BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP–heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

Fidelity of G protein β-subunit association by the G protein γ-subunit-like domains of RGS6, RGS7, and RGS11

Several regulators of G protein signaling (RGS) proteins contain a G protein γ-subunit-like (GGL) domain, which, as we have shown, binds to Gβ5 subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human RGS6. When RGS6 is coexpressed with different Gβ subunits, only RGS6 and Gβ5 interact. The expression of mRNA for RGS6 and Gβ5 in human tissues overlaps. Predictions of α-helical and coiled-coil character within GGL domains, coupled with measurements of Gβ binding by GGL domain mutants, support the contention that Gγ-like regions within RGS proteins interact with Gβ5 subunits in a fashion comparable to conventional Gβ/Gγ pairings. Mutation of the highly conserved Phe-61 residue of Gγ2 to tryptophan, the residue present in all GGL domains, increases the stability of the Gβ5/Gγ2 heterodimer, highlighting the importance of this residue to GGL/Gβ5 association.

A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage

Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. Much of our current understanding of checkpoints comes from genetic studies conducted in yeast. In the fission yeast Schizosaccharomyces pombe (Sp), SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints. The SpChk1 and SpCds1 protein kinases function downstream of SpRad3. SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint. SpCds1 functions in the DNA replication checkpoint and in the S phase DNA damage checkpoint. Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified. Here we report the identification of a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural, and functional similarity to SpCds1. HuCds1 was modified by phosphorylation and activated in response to ionizing radiation. It was also modified in response to hydroxyurea treatment. Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after hydroxyurea treatment. Like its fission yeast counterpart, human Cds1 phosphorylated Cdc25C to promote the binding of 14-3-3 proteins. These findings suggest that the checkpoint function of HuCds1 is conserved in yeast and mammals.

Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation

Electrical stimulation of neonatal cardiac myocytes produces hypertrophy and cellular maturation with increased mitochondrial content and activity. To investigate the patterns of gene expression associated with these processes, cardiac myocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation [c-fos, c-jun, JunB, nuclear respiratory factor 1 (NRF-1)], mitochondrial proliferation [cytochrome c (Cyt c), cytochrome oxidase], and mitochondrial differentiation [carnitine palmitoyltransferase I (CPT-I) isoforms] were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25–3 hr) and followed sequentially by c-jun (0.5–3 hr), JunB (0.5–6 hr), NRF-1 (1–12 hr), Cyt c (12–72 hr), and muscle-specific CPT-I (48–72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA, thus supporting the developmental fidelity of this pattern of gene regulation. Consistent with a transcriptional mechanism, electrical stimulation increased c-fos, β-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element, and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the NRF-1 and CRE sites inhibited the induction by electrical stimulation (5-fold and 2-fold, respectively) whereas mutation of the Sp-1 site maintained or increased the fold induction. This finding is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c and suggests that induction of these transcription factors is a prerequisite for the transcriptional activation of Cyt c expression. These results support a regulatory role for NRF-1 and possibly AP-1 in the initiation of mitochondrial proliferation.

Thermus thermophilus: A link in evolution of the tRNA-dependent amino acid amidation pathways

Thermus thermophilus possesses an aspartyl-tRNA synthetase (AspRS2) able to aspartylate efficiently tRNAAsp and tRNAAsn. Aspartate mischarged on tRNAAsn then is converted into asparagine by an ω amidase that differs structurally from all known asparagine synthetases. However, aspartate is not misincorporated into proteins because the binding capacity of aminoacylated tRNAAsn to elongation factor Tu is only conferred by conversion of aspartate into asparagine. T. thermophilus additionally contains a second aspartyl-tRNA synthetase (AspRS1) able to aspartylate tRNAAsp and an asparaginyl-tRNA synthetase able to charge tRNAAsn with free asparagine, although the organism does not contain a tRNA-independent asparagine synthetase. In contrast to the duplicated pathway of tRNA asparaginylation, tRNA glutaminylation occurs in the thermophile via the usual pathway by using glutaminyl-tRNA synthetase and free glutamine synthesized by glutamine synthetase that is unique. T. thermophilus is able to ensure tRNA aminoacylation by alternative routes involving either the direct pathway or by conversion of amino acid mischarged on tRNA. These findings shed light on the interrelation between the tRNA-dependent and tRNA-independent pathways of amino acid amidation and on the processes involved in fidelity of the aminoacylation systems.

A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete–scute complex, and antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.

