M.V. Volkenstein, evolutionary thinking and the structure of fitness landscapes.

High dimensional fitness landscapes are robustly dominated by saddle points, not isolated peaks. We present an argument to this effect that is reminiscent of May's complexity stability analysis and trace out the significance for the dynamics of speciation, the connection between the neutral and punctuated aspects of evolution and evolution on moving landscapes. The paper is written in honor of M.V. Volkenstein (October 23rd, 1912-February 18th, 1992), who devoted his last papers to uniting dynamics with evolutionary thinking.     

Analysis of regions of RAG-2 important for V(D)J recombination.

The recombinase activating genes RAG-1 and RAG-2 operate together to activate V(D)J recombination, and thus play an essential role in the generation of immune system diversity. As a first step in understanding the function of the RAG-2 protein, we have tested a series of deletion and insertion mutations for their ability to induce V(D)J joining of a variety of model substrates. Mutants were assayed for their ability to induce deletional and inversional V(D)J joining, thereby testing their proficiency at forming both signal and coding joints, and, in some cases, for their ability to carry out recombination of both extrachromosomal and integrated recombination substrates. All these reactions were affected similarly by any one mutation. Although the RAG-2 protein shows extensive evolutionary conservation across its length, we found that the carboxy-terminal portion of RAG-2, including an acidic region, is dispensable for all forms of recombination tested. In contrast, all mutations we created in the N-terminal region severely decreased recombination. Thus, the core active region required for V(D)J recombination is confined to the first three-quarters of the RAG-2 protein.     

Cryptic signals and the fidelity of V(D)J joining.

V(D)J recombination is responsible for the de novo creation of antigen receptor genes in T- and B-cell precursors. To the extent that lymphopoiesis takes place throughout an animal's lifetime, recombination errors present an ongoing problem. One type of aberrant rearrangement ensues when DNA sequences resembling a V(D)J joining signal are targeted by mistake. This study investigates the type of sequence likely to be subject to mistargeting, the level of joining-signal function associated with these sequences, and the number of such cryptic joining signals in the genome.     

Excision of the Drosophila mariner transposon Mos1. Comparison with bacterial transposition and V(D)J recombination

It has been proposed that the modern immune system has evolved from a transposon in an ancient vertebrate. While much is known about the mechanism by which bacterial transposable elements catalyze double-strand breaks at their ends, less is known about how eukaryotic transposable elements carry out these reactions. We have examined the mechanism by which mariner, a eukaryotic transposable element, performs DNA cleavage. We show that the nontransferred strand is cleaved initially, unlike prokaryotic transposons which cleave the transferred strand first. First strand cleavage is not tightly coupled to second strand cleavage and can occur independently of synapsis, as happens in V(D)J recombination but not in transposition of prokaryotic transposons. Unlike V(D)J recombination, however, second strand cleavage of mariner does not occur via a hairpin intermediate.     

A comparison of the energetics of annexin I and annexin V.

The annexins comprise a family of soluble Ca2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min-1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t1/2=61.8 degrees C, DeltaHcal=824 kJ mol-1 and DeltaCp=19 kJ mol-1 K-1. These parameters result in a stability value of DeltaG0D (20 degrees C)=51 kJ mol-1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded DeltaG0D (buffer) values of 48, 60 and 36 kJ mol-1 at 20, 12 and 5 degrees C, respectively. These DeltaG0D values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.     

Cooperation of the V1/V2 and V3 domains of human immunodeficiency virus type 1 gp120 for interaction with the CXCR4 receptor

Human immunodeficiency virus type 1 (HIV-1) entry is triggered by the interaction of the gp120 envelope glycoprotein with a cellular chemokine receptor, either CCR5 or CXCR4. We have identified different mutations in human CXCR4 that prevent efficient infection by one HIV-1 strain (NDK) but not another (LAI) and sought to define these strain-dependent effects at the gp120 level. The lack of activity toward the NDK strain of the HHRH chimeric CXCR4 in which the second extracellular loop (ECL2) derived from the rat CXCR4 and of CXCR4 with mutations at an aspartic acid in ECL2 (D193A and D193R) was apparently due to the sequence of the third variable loop (V3) of gp120, more precisely, to its C-terminal part. Indeed, substitution of the LAI V3 loop or only its C-terminal part in the NDK gp 120 context was sufficient to restore usage of the HHRH, D193A, and D193R receptors. The same result was achieved upon mutation of a single lysine residue of the NDK V3 loop to alanine (K319A) but not to arginine (K319R). These results provide a strong case for a direct interaction between the gp120 V3 loop and the ECL2 domain of CXCR4. By contrast, V3 substitutions had no effect on the inability of NDK to infect cells via a mutant CXCR4 in which the amino-terminal extracellular domain (NT) is deleted. In experiments with a set of chimeric NDK-LAI gp120s, the V1/V2 region from LAI gp120 was both necessary and sufficient for usage of the NT-deleted CXCR4. Different variable domains of gp120 can therefore cooperate for a functional interaction with CXCR4.     

