How do You Get a Hula Hoop onto DNA?

DNA synthesis relies on a hoop-shaped complex, or "clamp" around DNA to keep it on track.

Processivity is a big issue for biological polymerases. An RNA polymerase needs to stay on its template until it reaches the end. Otherwise, the mRNA might be truncated and the resulting protein would be incomplete. Such proteins frequently have an activity opposite to that which the complete protein has, either because of a particular domain structure that puts key domains at the end, or just because it gums up the works instead of being a well-oiled cog in those works.

Likewise for DNA polymerases that replicate genomes. DNA polymerases that do small repairs may jump in for only a few nucleotides, but for earnest replication of entire genomes, you need a polymerase that chugs along reliably, for long distances. Evolution has come up with an elegant, if obvious, solution- mate the polymerase to another protein complex that firmly encircles the DNA like a hula hoop and doesn't let go. But this leads to other questions ... how does such a complex assemble where it is supposed to, and what happens to it later on?

Whatever the answers to those questions, this solution has been around for a very long time. The structures of the replication-associated sliding clamp from bacteria and from humans look virtually identical:

Human (left), and E. coli (right) sliding clamp protein complexes that facilitate DNA replication. Where does the DNA go? Obviously through the middle. These proteins do not bind to the DNA in any sequence specific way, but have positive charges arrayed around the inner ring, to gently stay in contact with negatively charged DNA.

The clamp complexes assemble into extremely stable rings right after they are synthesized off the ribosomal assembly line. That means that they have to be pried apart again to get put on DNA. That is the job of "clamp loaders"- yet another protein complex that orchestrate the proper placement of these clamps. While the clamp proteins are pretty simple affairs- pure structures lacking any enzymatic activity- the clamp loaders are ATP-ases and quite dynamic. 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683903/

An extremely simplistic model of clamp loading, with the loader prying open the clamp to allow it to admit double stranded DNA.

The job of a clamp loader is to recognize the correct location in DNA, to bind a free clamp complex, to pry open that complex, transiently allowing the double stranded DNA to enter, to detect when the DNA has successfully been loaded into the clamp, and lastly to detach again from the resulting complex. It is a big job, obviously. What is the correct location? It is a fork where single stranded DNA meets double stranded DNA- i.e. where DNA replication has been primed by a special replication origin selection, opening, and priming process. Clamp loaders are also rings, with five highly related subunits all around. Their ATPase activity (active on each subunit) allows them to twist, which is how they manage to twist the clamp as well, to open it up.

A recent paper extended structural knowledge about clamp loaders. These authors used the new cryo-electron microscopy methods to obtain high resolution structures of these complexes in a variety of states. These states were made available by virtue of using a slowly hydrolyzing form of ATP that gave them the whole spectrum, then frozen and photographed with electrons. They capture beautifully the sequence of events, where the loader first tentatively binds a clamp, and then opens it up wide enough to accept DNA. The DNA binding groove is not open until the clamp is bound, enforcing this forward sequence of clamp binding, then DNA binding (availability), then clamp opening. The authors do not, however, provide reason to think that this opening is dependent on DNA being already bound, so it is possible that this opening complex can be futile- opening and closing repeatedly before encountering the right replication fork. This is probably unlikely in practice, though, because the clamp loader complexes are quite rare and probably have other interactions that position them at replication forks before this process even begins. Additionally, the loader + clamp complex may remain open as long as necessary, until it encounters the right fork location.

An early form of the clamp loading complex is shown, when it first binds a clamp. The five subunits of the clamp loader are shown in color, while the clamp is shown at bottom in gray. In B, the contacts between the two complexes are shown in color, on the clamp, which is composed of proteins called PCNA. The different structures are of various stably inhibited forms, and the A-gate is where DNA is bound during the loading process.

Later on, after the loader has expended some energy, it has more contacts apparent with the clamp (right) and has pried it open, wide enough to accept a strand of DNA.

With DNA, the whole complex contracts a little again, and you can see the continuous flow of DNA (yellow) through the cleft of the loader complex down through the clamp complex. 

The last step is closure, when the clamp closes fully around the DNA, and the loader complex unbinds. This seems to be when ATP is actually hydrolyzed, implying that the opened complex is the ATP binding state, the closed (free) loader complex is the ADP bound state, and that successful DNA binding to is what stimulates ATP hydrolysis. Finally, another structure- a closeup of the DNA as bound, shows that the end of the primer strand, which sits right at the crux of the DNA fork, is specifically bound with its last base flipped around into the protein. This provides part of the mechanism of how the clamp loader feels its way to the right place at replication forks, after the briefly interacting polymerases that create such primers have fallen off. It is likely that this clamp loader complex engages in interaction with the final processive DNA polymerase to help it find the fork and clamp, but notably, the same face of the clamp interacts with both the loader and with that polymerase, so the handoff can not involve both binding at the same time at the same place.

A blowup of just the DNA within the fully bound complex above. Note how the top base of the yellow primer strand is flipped out from the rest of the helix, due to interactions with the loader protein complex.

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DNA Mambo in the Nucleus

Some organizational principles for nuclear DNA to organize genes for local regulation.

There has been a long and productive line of research on the mechanisms of transcription from DNA to RNA- the process that reads the genome and translates its code into a running stream of instructions going out to the cell through development and all through life. This search has generally gone from the core of the process outwards to its regulatory apparatus. The opening of DNA by simple RNA polymerases was one of the first topics of study, followed by how the polymerase is positioned at the start site by "promoter" DNA sequences, with ever more ornate and distant surrounding machinery coming under scrutiny over time, as researchers climbed the evolutionary trajectory of life, from viruses and bacteria to mammals. 

