Chromosome Dynamics during Mitosis

The primary goal of mitosis is to partition duplicated chromosomes into daughter cells. Eukaryotic chromosomes are equipped with two distinct classes of intrinsic machineries, cohesin and condensins, that ensure their faithful segregation during mitosis. Cohesin holds sister chromatids together immediately after their synthesis during S phase until the establishment of bipolar attachments to the mitotic spindle in metaphase. Condensins, on the other hand, attempt to “resolve” sister chromatids by counteracting cohesin. The products of the balancing acts of cohesin and condensins are metaphase chromosomes, in which two rod-shaped chromatids are connected primarily at the centromere. In anaphase, this connection is released by the action of separase that proteolytically cleaves the remaining population of cohesin. Recent studies uncover how this series of events might be mechanistically coupled with each other and intricately regulated by a number of regulatory factors.In eukaryotic cells, genomic DNA is packaged into chromatin and stored in the cell nucleus, in which essential chromosomal processes, including DNA replication and gene expression, take place (Fig. 1, interphase). At the onset of mitosis, the nuclear envelope breaks down and chromatin is progressively converted into a discrete set of rod-shaped structures known as metaphase chromosomes (Fig. 1, metaphase). In each chromosome, a pair of sister kinetochores assembles at its centromeric region, and their bioriented attachment to the mitotic spindle acts as a prerequisite for equal segregation of sister chromatids. The linkage between sister chromatids is dissolved at the onset of anaphase, allowing them to be pulled apart to opposite poles of the cell (Fig. 1, anaphase). At the end of mitosis, the nuclear envelope reassembles around two sets of segregated chromatids, leading to the production of genetically identical daughter cells (Fig. 1, telophase).Open in a separate windowFigure 1.Overview of chromosome dynamics during mitosis. In addition to the crucial role of kinetochore–spindle interactions, an intricate balance between cohesive and resolving forces acting on sister chromatid arms (top left, inset) underlies the process of chromosome segregation. See the text for major events in chromosome segregation.Although the centromere–kinetochore region plays a crucial role in the segregation process, sister chromatid arms also undergo dynamic structural changes to facilitate their own separation. Conceptually, such structural changes are an outcome of two balancing forces, namely, cohesive and resolving forces (Fig. 1, top left, inset). The cohesive force holds a pair of duplicated arms until proper timing of separation, otherwise daughter cells would receive too many or too few copies of chromosomes. The resolving force, on the other hand, counteracts the cohesive force, reorganizing each chromosome into a pair of rod-shaped chromatids. From this standpoint, the pathway of chromosome segregation is regarded as a dynamic process, in which the initially robust cohesive force is gradually weakened and eventually dominated by the resolving force. Almost two decades ago, genetic and biochemical studies for the behavior of mitotic chromosomes converged productively, culminating in the discovery of cohesin (Guacci et al. 1997; Michaelis et al. 1997; Losada et al. 1998) and condensin (Hirano et al. 1997; Sutani et al. 1999), which are responsible for the cohesive and resolving forces, respectively. The subsequent characterizations of these two protein complexes have not only transformed our molecular understanding of chromosome dynamics during mitosis and meiosis, but also provided far-reaching implications in genome stability, as well as unexpected links to human diseases. In this article, I summarize recent progress in our understanding of mitotic chromosome dynamics with a major focus on the regulatory networks surrounding cohesin and condensin. I also discuss emerging topics and attempt to clarify outstanding questions in the field.     

Recombination,Pairing, and Synapsis of Homologs during Meiosis

Recombination is a prominent feature of meiosis in which it plays an important role in increasing genetic diversity during inheritance. Additionally, in most organisms, recombination also plays mechanical roles in chromosomal processes, most notably to mediate pairing of homologous chromosomes during prophase and, ultimately, to ensure regular segregation of homologous chromosomes when they separate at the first meiotic division. Recombinational interactions are also subject to important spatial patterning at both early and late stages. Recombination-mediated processes occur in physical and functional linkage with meiotic axial chromosome structure, with interplay in both directions, before, during, and after formation and dissolution of the synaptonemal complex (SC), a highly conserved meiosis-specific structure that links homolog axes along their lengths. These diverse processes also are integrated with recombination-independent interactions between homologous chromosomes, nonhomology-based chromosome couplings/clusterings, and diverse types of chromosome movement. This review provides an overview of these diverse processes and their interrelationships.The role of the meiotic program is to generate gametes having half the chromosome complement of the original progenitor cell. This task is accomplished by occurrence of a single round of DNA replication followed by two successive rounds of chromosome segregation. Homologs segregate to opposite poles at meiosis I, then sisters separate to opposite poles in meiosis II, analogously to mitosis (Fig. 1A).Open in a separate windowFigure 1.General features of meiosis. (A) At meiosis I, homologs segregate; at meiosis II, sisters segregate. At metaphase I (left), maternal (red) and paternal (black) chromosomes are held together by a chiasma comprising a reciprocal crossover (CO) plus connections along sister arms, which are released during segregation. (B) Monochiasmate bivalent of Locusta after bromodeoxyuridine (BrdU) incorporation. Differential staining of the sister chromatids confirms that exchange has occurred, for example, between red and purple chromatids in corresponding drawings. (From Jones 1987; reprinted, with permission, from Academic Press © 1987.) (C) Diplotene bivalent of grasshopper with three chiasmata (arrows) and corresponding drawing. (From Jones and Franklin 2006; reprinted, with permission, from Elsevier © 2006.) (D) Top: Meiotic prophase in rye microsporocytes; chromosomes are stained by hematoxylin (pictures by D.Z.). Bottom: corresponding timing of the recombination steps from double-strand breaks (DSBs) to COs; timing of intermediates as in budding yeast (Hunter 2007). SEI, Single-end invasion; dHJ, double Holliday junction; SDSA, synthesis-dependent strand annealing; NCO, noncrossover.During meiosis, a central role of recombination is to increase genetic diversity. However, recombination is also essential for two fundamental features unique to meiotic chromosome mechanics: pairing and segregation of homologous chromosomes (“homologs”). Pairing is mediated by the totality of programmed interhomolog recombinational interactions in association with chromosome structural axes (see below). Segregation is mediated specifically by the carefully chosen subset of those interactions that mature into crossover (CO) products. During segregation of homologs, just as for segregation of sister chromatids, the separating entities must be connected to one another such that regular bipolar alignment on the spindle results in tension on centromere/kinetochore complexes. When all segregating pairs are properly aligned and under tension, anaphase is triggered. Segregation of sisters is ensured by connections between sister centromere/kinetochore regions. Segregation of homologs is ensured by connections along chromosome arms that are provided by the combined effects of an interhomolog CO plus links between sisters (Fig. 1A). These connections can be seen cytologically as chiasmata (Fig. 1B,C). In organisms in which meiosis occurs without recombination, other features have evolved that hold homologs together to ensure regular segregation (Zickler and Kleckner 1998, 1999; reviewed in Stewart and Dawson 2008; Tsai and McKee 2011; Lake and Hawley 2012; Obeso et al. 2014).     