Kinetic analysis of the nicking and hairpin formation steps in V(D)J recombination

The complete cleavage phase of V(D)J recombination includes four phases: binding of the active RAG complexes to the 12- or 23-signals, nicking of the signals, synapsis of the two signals, and hairpin formation at both signals concurrently. We have done time courses for the complete cleavage phase of the V(D)J recombination reaction and quantitated the amount of active RAG enzyme. We have also formulated a kinetic model for the binding, nicking, synapsis, and hairpin formation phases. We have utilized free solution enzymatic measurements for the binding and nicking phases as we do mathematical simulations of the kinetic model. This permits iteration of rate constants for the synapsis and hairpin formation phases until the model fits the observed overall cleavage time course. This process yields a rate constant for the hairpin formation that is 0.004 min(-1), which corresponds to an average catalytic cycle time of 250 min. This value is exceedingly close to a measured value of this constant that relied on wash-out of an inhibitory cofactor. The agreement indicates that this is likely to be the rate of the hairpin step over a wide range of range of conditions and irrespective of the DNA sequence of the V, D or J coding end located adjacent to the signal. These findings indicate that, under optimal in vitro conditions, the core RAG proteins carry out nicking at a rate which is nearly 150-fold faster than hairpin formation. The physiologic implications of this and other kinetic inferences of these time courses are discussed.     

Chromatin remodeling at the Ig loci prior to V(D)J recombination.

Rearrangement of Ig H and L chain genes is highly regulated and takes place sequentially during B cell development. Several lines of evidence indicate that chromatin may modulate accessibility of the Ig loci for V(D)J recombination. In this study, we show that remodeling of V and J segment chromatin occurs before V(D)J recombination at the endogenous H and kappa L chain loci. In recombination-activating gene-deficient pro-B cells, there is a reorganization of nucleosomal structure over the H chain J(H) cluster and increased DNase I sensitivity of V(H) and J(H) segments. The pro-B/pre-B cell transition is marked by a decrease in the DNase I sensitivity of V(H) segments and a reciprocal increase in the nuclease sensitivity of Vkappa and Jkappa segments. In contrast, J(H) segments remain DNase I sensitive, and their nucleosomal organization is maintained in mu(+) recombination-activating gene-deficient pre-B cells. These results indicate that initiation of rearrangement is associated with changes in the chromatin structure of both V and J segments, whereas stopping recombination involves changes in only V segment chromatin. We further find an increase in histone H4 acetylation at both the H and kappa L chain loci at the pro-B cell stage. Although histone H4 acetylation appears to be an early change associated with B cell commitment, acetylation alone is not sufficient to promote subsequent modifications in Ig chromatin.     

V(D)J recombination: site-specific cleavage and repair

V(D)J recombination is a site-specific gene rearrangement process that contributes to the diversity of antigen receptor repertoires. Two lymphoid-specific proteins, RAG1 and RAG2, initiate this process at two recombination signal sequences. Due to the recent development of an in vitro assay for V(D)J cleavage, the mechanism of cleavage has been elucidated clearly. The RAG complex recognizes a recombination signal sequence, makes a nick at the border between signal and coding sequence, and carries out a transesterification reaction, resulting in the production of a hairpin structure at the coding sequence and DNA double-strand breaks at the signal ends. RAG1 possesses the active site of the V(D)J recombinase although RAG2 is essential for signal binding and cleavage. After DNA cleavage by the RAG complex, the broken DNA ends are rejoined by the coordinated action of DNA double-strand break repair proteins as well as the RAG complex. The junctional variability resulting from imprecise joining of the coding sequences contributes additional diversity to the antigen receptors.     

Differential Effects of CSF-1R D802V and KIT D816V Homologous Mutations on Receptor Tertiary Structure and Allosteric Communication

The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.     

Bovine coagulation factor V visualized with electron microscopy. Ultrastructure of the isolated activated forms and of the activation fragments