Organization of G Proteins and Adenylyl Cyclase at the Plasma Membrane

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein α and β subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein α subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation

High affinity antibodies are generated in mice and humans by means of somatic hypermutation (SHM) of variable (V) regions of Ig genes. Mutations with rates of 10−5–10−3 per base pair per generation, about 106-fold above normal, are targeted primarily at V-region hot spots by unknown mechanisms. We have measured mRNA expression of DNA polymerases ι, η, and ζ by using cultured Burkitt's lymphoma (BL)2 cells. These cells exhibit 5–10-fold increases in heavy-chain V-region mutations targeted only predominantly to RGYW (R = A or G, Y = C or T, W = T or A) hot spots if costimulated with T cells and IgM crosslinking, the presumed in vivo requirements for SHM. An ∼4-fold increase pol ι mRNA occurs within 12 h when cocultured with T cells and surface IgM crosslinking. Induction of pols η and ζ occur with T cells, IgM crosslinking, or both stimuli. The fidelity of pol ι was measured at RGYW hot- and non-hot-spot sequences situated at nicks, gaps, and double-strand breaks. Pol ι formed T⋅G mispairs at a frequency of 10−2, consistent with SHM-generated C to T transitions, with a 3-fold increased error rate in hot- vs. non-hot-spot sequences for the single-nucleotide overhang. The T cell and IgM crosslinking-dependent induction of pol ι at 12 h may indicate an SHM “triggering” event has occurred. However, pols ι, η, and ζ are present under all conditions, suggesting that their presence is not sufficient to generate mutations because both T cell and IgM stimuli are required for SHM induction.

Participation of recombination proteins in rescue of arrested replication forks in UV-irradiated Escherichia coli need not involve recombination

Alternative reproductive cycles make use of different strategies to generate different reproductive products. In Escherichia coli, recA and several other rec genes are required for the generation of recombinant genomes during Hfr conjugation. During normal asexual reproduction, many of these same genes are needed to generate clonal products from UV-irradiated cells. However, unlike conjugation, this latter process also requires the function of the nucleotide excision repair genes. Following UV irradiation, the recovery of DNA replication requires uvrA and uvrC, as well as recA, recF, and recR. The rec genes appear to be required to protect and maintain replication forks that are arrested at DNA lesions, based on the extensive degradation of the nascent DNA that occurs in their absence. The products of the recJ and recQ genes process the blocked replication forks before the resumption of replication and may affect the fidelity of the recovery process. We discuss a model in which several rec gene products process replication forks arrested by DNA damage to facilitate the repair of the blocking DNA lesions by nucleotide excision repair, thereby allowing processive replication to resume with no need for strand exchanges or recombination. The poor survival of cellular populations that depend on recombinational pathways (compared with that in their excision repair proficient counterparts) suggests that at least some of the rec genes may be designed to function together with nucleotide excision repair in a common and predominant pathway by which cells faithfully recover replication and survive following UV-induced DNA damage.

Accuracy of lesion bypass by yeast and human DNA polymerase η

DNA polymerase η (Polη) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Polη in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Polη replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Polη, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of ≈ 10−2 to 10−3 opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Polη in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Polη to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Polη extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of ≈ 10−2 to 10−3 relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Polη would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3′→5′ exonuclease. Replication through a TT dimer by Polη then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development

Runx (Cbfa/AML) transcription factors are critical for tissue-specific gene expression. A unique targeting signal in the C terminus directs Runx factors to discrete foci within the nucleus. Using Runx2/CBFA1/AML3 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (Runx2ΔC) do not form bone due to maturational arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2ΔC protein enters the nucleus and retains normal DNA binding activity, but shows complete loss of intranuclear targeting. These results demonstrate that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. We conclude that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development.

Replication infidelity during a single cycle of Ty1 retrotransposition.

Retroviruses undergo a high frequency of genetic alterations during the process of copying their RNA genomes. However, little is known about the replication fidelity of other elements that transpose via reverse transcription of an RNA intermediate. The complete sequence of 29 independently integrated copies of the yeast retrotransposon Ty1 (173,043 nt) was determined, and the mutation rate during a single cycle of replication was calculated. The observed base substitution rate of 2.5 x 10(-5) bp per replication cycle suggests that this intracellular element can mutate as rapidly as retroviruses. The pattern and distribution of errors in the Ty1 genome is nonrandom and provides clues to potential in vivo molecular mechanisms of reverse transcriptase-mediated error generation, including heterogeneous RNase H cleavage of Ty1 RNA, addition of terminal nontemplated bases, and transient dislocation and realignment of primer-templates. Overall, analysis of errors generated during Ty1 replication underscores the utility of a genetically tractable model system for the study of reverse transcriptase fidelity.

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

A thymidine triphosphate shape analog lacking Watson–Crick pairing ability is replicated with high sequence selectivity

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5′-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo− mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F–A pair is associated with a brief pause, whereas that beyond A in the inverted A–F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A–F/F–A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.

BRCA1 is associated with the centrosome during mitosis

Centrosomes and their associated microtubules direct events during mitosis and control the organization of animal cell structures and movement during interphase. The centrosome replicates during the cell cycle, directs the assembly of bipolar mitotic spindles, and plays an important role in maintaining the fidelity of cell division. Recently, tumor suppressors such as p53 and retinoblastoma protein pRB have been localized to the centrosome in a cell cycle-dependent manner. Immunofluorescence microscopy and analysis of isolated centrosomes now provide evidence that BRCA1 protein, a suppressor of tumorigenesis in breast and ovary, also is associated with centrosomes during mitosis. Our results indicate that BRCA1 localizes with the centrosome during mitosis and coimmunoprecipitates with γ-tubulin, a centrosomal component essential for nucleation of microtubules. Furthermore, γ-tubulin associates preferentially with a hypophosphorylated form of BRCA1.