Holliday Junction Resolvases

Four-way DNA intermediates, called Holliday junctions (HJs), can form during meiotic and mitotic recombination, and their removal is crucial for chromosome segregation. A group of ubiquitous and highly specialized structure-selective endonucleases catalyze the cleavage of HJs into two disconnected DNA duplexes in a reaction called HJ resolution. These enzymes, called HJ resolvases, have been identified in bacteria and their bacteriophages, archaea, and eukaryotes. In this review, we discuss fundamental aspects of the HJ structure and their interaction with junction-resolving enzymes. This is followed by a brief discussion of the eubacterial RuvABC enzymes, which provide the paradigm for HJ resolvases in other organisms. Finally, we review the biochemical and structural properties of some well-characterized resolvases from archaea, bacteriophage, and eukaryotes.Homologous recombination (HR) is an essential process that promotes genetic diversity during meiosis (see Lam and Keeney 2014; Zickler and Kleckner 2014). However, in somatic cells, HR plays a key role in conserving genetic information by facilitating DNA repair, thereby ensuring faithful genome duplication and limiting the divergence of repetitive DNA sequences (see Mehta and Haber 2014). As shown in Figure 1, HR is initiated by a DNA double-strand break, the ends of which are resected to produce single-stranded (ss) 3′-overhangs (see Symington 2014). Homologous strand invasion by one of the 3′ overhangs (e.g., one catalyzed by Escherichia coli RecA or human RAD51) leads to the formation of a displacement loop (D-loop) (see Morrical 2014). The invading 3′ end of the D-loop can then be extended by a DNA polymerase, which uses the homologous strand as a template for DNA synthesis. Recombination then proceeds in one of several different ways, some of which involve second-end capture, such that the other resected 3′ end anneals to the displaced strand of the D-loop (Szostak et al. 1983). In the resulting recombination intermediate, the two interacting DNAs are linked by nicked Holliday junctions (HJs). Additional DNA synthesis and nick ligation lead to the formation of a double Holliday junction (dHJ) intermediate. In eukaryotes, dHJs are removed primarily by “dissolution” (Fig. 1, bottom left) (see Bizard and Hickson 2014). This pathway involves the combined activities of a DNA helicase and a type IA topoisomerase, which catalyze branch migration and decatenation of the dHJ into noncrossover products (Manthei and Keck 2014). In somatic cells, this is essential for the avoidance of sister-chromatid exchanges (SCEs) and loss of heterozygosity. Alternatively, dHJs can be processed by “resolution” in reactions mediated by canonical or noncanonical mechanisms of endonuclease-mediated cleavage into either crossover or noncrossover products (Fig. 1, bottom middle and right).Open in a separate windowFigure 1.Pathways for the formation and processing of Holliday junctions. Resected DNA double-strand breaks invade homologous duplex DNA to create a joint molecule, or displacement-loop structure. The invading 3′ end then serves as a primer for DNA synthesis, leading to second end capture and the formation of a double Holliday junction. In eukaryotes, these structures are removed by “dissolution” (bottom left panel) or “resolution” (bottom middle and right panels). Canonical Holliday junction resolvases introduce a pair of symmetrical and coordinated nicks across one of the helical axes (bottom middle panel) to generate nicked DNA duplexes that can be directly ligated. Alternatively, noncanonical resolvases cleave Holliday junctions with asymmetric nicks to produce gapped and flapped DNA duplexes that require further processing prior to ligation (bottom right panel). *Mitochondrial Holliday junction resolvase.     

Break-Induced DNA Replication

Recombination-dependent DNA replication, often called break-induced replication (BIR), was initially invoked to explain recombination events in bacteriophage but it has recently been recognized as a fundamentally important mechanism to repair double-strand chromosome breaks in eukaryotes. This mechanism appears to be critically important in the restarting of stalled and broken replication forks and in maintaining the integrity of eroded telomeres. Although BIR helps preserve genome integrity during replication, it also promotes genome instability by the production of loss of heterozygosity and the formation of nonreciprocal translocations, as well as in the generation of complex chromosomal rearrangements.The break-copy mode of recombination (as opposed to break-join), was initially proposed by Meselson and Weigle (1961). Break-copy recombination, now more commonly known as recombination-dependent DNA replication or break-induced replication (BIR), is believed to account for restarting replication at broken replication forks and may also play a central role in the maintenance of telomeres in the absence of telomerase. BIR has been studied in various model systems and has been invoked to explain chromosome rearrangements in humans. This review focuses primarily on mechanistic studies in Escherichia coli and its bacteriophages, T4 and λ, in the budding yeasts Saccharomyces cerevisiae and Kluyveromyces lactis and on apparently similar, but less well-documented, mechanisms in mammalian cells.Homology-dependent repair of DNA double-strand breaks (DSBs) occur by three major repair pathways (Pâques and Haber 1999) (Fig. 1). When both ends of the DNA share substantial homology with a donor template (a sister chromatid, a homologous chromosome, or an ectopically located segment), repair occurs almost exclusively by gene conversion (GC). If the DSB is flanked by direct repeats, then a second repair process, single-strand annealing (SSA), can occur as 5′ to 3′ resection of the DSB ends exposes complementary sequences that can anneal to each other and repair the break by the formation of a deletion. However, when only one DSB end shares homology with a donor sequence, repair occurs by BIR. There are two BIR pathways, one dependent on Rad51 recombinase and the other independent of Rad51.Open in a separate windowFigure 1.Three major repair pathways of homology-dependent recombination. Noncrossover (NCO) and crossover (CO) events are indicated. Black triangles represent resolution of Holliday junctions (HJs). Dashed lines represent new DNA synthesis. GC, gene conversion; SSA, single-strand annealing; BIR, break-induced replication.     

Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity.