Single chain bovine factor V (Mr = 330,000) was isolated and visualized by means of high resolution transmission electron microscopy of negatively stained samples. Both factor Va, activated by thrombin or by the factor V activator from Russell's viper venom, and the isolated fragments, D (Mr = 105,000), C1 (Mr = 150,000), and F1F2 (Mr = 72,000), were studied. Single chain factor V appeared as a multidomain structure with three globular domains of similar size (diameter approximately 80 A), and oriented around a somewhat larger central domain (diameter approximately 140 A). The distance between the center of the molecule and the center of each of the peripheral domains was 120 A and the maximum length of factor V was 300 A. The structure was essentially identical with that recently shown for human single chain factor V (Dahlb?ck, B. (1985) J. Biol. Chem. 260, 1347-1349). Isolated thrombin-activated factor Va (containing fragments D and F1F2) was composed of two domains of similar size, each of which was approximately 80 A in diameter and corresponded in size and shape to the peripheral domains seen in intact factor V. The isolated activation fragment C1 appeared as an irregular structure with an approximate diameter of 140 A and corresponded in size and shape to the larger central domain in intact factor V. The activator from Russell's viper venom only cleaves the bond(s) between C1 and F1F2, which results in two fragments, a larger fragment (Mr = 220,000) bearing the D, E, and C1 region and a smaller one corresponding to the F1F2 fragment. The venom-activated factor Va in the electron microscope demonstrated a multidomain structure similar in size and shape to that obtained with intact factor V. A model for factor V and the molecular events involved in activation is proposed.     

The structure of V alpha and J alpha segments in the mouse.

Antigen receptors on most T-cells are heterodimeric glycoproteins, comprised of an alpha chain and a beta chain. These chains are encoded by discontiguous variable (V), diversity (D) and joining (J) gene segments that rearrange to produce a contiguous and functional alpha or beta chain gene. To investigate the size and diversity of the germline repertoire of alpha-chain gene segments, we have characterized and sequenced 20 alpha chain cDNAs. Among these cDNA clones, we have found 4 J alpha and 4 V alpha sequences that have not yet been described. The relationship of these "new" gene segments to those already characterized is discussed.     

Strand breaks without DNA rearrangement in V (D)J recombination.

Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previously been shown to deleteriously affect the V(D)J recombination process. Cassettes containing a point mutation in one of the two signal sequences inhibited rearrangement in wild-type cells. In contrast, scid cells continued to rearrange these cassettes with the characteristic scid deletional phenotype. Using these mutated templates, we identified junctional modifications at the wild-type signal sequences that had arisen from strand breaks which were not associated with overall V(D)J rearrangements. Neither cell type was able to rearrange constructs which contained only a single, nonmutated, signal sequence. In addition, scid and wild-type cell lines harboring cassettes with mutations in both signal sequences did not undergo rearrangement, suggesting that at least one functional signal sequence was required for all types of V(D)J recombination events. Analysis of these signal sequence mutations has provided insights into intermediates in the V(D)J rearrangement pathway in wild-type and scid pre-B cells.     

Identification of a novel target of D/V signaling in Drosophila wing disc: Wg-independent function of the organizer

Growth and patterning during Drosophila wing development are mediated by signaling from its dorso-ventral (D/V) organizer. Wingless is expressed in the D/V boundary and functions as a morphogen to activate target genes at a distance. Wingless pathway and thereby D/V signaling is negatively regulated by the homeotic gene Ultrabithorax (Ubx) to mediate haltere development. In an enhancer-trap screen to identify genes that show differential expression between wing and haltere discs, we identified CG32062, which codes for a RNA-binding protein. In wing discs, CG32062 is expressed only in non-D/V cells. CG32062 expression in non-D/V cells is dependent on Notch-mediated signaling from the D/V boundary. However, CG32062 expression is independent of Wingless function, thus providing evidence for a second long-range signaling mechanism of the D/V organizer. In haltere discs, CG32062 is negatively regulated by Ubx. The non-cell autonomous nature of Ubx-mediated repression of CG32062 expression suggests that the novel component of D/V signaling is also negatively regulated during haltere specification.     

Unveiling the Unfolding Pathway of F5F8D Disorder-Associated D81H/V100D Mutant of MCFD2 via Multiple Molecular Dynamics Simulations

Combined factor deficiency (F5F8D) is a rare autosomal recessive disorder caused by mutations in the LMAN1 or MCFD2 genes. It has been proposed that this pathogenic process occurs via a multi-step pathway involving metal loss, EF-hand-Ca21 dissociation and assembly of misfolded MCFD2-LMAN1 complex. Here, we have investigated the solution conformations of the MCFD2((D81H,V100D)) protein mutant through extensive molecular dynamics (MD) simulations. The V100D, one of the many MCFD2 mutations known to be associated to F5F8D, is difficult to be reconciled with the pathway model because it is located far from the metal sites and the MCFD2/LMAN1 interface. Consequently, an inspection of all the steps involved in D81H/V100D MCFD2 misfolding is expected to provide hints in the understanding of the molecular basis of the disease. A comparison with parallel studies carried out for the Wild-Type (WT) MCFD2 pointed out that the mutation decreases the affinity of the protein for the Ca21 ion. Multiple explicit solvents MD simulations (50_ns) performed on the two proteins revealed that in the WT protein, stable H-bond network and compact hydrophobic core region are created thus confirming a pivotal role of this region in driving the biophysical properties of the entire protein. In fact it is shown that the V100D mutation, although located far away the EF-hand domain, may induce subtle modification in the structural core of MCFD2 leading to the loosening of metal binding and to the formation of metastable intermediate states along the unfolding pathway. The native-like hydrophobic cluster formed near the V100 residue in the wild-type protein is disrupted by the negatively charged Asparagine residue. Furthermore, the presence of the D81H mutation in the EF-1 hand domain may also increase the protein unfolding rate and consequently prevent the formation of the MCFD2-LMAN1 complex. The detailed structural insights obtained from our large-scale simulations complement the clinical features and offer useful insights into the mechanism behind MCFD2 protein misfolding.     

