Its knockdown expression in neurons was shown to change spine morphology and synaptic functions. To further understand the molecular mechanisms by which Stau2 modulates synaptic function in neurons, it is important to identify and characterize protein co-factors that regulate the fate of Stau2-containing mRNPs. To this end, a proteomic approach was used to identify co-immunoprecipitated proteins in Staufen2-containing mRNPs isolated from embryonic rat brains. The proteomic approach identified mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (Ξ±- and Ξ²-tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. PABPC1 and YB1 proteins formed puncta in dendrites of embryonic rat hippocampal neurons. However, they poorly co-localized with Stau2 in the large dendritic complexes suggesting that they are rather components of Stau2-containing mRNA particles. All together, these results represent a further step in the characterization of Stau2-containing mRNPs in neurons and provide new tools to study and understand how Stau2-containing mRNPs are transported, translationally silenced during transport and/or locally expressed according to cell needs
SUMO-Interacting Motifs of Human TRIM5Ξ± are Important for Antiviral Activity
Human TRIM5Ξ± potently restricts particular strains of murine leukemia viruses
(the so-called N-tropic strains) but not others (the B- or NB-tropic strains)
during early stages of infection. We show that overexpression of SUMO-1 in human
293T cells, but not in mouse MDTF cells, profoundly blocks N-MLV infection. This
block is dependent on the tropism of the incoming virus, as neither B-, NB-, nor
the mutant R110E of N-MLV CA (a B-tropic switch) are affected by SUMO-1
overexpression. The block occurred prior to reverse transcription and could be
abrogated by large amounts of restricted virus. Knockdown of TRIM5Ξ± in 293T
SUMO-1-overexpressing cells resulted in ablation of the SUMO-1 antiviral
effects, and this loss of restriction could be restored by expression of a human
TRIM5Ξ± shRNA-resistant plasmid. Amino acid sequence analysis of human
TRIM5Ξ± revealed a consensus SUMO conjugation site at the N-terminus and
three putative SUMO interacting motifs (SIMs) in the B30.2 domain. Mutations of
the TRIM5Ξ± consensus SUMO conjugation site did not affect the antiviral
activity of TRIM5Ξ± in any of the cell types tested. Mutation of the SIM
consensus sequences, however, abolished TRIM5Ξ± antiviral activity against
N-MLV. Mutation of lysines at a potential site of SUMOylation in the CA region
of the Gag gene reduced the SUMO-1 block and the TRIM5Ξ± restriction of
N-MLV. Our data suggest a novel aspect of TRIM5Ξ±-mediated restriction, in
which the presence of intact SIMs in TRIM5Ξ±, and also the SUMO conjugation
of CA, are required for restriction. We propose that at least a portion of the
antiviral activity of TRIM5Ξ± is mediated through the binding of its SIMs to
SUMO-conjugated CA
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