The evolution of modern cells is arguably the most challenging and important problem the field of biology has ever faced …—Carl R. Woese(Woese 2002)

Current models may accept that all modern eukaryotic cells arose from a single common ancestor (the cenancestral eukaryote), the nature of which is—owing to the lack of direct living or fossil descendants—still highly under debate (de Duve 2007). The chimeric nature of eukaryotic genomes with eubacterial and archaebacterial shares led to a discussion about the origin of this first “proto-eukaryote.” Several models exist (see Fig. 1), which either place the evolution of the nucleus before or after the emergence of the mitochondrion (outlined in Koonin 2010; Martijn and Ettema 2013). According to the different postulated scenarios (summarized in Embley and Martin 2006), eukaryotes in the latter case might have evolved by endosymbiosis between a hydrogen-producing, oxygen-producing, or sulfur-dependent α-proteobacterium and an archaebacterial host (Fig. 1C). The resulting mitochondriate prokaryote would have evolved the nucleus subsequently. In other scenarios (Fig. 1B), the cenancestral eukaryote emerged by cellular fusion or endosymbiosis of a Gram-negative, maybe hydrogen-producing, eubacterium and a methanogenic archaebacterium or eocyte, leading to a primitive but nucleated amitochondrial (archezoan) cell (Embley and Martin 2006, and references therein). As a third alternative, Cavalier-Smith (2002) suggested a common eubacterial ancestor for eukaryotes and archaebacteria (the Neomuran hypothesis) (Fig. 1A).Open in a separate windowFigure 1.Evolution of the last common ancestor of all eukaryotic cells. A schematic depiction of the early eukaryogenesis. Because of the lack of living and fossil descendants, several opposing models are discussed (A–C). The anticipated order of events is shown as a flow chart. For details, see text. (Derived from Embley and Martin 2006; Koonin 2010.)     

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and single-strand annealing pathways—DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species.A central step in the process of homologous recombination is the formation of heteroduplex DNA. In this article, heteroduplex DNA is defined as double-stranded DNA that arose from recombination, in which the two strands are derived from different parental DNA molecules or regions. The two strands of the heteroduplex may be fully complementary in sequence, or may contain small regions of noncomplementarity embedded within their otherwise complementary sequences. In either case, Watson-Crick base pairs must stabilize the heteroduplex to the extent that it can exist as free DNA following the dissociation of the recombination proteins that promoted its formation.The ability to form heteroduplex DNA using strands from two different parental DNA molecules lies at the heart of fundamental biological processes that control genome stability in individual organisms, inheritance of genetic information by their progeny, and genetic diversity within the resulting populations (Amunugama and Fishel 2012). During meiosis, the formation of heteroduplex DNA facilitates crossing-over and allelic exchange between homologous chromosomes; this process ensures that progeny are not identical clones of their parents and that sexual reproduction between individuals will result in a genetically diverse population (see Lam and Keeney 2015; Zickler and Kleckner 2015). Heteroduplex DNA generated by meiotic COs also ensures proper segregation of homologous chromosomes, so that each gamete receives a complete but genetically distinct set of chromosomes (Bascom-Slack et al. 1997; Gerton and Hawley 2005). In mitotic cells, heteroduplex DNA formation between sister chromatids is essential for homology-directed repair (HR) of DNA double-strand breaks (DSBs), stalled replication forks, and other lesions (Maher et al. 2011; Amunugama and Fishel 2012; Mehta and Haber 2014). Prokaryotic organisms also generate heteroduplex DNA to perform HR transactions, and to promote genetic exchanges, such as occur during bacterial conjugation (Cox 1999; Thomas and Nielsen 2005).Fundamentally, heteroduplex DNA generation involves the formation of tracts of Watson-Crick base pairs between strands of DNA derived from two different progenitor (parental) DNA molecules. Mechanistically, the DNA transactions giving rise to heteroduplex may involve two, three, or four strands of DNA (Fig. 1). DNA annealing refers to heteroduplex formation from two complementary (or nearly complementary) molecules or regions of single-stranded DNA (ssDNA) (Fig. 1A). DNA annealing may occur spontaneously, but it is promoted in vivo by certain classes of annealing proteins. Three-stranded reactions yielding heteroduplex DNA proceed by a different mechanism referred to as DNA pairing, strand invasion, or strand exchange. These reactions involve the invasion of a duplex DNA molecule by homologous (or nearly homologous) ssDNA. The invading DNA may be completely single stranded, as is often the case in in vitro assays for DNA-pairing activity (Fig. 1B) (Cox and Lehman 1981). Under physiological conditions, however, the invading ssDNA is contained as a single-stranded tail or gap within a duplex (Fig. 1C,D). DNA-pairing reactions are promoted by DNA-pairing proteins of the RecA family (Bianco et al. 1998), and proceed via the formation of D-loop or joint molecule intermediates that contain the heteroduplex DNA (Fig. 1B–D). Three-stranded reactions may also be promoted by exonuclease/annealing protein complexes found in certain viruses. Four-stranded reactions generating heteroduplex DNA involve branch migration of a Holliday junction (Fig. 1D). In practice, a four-stranded reaction must be initiated by a three-stranded pairing reaction catalyzed by a DNA-pairing protein, after which the heteroduplex is extended into duplex regions through the action of the DNA-pairing protein or of an associated DNA helicase/translocase (Das Gupta et al. 1981; Kim et al. 1992; Tsaneva et al. 1992).Open in a separate windowFigure 1.Common DNA annealing and pairing reactions. (A) Simple annealing between two complementary molecules of single-stranded DNA to form a heteroduplex. (B) Three-stranded DNA-pairing reaction of the type used for in vitro assays of RecA-family DNA-pairing proteins. The single-stranded circle is homologous to the linear duplex. Formation of heteroduplex (red strand base-paired to black) requires protein-promoted invasion of the duplex by the ssDNA to form a joint molecule or D-loop (i). The length of the heteroduplex may be extended by branch migration (ii). (C) Three-stranded DNA-pairing reaction of the type used for high-fidelity repair of DNA DSBs in vivo. The invading strand is the ssDNA tail of a resected DSB. The 3′ end of the invading strand is incorporated into the heteroduplex within the D-loop intermediate. (D) Example of a four-stranded DNA-pairing transaction that is initiated by a three-stranded pairing event and extended by branch migration. The ssDNA in a gapped duplex serves as the invading strand to generate a joint molecule (i), reminiscent of the reaction shown in panel B. Protein-directed branch migration may proceed into the duplex region adjacent to the original gap, generating α-structure intermediates (ii), or eventually a complete exchange of strands (iii).     

