Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.     

RAD51-associated protein 1 (RAD51AP1) interacts with the meiotic recombinase DMC1 through a conserved motif

Homologous recombination (HR) reactions mediated by the RAD51 recombinase are essential for DNA and replication fork repair, genome stability, and tumor suppression. RAD51-associated protein 1 (RAD51AP1) is an important HR factor that associates with and stimulates the recombinase activity of RAD51. We have recently shown that RAD51AP1 also partners with the meiotic recombinase DMC1, displaying isoform-specific interactions with DMC1. Here, we have characterized the DMC1 interaction site in RAD51AP1 by a series of truncations and point mutations to uncover a highly conserved WVPP motif critical for DMC1 interaction but dispensable for RAD51 association. This RAD51AP1 motif is reminiscent of the FVPP motif in the tumor suppressor protein BRCA2 that mediates DMC1 interaction. These results further implicate RAD51AP1 in meiotic HR via RAD51 and DMC1.     

Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress

Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.     

The unstructured C-terminal tail of yeast Dpb11 (human TopBP1) protein is dispensable for DNA replication and the S phase checkpoint but required for the G2/M checkpoint

Budding yeast Dpb11 (human TopBP1, fission yeast Cut5) is an essential protein required for replisome assembly and for the DNA damage checkpoint. Previous studies with the temperature-sensitive dpb11-1 allele, truncated at amino acid 583 of the 764-amino acid protein, have suggested the model that Dpb11 couples DNA replication to the replication checkpoint. However, the dpb11-1 allele shows distinct replication defects even at permissive temperatures. Here, we determine that the 1-600-amino acid domain of DPB11 is both required and sufficient for full replication function of Dpb11 but that this domain is defective for activation of the principal checkpoint kinase Mec1 (human ataxia telangiectasia and Rad3-related) in vitro and in vivo. Remarkably, mutants of DPB11 that leave its replication function intact but abrogate its ability to activate Mec1 are proficient for the replication checkpoint, but they are compromised for the G(2)/M DNA damage checkpoint. These data suggest that replication checkpoint defects may result indirectly from defects in replisome assembly. Two conserved aromatic amino acids in the C terminus of Dpb11 are critical for Mec1 activation in vitro and for the G(2)/M checkpoint in yeast. Together with aromatic motifs identified previously in the Ddc1 subunit of 9-1-1, another activator of Mec1 kinase, they define a consensus structure for Mec1 activation.     

Host protein Ku70 binds and protects HIV-1 integrase from proteasomal degradation and is required for HIV replication

HIV-1 integrase (IN) is a key viral enzymatic protein acting in several viral replication steps, including integration. IN has been shown to be an unstable protein degraded by the N-end rule pathway through the host ubiquitin-proteasome machinery. However, it is still not fully understood how this viral protein is protected from the host ubiquitin-proteasome system within cells during HIV replication. In the present study, we provide evidence that the host protein Ku70 interacts with HIV-1 IN and protects it from the Lys(48)-linked polyubiquitination proteasomal pathway. Moreover, Ku70 is able to down-regulate the overall protein polyubiquitination level within the host cells and to specifically deubiquitinate IN through their interaction. Mutagenic studies revealed that the C terminus of IN (residues 230-288) is required for IN binding to the N-terminal part of Ku70 (Ku70(1-430)), and their interaction is independent of Ku70/80 heterodimerization. Finally, knockdown of Ku70 expression in both virus-producing and target CD4(+) T cells significantly disrupted HIV-1 replication and rendered two-long terminal repeat circles and integration undetectable, indicating that Ku70 is required for both the early and the late stages of the HIV-1 life cycle. Interestingly, Ku70 was incorporated into the progeny virus in an IN-dependent way. We proposed that Ku70 may interact with IN during viral assembly and accompany HIV-1 IN upon entry into the new target cells, acting to 1) protect IN from the host defense system and 2) assist IN integration activity. Overall, this report provides another example of how HIV-1 hijacks host cellular machinery to protect the virus itself and to facilitate its replication.     

Enabling association of the GINS protein tetramer with the mini chromosome maintenance (Mcm)2-7 protein complex by phosphorylated Sld2 protein and single-stranded origin DNA

The Cdc45-Mcm2-7-GINS (CMG) complex is the replication fork helicase in eukaryotes. Synthetic lethal with Dpb11-1 (Sld2) is required for the initiation of DNA replication, and the S phase cyclin-dependent kinase (S-CDK) phosphorylates Sld2 in vivo. We purified components of the replication initiation machinery and studied their interactions in vitro. We found that unphosphorylated or CDK-phosphorylated Sld2 binds to the mini chromosome maintenance (Mcm)2-7 complex with similar efficiency. Sld2 interaction with Mcm2-7 blocks the interaction between GINS and Mcm2-7. The interaction between CDK-phosphorylated Sld2 and Mcm2-7 is substantially inhibited by origin single-stranded DNA (ssDNA). Furthermore, origin ssDNA allows GINS to bind to Mcm2-7 in the presence of CDK-phosphorylated Sld2. However, unphosphorylated Sld2 blocks the interaction between GINS and Mcm2-7 even in the presence of origin ssDNA. We identified a mutant of Sld2 that does not bind to DNA. When this mutant is expressed in yeast cells, cell growth is severely inhibited with very slow progression into S phase. We propose a model wherein Sld2 blocks the interaction between GINS and Mcm2-7 in vivo. Once origin ssDNA is extruded from the Mcm2-7 ring and CDK phosphorylates Sld2, the origin ssDNA binds to CDK-phosphorylated Sld2. This event may allow the interaction between GINS and Mcm2-7 in vivo. Thus, CDK phosphorylation of Sld2 may be important to release Sld2 from Mcm2-7, thereby allowing GINS to bind Mcm2-7. Furthermore, origin ssDNA may stimulate the formation of the CMG complex by alleviating inhibitory interactions between Sld2 with Mcm2-7.