The nicking step in V(D)J recombination is independent of synapsis: implications for the immune repertoire

In all of the transposition reactions that have been characterized thus far, synapsis of two transposon ends is required before any catalytic steps (strand nicking or strand transfer) occur. In V(D)J recombination, there have been inconclusive data concerning the role of synapsis in nicking. Synapsis between two 12-substrates or between two 23-substrates has not been ruled out in any studies thus far. Here we provide the first direct tests of this issue. We find that immobilization of signals does not affect their nicking, even though hairpinning is affected in a manner reflecting its known synaptic requirement. We also find that nicking is kinetically a unireactant enzyme-catalyzed reaction. Time courses are no different between nicking seen for a 12-substrate alone and a reaction involving both a 12- and a 23-substrate. Hence, synapsis is neither a requirement nor an effector of the rate of nicking. These results establish V(D)J recombination as the first example of a DNA transposition-type reaction in which catalytic steps begin prior to synapsis, and the results have direct implications for the order of the steps in V(D)J recombination, for the contribution of V(D)J recombination nicks to genomic instability, and for the diversification of the immune repertoire.     

The Discovery of Error-prone DNA Polymerase V and Its Unique Regulation by RecA and ATP

My career pathway has taken a circuitous route, beginning with a Ph.D. degree in electrical engineering from The Johns Hopkins University, followed by five postdoctoral years in biology at Hopkins and culminating in a faculty position in biological sciences at the University of Southern California. My startup package in 1973 consisted of $2,500, not to be spent all at once, plus an ancient Packard scintillation counter that had a series of rapidly flashing light bulbs to indicate a radioactive readout in counts/minute. My research pathway has been similarly circuitous. The discovery of Escherichia coli DNA polymerase V (pol V) began with an attempt to identify the mutagenic DNA polymerase responsible for copying damaged DNA as part of the well known SOS regulon. Although we succeeded in identifying a DNA polymerase, one that was induced as part of the SOS response, we actually rediscovered DNA polymerase II, albeit in a new role. A decade later, we discovered a new polymerase, pol V, whose activity turned out to be regulated by bound molecules of RecA protein and ATP. This Reflections article describes our research trajectory, includes a review of key features of DNA damage-induced SOS mutagenesis leading us to pol V, and reflects on some of the principal researchers who have made indispensable contributions to our efforts.     

CpG methylated minichromosomes become inaccessible for V(D)J recombination after undergoing replication.

The physical parameters controlling the accessibility of antigen receptor loci to the V(D)J recombination activity are unknown. We have used minichromosome substrates to study the role that CpG methylation might play in controlling V(D)J recombination site accessibility. We find that CpG methylation decreases the V(D)J recombination of these substrates more than 100-fold. The decrease correlates with a considerable increase in resistance to endonuclease digestion of the methylated minichromosome DNA. The minichromosomes acquire resistance to both the intracellular V(D)J recombinase and exogenous endonuclease only after DNA replication. Therefore, CpG methylation specifies a chromatin structure that, upon DNA replication, is resistant to eukaryotic site-specific recombination. These findings are important to V(D)J recombination as well as to the chromatin assembly of methylated DNA during replication.     

Menage á trois: Double strand break repair,V(D)J recombination and DNA-PK

All organisms possess mechanisms to repair double strand breaks (dsbs) generated in their DNA by damaging agents. Site-specific dsbs are also introduced during V(D)J recombination. Four complementation groups of radiosensitive rodent mutants are defective in the repair of dsbs, and are unable to carry out V(D)J recombination effectively. The immune defect in Severe Combined Immunodeficient (scid) mice also results from an inability to undergo effective V(D)J recombination, and scid cell lines display a repair defect and belong to one of these complementation groups. These findings indicate a mechanistic overlap between the processes of DNA repair and V(D)J recombination. Recently, two of the genes defined by these complementation groups have been identified and shown to encode components of DNA-dependent protein kinase (DNA-PK). We review here the three fields which have become linked by these findings, and discuss the involvement of DNA-PK in dsb rejoining and in V(D)J recombination.