Apoptotic and Nonapoptotic Caspase Functions in Animal Development

A developing animal is exposed to both intrinsic and extrinsic stresses. One stress response is caspase activation. Caspase activation not only controls apoptosis but also proliferation, differentiation, cell shape, and cell migration. Caspase activation drives development by executing cell death or nonapoptotic functions in a cell-autonomous manner, and by secreting signaling molecules or generating mechanical forces, in a noncell autonomous manner.Programmed cell death or apoptosis occurs widely during development. During C. elegans development, 131 cells die by caspase CED-3-dependent apoptosis; however, ced-3 mutants do not show significant developmental defects (Ellis and Horvitz 1986). In contrast, studies on caspase mutants in mouse and Drosophila have revealed caspases’ roles in development. During development, cells are exposed to extrinsic and intrinsic stresses, and caspases are activated as one of multiple stress responses that ensure developmental robustness (Fig. 1). Caspases actively regulate animal development through both apoptosis and nonapoptotic functions that involve cell–cell communication in developing cell communities (Miura 2011). This chapter focuses on the in vivo roles of caspases in development and regeneration.Open in a separate windowFigure 1.Caspase activation during development. An embryo undergoes intrinsic and extrinsic stress, which activates caspases to execute both apoptotic and nonapoptotic functions, including cell differentiation and dendrite pruning. Apoptotic cells affect the shape and behavior of their neighboring cells. Caspase-activated cells are shown in dark gray.     

Schwann Cells: Development and Role in Nerve Repair

Schwann cells develop from the neural crest in a well-defined sequence of events. This involves the formation of the Schwann cell precursor and immature Schwann cells, followed by the generation of the myelin and nonmyelin (Remak) cells of mature nerves. This review describes the signals that control the embryonic phase of this process and the organogenesis of peripheral nerves. We also discuss the phenotypic plasticity retained by mature Schwann cells, and explain why this unusual feature is central to the striking regenerative potential of the peripheral nervous system (PNS).The myelin and nonmyelin (Remak) Schwann cells of adult nerves originate from the neural crest in well-defined developmental steps (Fig. 1). This review focuses on embryonic development (for additional information on myelination, see Salzer 2015). We also discuss how the ability to change between differentiation states, a characteristic attribute of developing cells, is retained by mature Schwann cells, and explain how the ability of Schwann cells to change phenotype in response to injury allows the peripheral nervous system (PNS) to regenerate after damage.Open in a separate windowFigure 1.Main transitions in the Schwann cell precursor (SCP) lineage. The diagram shows both developmental and injury-induced transitions. Black uninterrupted arrows, normal development; red arrows, the Schwann cell injury response; stippled arrows, postrepair reformation of myelin and Remak cells. Embryonic dates (E) refer to mouse development. (Modified from Jessen and Mirsky 2012; reprinted, with permission and with contribution from Y. Poitelon and L. Feltri.)     

The Structure and Function of the Eukaryotic Ribosome

Structures of the bacterial ribosome have provided a framework for understanding universal mechanisms of protein synthesis. However, the eukaryotic ribosome is much larger than it is in bacteria, and its activity is fundamentally different in many key ways. Recent cryo-electron microscopy reconstructions and X-ray crystal structures of eukaryotic ribosomes and ribosomal subunits now provide an unprecedented opportunity to explore mechanisms of eukaryotic translation and its regulation in atomic detail. This review describes the X-ray crystal structures of the Tetrahymena thermophila 40S and 60S subunits and the Saccharomyces cerevisiae 80S ribosome, as well as cryo-electron microscopy reconstructions of translating yeast and plant 80S ribosomes. Mechanistic questions about translation in eukaryotes that will require additional structural insights to be resolved are also presented.All ribosomes are composed of two subunits, both of which are built from RNA and protein (Figs. ​(Figs.11 and ​and2).2). Bacterial ribosomes, for example of Escherichia coli, contain a small subunit (SSU) composed of one 16S ribosomal RNA (rRNA) and 21 ribosomal proteins (r-proteins) (Figs. ​(Figs.1A1A and ​and1B)1B) and a large subunit (LSU) containing 5S and 23S rRNAs and 33 r-proteins (Fig. 2A). Crystal structures of prokaryotic ribosomal particles, namely, the Thermus thermophilus SSU (Schluenzen et al. 2000; Wimberly et al. 2000), Haloarcula marismortui and Deinococcus radiodurans LSU (Ban et al. 2000; Harms et al. 2001), and E. coli and T. thermophilus 70S ribosomes (Yusupov et al. 2001; Schuwirth et al. 2005; Selmer et al. 2006), reveal the complex architecture that derives from the network of interactions connecting the individual r-proteins with each other and with the rRNAs (Brodersen et al. 2002; Klein et al. 2004). The 16S rRNA can be divided into four domains, which together with the r-proteins constitute the structural landmarks of the SSU (Wimberly et al. 2000) (Fig. 1A): The 5′ and 3′ minor (h44) domains with proteins S4, S5, S12, S16, S17, and S20 constitute the body (and spur or foot) of the SSU; the 3′ major domain forms the head, which is protein rich, containing S2, S3, S7, S9, S10, S13, S14, and S19; whereas the central domain makes up the platform by interacting with proteins S1, S6, S8, S11, S15, and S18 (Fig. 1B). The rRNA of the LSU can be divided into seven domains (including the 5S rRNA as domain VII), which—in contrast to the SSU—are intricately interwoven with the r-proteins as well as each other (Ban et al. 2000; Brodersen et al. 2002) (Fig. 2A). Structural landmarks on the LSU include the central protuberance (CP) and the flexible L1 and L7/L12 stalks (Fig. 2A).Open in a separate windowFigure 1.The bacterial and eukaryotic small ribosomal subunit. (A,B) Interface (upper) and solvent (lower) views of the bacterial 30S subunit (Jenner et al. 2010a). (A) 16S rRNA domains and associated r-proteins colored distinctly: b, body (blue); h, head (red); pt, platform (green); and h44, helix 44 (yellow). (B) 16S rRNA colored gray and r-proteins colored distinctly and labeled. (C–E) Interface and solvent views of the eukaryotic 40S subunit (Rabl et al. 2011), with (C) eukaryotic-specific r-proteins (red) and rRNA (pink) shown relative to conserved rRNA (gray) and r-proteins (blue), and with (D,E) 18S rRNA colored gray and r-proteins colored distinctly and labeled.Open in a separate windowFigure 2.The bacterial and eukaryotic large ribosomal subunit. (A) Interface (upper) and solvent (lower) views of the bacterial 50S subunit (Jenner et al. 2010b), with 23S rRNA domains and bacterial-specific (light blue) and conserved (blue) r-proteins colored distinctly: cp, central protuberance; L1, L1 stalk; and St, L7/L12 stalk (or P-stalk in archeaa/eukaryotes). (B–E) Interface and solvent views of the eukaryotic 60S subunit (Klinge et al. 2011), with (B) eukaryotic-specific r-proteins (red) and rRNA (pink) shown relative to conserved rRNA (gray) and r-proteins (blue), (C) eukaryotic-specific expansion segments (ES) colored distinctly, and (D,E) 28S rRNA colored gray and r-proteins colored distinctly and labeled.In contrast to their bacterial counterparts, eukaryotic ribosomes are much larger and more complex, containing additional rRNA in the form of so-called expansion segments (ES) as well as many additional r-proteins and r-protein extensions (Figs. 1C–E and ​and2C–E).2C–E). Compared with the ∼4500 nucleotides of rRNA and 54 r-proteins of the bacterial 70S ribosome, eukaryotic 80S ribosomes contain >5500 nucleotides of rRNA (SSU, 18S rRNA; LSU, 5S, 5.8S, and 25S rRNA) and 80 (79 in yeast) r-proteins. The first structural models for the eukaryotic (yeast) ribosome were built using 15-Å cryo–electon microscopy (cryo-EM) maps fitted with structures of the bacterial SSU (Wimberly et al. 2000) and archaeal LSU (Ban et al. 2000), thus identifying the location of a total of 46 eukaryotic r-proteins with bacterial and/or archaeal homologs as well as many ES (Spahn et al. 2001a). Subsequent cryo-EM reconstructions led to the localization of additional eukaryotic r-proteins, RACK1 (Sengupta et al. 2004) and S19e (Taylor et al. 2009) on the SSU and L30e (Halic et al. 2005) on the LSU, as well as more complete models of the rRNA derived from cryo-EM maps of canine and fungal 80S ribosomes at ∼9 Å (Chandramouli et al. 2008; Taylor et al. 2009). Recent cryo-EM reconstructions of plant and yeast 80S translating ribosomes at 5.5–6.1 Å enabled the correct placement of an additional six and 10 r-proteins on the SSU and LSU, respectively, as well as the tracing of many eukaryotic-specific r-protein extensions (Armache et al. 2010a,b). The full assignment of the r-proteins in the yeast and fungal 80S ribosomes, however, only became possible with the improved resolution (3.0–3.9 Å) resulting from the crystal structures of the SSU and LSU from Tetrahymena thermophila (Klinge et al. 2011; Rabl et al. 2011) and the Saccharomyces cerevisiae 80S ribosome (Figs. ​(Figs.1D,E1D,E and ​and2D,E)2D,E) (Ben-Shem et al. 2011).     

How Genes Have Illuminated the History of Early Americans and Latino Americans

The American continent currently accounts for ∼15% of the world population. Although first settled thousands of years ago and fitting its label as “the New World,” the European colonial expansion initiated in the late 15th century resulted in people from virtually every corner of the globe subsequently settling in the Americas. The arrival of large numbers of immigrants led to a dramatic decline of the Native American population and extensive population mixing. A salient feature of the current human population of the Americas is, thus, its great diversity. The genetic variation of the Native peoples that recent immigrants encountered had been shaped by demographic events acting since the initial peopling of the continent. Similarly, but on a compressed timescale, the colonial history of the Americas has had a major impact on the genetic makeup of the current population of the continent. A range of genetic analyses has been used to study both the ancient settlement of the continent and more recent history of population mixing. Here, I show how these two strands of research overlap and make use of results from other scientific disciplines to produce a fuller picture of the settlement of the continent at different time periods. The biological diversity of the Americas also provides prominent examples of the complex interaction between biological and social factors in constructing human identities and of the difficulties in defining human populations.A multiplicity of research approaches have been used to explore the original settlement of the American continent, often focusing on three prominent questions: (1) the route of entry of the initial settlers, (2) their time of arrival, and (3) the pattern of subsequent migration. These questions have been approached with variable degrees of success using various types of genetic markers examined in “Native” populations, defined on anthropological grounds (particularly language). Early studies used information from blood groups and proteins (Cavalli-Sforza et al. 1994) and were followed by DNA analyses mainly of mitochondrial DNA (mtDNA) (Forster et al. 1996; Tamm et al. 2007; Fagundes et al. 2008; Kitchen et al. 2008) and the Y chromosome (Lell et al. 1997; Bianchi et al. 1998; Karafet et al. 1999; Bortolini et al. 2003). The more recent studies have examined the human genome at increasing levels of resolution, from analyses with restricted sets of markers (Wang et al. 2007; Ray et al. 2010) to ongoing studies based on full genome sequences. Although a range of scenarios for the initial peopling of the Americas have been envisaged, genetic evidence points to the continent being settled by people migrating into the northwestern tip of the continent from Asia. This migration would have been facilitated by the existence, at that time, of a land bridge connecting Siberia to Alaska, which later was submerged beneath the Bering Strait by the rising sea level at the end of the last glaciation, around 15,000 years ago (Fiedel 2000). Genetic support for an American settlement from Eastern Siberia includes the finding that Native Americans are genetically most similar to North Asians (Cavalli-Sforza et al. 1994; Wang et al. 2007) and the existence of a gradient of declining genetic diversity from northwest North America southward (Wang et al. 2007; Reich et al. 2012). This gradient extends beyond that seen in the “Old World” for populations at increasing distance from Africa, possibly resulting from a sequence of population contractions that occurred as small groups of humans moved from settled areas into uninhabited territories (Ramachandran et al. 2005; Handley et al. 2007; Wang et al. 2007). The American continent, being the last major landmass to have been settled by humans, shows a low genetic diversity as compared with all other continents (Wang et al. 2007).Estimating the date of the initial settlement of the Americas has proven a difficult and contentious issue. Geological information provides a key reference point in that because of extensive ice sheets covering North America at the peak of the last glaciation (around 20,000 years ago), the continent would have been impenetrable then (Fig. 1). Therefore, this leaves two broad opportunities for settlement: before or after this last glacial maximum (LGM). Calculating the time of initial settlement of the continent from genetic information requires a number of assumptions of which the exact validity is difficult to assess, including variation in factors such as population demography, mutation rates, and the influence of selection. Perhaps, not surprisingly, the range of genetic estimates for the time of human settlement of America is quite wide, extending to both sides of the glacial maximum. It is, however, encouraging that most of the recent estimates, based on increasingly larger amounts of data and more sophisticated statistical methods, point to a settlement not long after the LGM. These estimates show greater consistency with the archaeological evidence, which, although itself not devoid of controversy, points to a human presence in the Americas by ∼14,000 years ago.Open in a separate windowFigure 1.First peopling of the American continent. Settlement is thought to have occurred from Eastern Siberia through several waves of migration (arrows) across a land bridge connecting Siberia to Alaska, existing at the time. Crossing was impossible during the last glacial maximum (LGM) (∼20,000 years ago) because of glaciers covering a large part of North America. Most genetic studies of contemporary Native Americans point to a settlement of the continents soon after the LGM, subsequent to the retreat of the ice sheets. Although classical studies associated initial settlement with the Clovis archaeological complex of North America (∼13,000 years ago), older sites have been identified, including Monte Verde in South America (dated at ∼15,000 years ago). The Native American populations placed on this map are those included in the phylogenetic tree shown in Figure 3. Analysis of genetic data from these populations is consistent with the important role of the coast during the initial settlement of the continent (Reich et al. 2012).The pattern of migration into the continent has also been the subject of considerable disagreement. An influential model put forward in the mid-1980s posited that the settlement of the continent occurred in three sequential migratory waves from Asia, corresponding to the three major linguistic stocks in which the linguist Joseph Greenberg classified Native American languages (Greenberg et al. 1986; Greenberg 1987; Ruhlen 1991). The first migration would have given rise to a very large Amerind linguistic family comprising populations living all over the continent, whereas two subsequent migrations, restricted to North America and the Arctic, would be associated with populations speaking languages of the Na-Dene and Eskimo-Aleut linguistic families, respectively. Although early blood group and protein data were interpreted in support of the Greenberg model (Cavalli-Sforza et al. 1994), subsequent mtDNA and Y-chromosome analyses have been mostly interpreted as indicative of a single migration wave into the continent (Bonatto and Salzano 1997; Tamm et al. 2007; Fagundes et al. 2008; Kitchen et al. 2008). The recent genome-wide surveys of diversity with increasing resolution have, however, provided a different view. These are inconsistent with the single migration model and are more in line with the occurrence of multiple migrations (Fig. 1). Particularly strong support for several ancient migrations comes from a study based on a large survey of populations and using data for hundreds of thousands of genetic markers. With this type of data, it is possible to estimate the ancestry of every segment of DNA along the genome and state whether such a segment is of African, European, or Native American origin. Analyses can then focus only on the Native American segments of the genome (Fig. 2). This means that Native American individuals, and populations, that previously had to be excluded from study because of admixture with non-Natives can now be included, facilitating a more extensive population survey and reducing bias. These recent data have provided strong evidence that the Eskimo-Aleut, Na-Dene, and Amerind linguistic groups show evidence of differential gene flow from Asia, inconsistent with stemming from a discrete single colonization event with no subsequent migration (Fig. 1). Noticeably, although North American populations show evidence of multiple episodes of gene exchange with Asia, Native populations from Mexico to the Southern tip of South America appear to stem from one colonization wave with no subsequent Asian gene flow. This observation agrees with the highly controversial proposal of grouping widely separated Native American languages into a single “Amerindian” linguistic family (Greenberg 1987; Ruhlen 1991). These data also confirm the correlation of population diversity with distance from the Bering Strait, in agreement with settlement in a north-to-south direction. Interestingly, this correlation increases when considering the coasts as facilitators of population movement, suggesting an important role of the coast during the initial population dispersals on the continent. A phylogenetic tree relating the Native American populations examined in that survey is also consistent with the north-to-south settlement of the continent, as it shows a sequence of major population splits separating groups of populations mostly along a north-to-south axis (Figs. 1 and ​and3).3). Consistent with some degree of parallel evolution for languages and genes (Cavalli-Sforza et al. 1994), resulting from population separation followed by relative isolation, the major clusters of populations in this genetic tree show a broad correspondence with the linguistic affiliation of the populations (Fig. 3).Open in a separate windowFigure 2.Inference of local ancestry along the two copies of chromosome 1 in an admixed Native American individual. The height of the thick line indicates local ancestry as the number of chromosome copies at that position that are estimated to be Native American (0, 1, or 2). Numbers on the x-axis refer to the position along chromosome 1 (in kilobases) of the genetic markers allowing inference of local ancestry. (Modified from data in Reich et al. 2012.)Open in a separate windowFigure 3.Phylogenetic tree relating representative African, European, Oceanian, East Asian, and Native American populations based on high-density genetic marker data. For this analysis, Native American data used was restricted to genome segments of confirmed Native ancestry (determined as in Fig. 2). The tree branches are color-coded to represent the linguistic affiliation of the populations, as shown in the inset. Numbers in parentheses refer to sample size in each population. The length of the branches on this tree is proportional to a measure of genetic differentiation (F_ST). (From Reich et al. 2012; reprinted, with permission, from the author.)The recent study by Reich et al. (2012) illustrates the potential of high-density genotyping for extending studies focused on the original settlement of the Americas to Native individuals with evidence of admixture with recent immigrants. This admixture is extensive across the continent and involves not only Natives but also the general population, particularly in the countries of what is now referred to as Latin America. Historically, a major driver behind population mixing in this region was the fact that immigrants from Spain and Portugal, particularly in the early phases of the colonial expansion, were mostly men (Boyd-Bowman 1973). It is well documented that many Conquistadors had children with Native women, the most famous example possibly being that of the Conquistador of Mexico, Hernán Cortez, and the Nahua woman known as “Malinche” (Fig. 4). This “sex-biased” pattern of mating between immigrant men and Native women had been alluded to by historians (Morner 1967), but it was only with mtDNA and Y-chromosome studies that the full genetic impact of this feature became apparent. Because mtDNA and the Y chromosome are only transmitted by mothers and fathers, respectively, they allow the inference of the maternal (mtDNA) and paternal (Y-chromosome) ancestry of individuals. One of the first such studies was performed in Antioquia (Colombia), a population traditionally considered as mainly of Spanish descent. Consistent with this view, it was found that >90% of men in Antioquia had Y-chromosome lineages of European origin (Carvajal-Carmona et al. 2000). Surprisingly, when examining their mtDNA, a sharply different picture was observed. In 90% of individuals, maternal ancestry was Native American (Carvajal-Carmona et al. 2000). Similar observations have now been made in many Latin American countries (Alves-Silva et al. 2000; Green et al. 2000; Carvalho-Silva et al. 2001; Marrero et al. 2007), although with a considerable variation in ancestry proportions between them (Fig. 5). These studies, in addition, show a higher African ancestry with mtDNA than the Y chromosome, indicating that, historically, admixture with Africans has also mostly involved African women.Open in a separate windowFigure 4.The Native American woman known as “Malinche” or Malintzin (her Nahuatl name) was the interpreter and mistress of the Spanish Conquistador, Hernán Cortés. In 1523, she gave him a son, Martín, who is one of the first recorded individuals of mixed Native–European ancestry born in the Americas. Such offspring between immigrant men and Native women were a common occurrence in early colonial Latin America. The drawing shown is from the late 16th century “Codex Tlaxcala” and represents a meeting between the Mexican ruler, Moctezuma, and Hernán Cortés, with Malintzin (on the right) translating.Open in a separate windowFigure 5.Proportion of Native American, European, and African ancestry in 13 Latin American populations estimated using mtDNA and Y-chromosome markers. Samples from urban centers in five countries were examined (Mexico—Mexico City; Guatemala—Oriente; Costa Rica—Central Valley of Costa Rica [CVCR]; Colombia—Peque, Medellín, and Cundinamarca; Chile—Paposo and Quetalmahue; Argentina—Salta, Tucuman, and Tacamarca; and Brazil—Rio Grande do Sul [RGS]). Ancestry proportion (fraction of the pie chart) is color-coded: African (green), European (blue), and Native American (red). (Data for 20 individuals per population are from Wang et al. 2007, Yang et al. 2010, and NN Yang et al. unpubl.)The large variation in ancestry seen across Latin America relates to differences in pre-Columbian Native population density and the pattern of recent immigration into specific regions of the continent. For instance, most studies performed so far have mainly focused on areas with little documented African immigration and consistently show a relatively low African genetic ancestry. In these population samples, the variation in individual European and Native American ancestry is very large, to the extent that it overlaps with that seen in Native population samples (Fig. 6). The variation in individual ancestry seen in these samples thus effaces their designation as “Native” or “non-Native.” This observation punctuates the interest of incorporating admixed Latin American populations, traditionally considered non-Native, into studies on the initial settlement of the continent. Similar to what has been performed in the recent survey by Reich et al. (2012), the inference of ancestry of each genome segment in Latin Americans could be used to focus solely on Native American segments of the genome. This is an avenue of research that is just beginning to be explored and shows great potential for the future. It promises to be of particular importance for the analysis of regions where anthropologically recognizable Native populations and individuals are virtually nonexistent, as they have been absorbed into the current mixed population. This is the case for the many areas that were relatively sparsely populated in pre-Columbian times and, subsequently, received a large flow of immigrants, such as from the Caribbean and many parts of North and South America. Consequently, estimation of individual ancestry along the genome will facilitate denser demographic history analyses across the Americas, as well as a reexamination of the original settlement of the continent based on a more comprehensive population sampling.Open in a separate windowFigure 6.Distribution of individual Native ancestry estimated in samples from Native Americans (shown in blue) and two Latin American urban centers (850 residents of Medellín, Colombia shown in red, and 220 residents of Mexico City, Mexico shown in green). For each of the three population samples, the x-axis indicates the proportion of Native ancestry (in 0.1 unit intervals) and the y-axis indicates the proportion of individuals with that ancestry estimate. The Native American group includes 225 individuals from North, Central, and South America. Ancestry proportions were estimated using autosomal genetic markers. (Data from Florez et al. 2009, Campbell et al. 2011, and Reich et al. 2012.)Other than being informative for addressing questions of population history, the study of Latin American populations promises to facilitate the genetic characterization of biological attributes differentiated among the populations that participated in admixture on the continent. For instance, a range of facial features differ between Native Americans and Europeans, and the genetic study of admixed Latin Americans promises to help in the identification of genes explaining variation in facial appearance. Such research is of interest for understanding disorders of craniofacial development and could also have forensic applications. Another example is type 2 diabetes (T2D), a disease that has a very high frequency in Native Americans and for which a higher risk is associated with increased Native American ancestry. This observation led to the proposal of the “thrifty genotype” hypothesis, which posits that the increased risk of T2D in Native Americans results from genetic adaption to a low-calorie/high-exercise way of life that became detrimental with the recent change to a high-calorie/low-exercise lifestyle (Pollard 2008). The study of large, carefully characterized samples from Latin American populations offers a unique opportunity for conducting a detailed assessment of this hypothesis. The identification of genes explaining the variable frequency of diseases between populations (such as T2D) will be an important step forward in the development of novel, more effective (even individualized) disease-management strategies that account for human population diversity.The overlap of individual genetic ancestry estimates, seen in Latin and Native American populations (Fig. 5), raises the question of the relationship of these estimates to the perception that individuals have of their own ancestry. A recent analysis of a large sample of individuals from five Latin American countries found a highly significant correlation between self-perception and genetically estimated ancestry (Fig. 7). However, this study also found evidence that self-perception is biased. A particularly clear bias involves pigmentation: individuals with greater pigmentation tend to overestimate their Native and African ancestry, whereas individuals with lighter pigmentation tend to overestimate their European ancestry (AR Ruiz-Linares, in press). Statistically significant differences were also observed between countries, pointing to the influence of social factors in self-perception. Consistent with this observation, social scientists have argued that, in Latin America, self-identification as Native or non-Native is often strongly influenced by social cues (Wade 2010).Open in a separate windowFigure 7.Box plots displaying the relationship of individual genetic ancestry estimates to self-perceived ancestry in 7342 Latin Americans (from Mexico, Colombia, Peru, Chile, and Brazil). Self-perception was categorized into 20% bands for African, European, and Native American ancestry. There is a highly significant correlation between the genetic estimate and self-perception for each continental ancestry component. However, there is a trend at higher Native American and African ancestry for self-perception to exceed the genetic estimates. Correspondingly, at lower European ancestry, there is a trend for the genetic estimates to exceed self-perception. Further analyses show that pigmentation impacts on these differences. Individuals with lower skin pigmentation tend to overestimate their European ancestry, whereas individuals with higher pigmentation overestimate their Native American and African ancestries. Orange lines indicate the median and the blue boxes are delimited by the 25th and 75th percentiles. (From AR Ruiz-Linares et al., in press; with permission from the author.)The insights into the initial settlement of the continent provided by the genetic study of Native Americans illustrate the fact that a population sampling that maximizes diversity based on anthropological grounds (such as language) can facilitate investigations concerned with the first settlement of the continent. However, the analysis of intercontinental admixture both in Native and non-Native Latin American populations show some of the complexities of defining human groups, with population labels suggesting a potentially misleading genetic singularity. The biological reality is that of a gradient in the genetic makeup of these populations (and individuals) involving various degrees of mixture between the initial settlers of the continent and more recent immigrants. The genetic diversity of Latin Americans is, thus, a prominent example of the fuzzy meaning of the labels used to refer to human populations. Although these labels can assist in study design and facilitate certain historical inferences, ethnicity, race, and other such terms are social constructs devoid of a clear-cut biological meaning.     

The Dissolution of Double Holliday Junctions

Double Holliday junctions (dHJS) are important intermediates of homologous recombination. The separate junctions can each be cleaved by DNA structure-selective endonucleases known as Holliday junction resolvases. Alternatively, double Holliday junctions can be processed by a reaction known as “double Holliday junction dissolution.” This reaction requires the cooperative action of a so-called “dissolvasome” comprising a Holliday junction branch migration enzyme (Sgs1/BLM RecQ helicase) and a type IA topoisomerase (Top3/TopoIIIα) in complex with its OB (oligonucleotide/oligosaccharide binding) fold containing accessory factor (Rmi1). This review details our current knowledge of the dissolution process and the players involved in catalyzing this mechanistically complex means of completing homologous recombination reactions.For decades, homologous recombination (HR) was defined as a mechanism for the production of new allelic combinations during meiosis because it can generate so-called crossing-over (see Mehta and Haber 2014). Crossovers are likely generated by the asymmetric cleavage of a key intermediate in HR, the dHJ, by the action of structure-selective endonucleases called “resolvases” (Fig. 1A) (see Wyatt and West 2014). In addition to its essential function during meiosis, HR has proven to be a crucial DNA repair pathway in mitotic cells. Precisely because it has the potential to generate crossing-over, the resolution of dHJ by resolvases affords a high risk of genomic instability in these circumstances. Indeed, when HR is engaged between two homologous chromosomes or two homeologous sequences, dHJ resolution could lead, respectively, to loss of heterozygosity or gross chromosomal rearrangements. Thus, an alternative mechanism allowing dHJ processing without crossing-over would appear essential when HR is used for DNA repair. Such a mechanism, termed dHJ dissolution, is thought to be a major route for dissipation of dHJs arising from HR repair (LaRocque et al. 2011; Krejci et al. 2012). During dHJ dissolution, the two HJs are branch migrated toward one another until they form a hemicatenated intermediate that can be decatenated by a topoisomerase (Fig. 1B). This sophisticated reaction is performed by the so-called “dissolvasome” complex composed of a specific RecQ helicase (BLM in humans/Sgs1 in budding yeast) and a type IA topoisomerase known as topoisomerase III (Fig. 2; for general reviews about RecQ helicases and topoisomerases, see Champoux 2001; Wang 2002; Bachrati and Hickson 2003; Viard and de la Tour 2007; Chu and Hickson 2009; Vindigni and Hickson 2009.Open in a separate windowFigure 1.Double Holliday junction processing pathways. (A) During HJ resolution, each HJ of a dHJ is cleaved by a structure-selective endonuclease (resolvase). Depending on the combination of cleavage orientations, which can be asymmetric or symmetric, this process can generate both crossover and noncrossover products. In contrast, during dissolution (B), each strand engaged in the dHJ is reassociated with its original complementary strand, preventing exchange of genetic material between the two homologous sequences (and hence generating exclusively noncrossover products). DHJ dissolution (B) is initiated by migration of the HJs toward one another. The fusion/collapse of the two HJs results in a hemicatenated intermediate. Decatenation of this intermediate regenerates the original DNA species present before the initiation of HR.Open in a separate windowFigure 2.Domain organization of RecQ helicases, topoisomerases IA, and RMI proteins. (A) Most of the RecQ helicase members share a superfamily 2 helicase domain (SF2), a RecQ conserved domain (RQC), and a helicase and RNase D carboxy-terminal domain (HRDC). Besides this “RecQ core” domain, some RecQ helicases contain amino-terminal and carboxy-terminal extensions that vary in size, sequence, and functionality (e.g., SLD2 homology domain in RECQ4, and a signature motif in the carboxy-terminal domain of RECQ5). The hatched boxes denote partially degenerate RQC domains. BLM/Sgs1 helicases share a common domain organization, including an amino-terminal extension that includes domains for interaction with both TopoIII/RMI1 (TR) and replication protein A (RPA), in addition to a region that has been proposed to be required for DNA strand exchange (SE) activity. (B) All type IA topoisomerases contain a conserved catalytic domain (topoisomerase IA). Some topoisomerase IA enzymes also exhibit a carboxy-terminal extension, frequently composed of zinc finger motifs (black boxes), which is believed to mediate protein–DNA and protein–protein interactions. The contribution of the carboxy-terminal extension to dissolution is unknown. The regions interacting with other components of the dissolvasome are unknown. (C) In RMI1 proteins, only the DUF1676 and the OB-fold domain 1 (OB1) are conserved from yeast to human. The OB1 associates with both BLM/Sgs1 and topoisomerase III (BT/ST). In addition, human RMI1 exhibits a carboxy-terminal extension, composed of a middle region, which mediates RPA binding, and a second OB fold (OB2), which is able to associate with RMI2. RMI2 is also an OB-fold protein (OB3) that stably associates with the dissolvasome in human cells. In total, therefore, the human RMI1/2 complex contains three OB folds.In this review, we first take a historical look at the experimental evidence that led some groups to formulate the proposal that a reaction akin to dissolution must exist, and which then led Wu and Hickson (2003) to confirm its existence by reconstitution of the dissolution reaction in vitro using purified proteins. Following that, we will review the individual and combined roles of the components of what we will term the dHJ dissolvasome. Although many mechanistic aspects of dHJ dissolution remain obscure, several biochemical studies have provided a general understanding of this conceptually simple, but mechanistically complex, reaction.