But how this process fits into the larger structure of the nucleus, and how it is globally organized eukaryotes has long been an intriguing question, and tools are finally available to bring this level of organization into focus. For example, genes are known to be activated by direct contact with "enhancer" elements located thousands, even many tens of thousands, of basepairs away on the DNA- so why can't those enhancers activate other genes elsewhere in the nucleus, rather than the genes they are nearest to on the one-dimensional DNA? The nucleus is a small place with a lot of DNA. Roughly 1/100 of its physical space is taken up by DNA, and it is highly likely that such enhancers could be closer in 3-D space to other genes than the ones they are supposed to regulate, if everything were arranged randomly. Similarly, how do such enhancer elements find their proper targets, amid the welter of other DNA and proteins? A hundred thousand base pairs is long enough to traverse the entire nucleus.

So there has to be some organization, and new techniques have come along to illuminate it. These are crosslinking methods where the cells are treated with a chemical to crosslink / freeze a fraction of protein and DNA interactions in place, then enzymes are introduced to chop everything up, to various degrees of completeness. What is left are little clumps of DNA and protein that hopefully include distant cross-links, between enhancers and promoters, between key organizational sites and the genes they interact with, etc. Then comes the sequencing magic. These clumped stray DNAs are diluted and ligated together (only to local ends), amplified and sequenced, generating a slew of DNA sequences. Those hybrid sequences can be interpreted, (given the known sequence of the reference genome), to say whether some genomic location X got tangled up with some other location Y, reflecting their 3-D interaction in the cell when it was originally treated.

A recent paper pushed this method forward a bit, with finer-grained enzymatic digestion and deeper sequencing, to come up with the most detailed look ever at a drosophila genome, and at some particular genes that have long held interest as key regulators of development. This refined detail, plus some experiments mutating some of the key DNA sites involved, allowed them to come up with a new class of organizing elements and a theory of how the nuclear tangle works.

Long range contacts in the Antennapedia locus of flies. Micro-C refers to the crosslinking and sequencing method that maps long-range DNA contacts mediated by proteins. Pyramids in the top diagram map binary location-to-location contacts. Local contacts generally predominate over distant ones, but a few distant connections are visible, such as between the ends of the ftz gene. TAD stands for topologically associating domain, mapping out the connections seen above between pink sites. This line also lists the genes residing in each zone (Deformed, micro RNA 10, Sex combs reduced, fushi terazu, and Antennapedia promoters P1 and P2). The contacts track shows where the authors map specific sites where organizing factors (including Trl (trithorax-like) and CP190 (centrosomal protein of 190 kDa)) bind. The overall idea is that there are two kinds of contacts, boundaries and tethers. Boundaries insulate one region from the next, preventing regulatory spill-over to the wrong gene. Tethers serve as pro-regulatory staging points, helping enhancers contact their proper promoter targets, even though the tether complex does not itself promote RNA transcription.

Insulator elements have been recognized for some time. These are locations that seem to block regulatory interactions across them, thus defining, between two such sites, a topologically associated domain, (TAD). How they work is not entirely clear, but they may stitch themselves to the nuclear membrane. They are thought to interact with a DNA pump called cohesin to extrude a loop of DNA between two insulator sites, thereby keeping that DNA clear of other interactions, at least temporarily, and locally clumped. The authors claim to find a new element called a distal tethering element (DTE), which works like an enhancer in promoting interaction between distant activating regulatory sites and genes, but doesn't actually activate. They just structure the region so that when a signal comes, the gene is ready to be activated efficiently. 

One theory of how insulator elements work. The insulator sites "CTCF motif" are marked on the DNA with dark blue arrow heads. They control the boundaries of action by the protein complex cohesin, which forms dimeric doughnuts around DNA and can pump DNA. Cohesins are central to the mechanisms of meiosis and mitosis. The net effect is to produce a segregated region of DNA as portrayed at the bottom, which should have a much higher rate of local interactions (as seen in the Micro-C method) than distant interactions.

At the largest scale, these authors claim that there are, in the whole fly genome and at this particular (early) point in development, 2034 insulator locations (TADs) and 620 tethering elements (TEs or DTEs). They show that DTEs in the locus they study closely play an active role in turning the nearby genes on at early times in development, and in directing activation from enhancers near the DTE, rather than ones farther away. What binds to the DTEs? So-called "pioneer" regulatory factors(such as Zelda) that have the power to make way through nucleosomes and other chromatin proteins to bind their target DNA. The authors say that these tether sites, once set up, are then stable on a permanent basis, through all developmental stages, even though the genes they assist may only be active transiently. 

The "poised" nature of some genes had been observed long ago, so it is not entirely surprising to see this mechanism get fleshed out a little, as a structural connection that is made between genes and their regulatory sites in advance of the actual activator proteins arriving at the associated enhancers and turning them on.

 

Final model: the normal case around the Antennapedia locus is shown at top, with insulator sites shown in pink, and tethering sites shown in teal. If one of the tethering elements is removed (middle), then the enhancer EE has less effect on the gene Scr, whose expression is reduced. If an insulator is removed (bottom), the re-organized domain allows the ftz gene's regulators, including the enhancer AE1, to affect Scr expression, altering its timing and location of expression.

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Origins in the Other: Moses the Egyptian

Stray notes on what Judaism owes to Egypt.

It is a bitter historical irony that Jesus was a Jew, (as were all the founding Christians), yet his religion was taken up by non-Jewish communities who turned its stories against its originators, casting Jews as the betrayers, stubborn heretics, and generally the other, with disastrous consequences. Well, something similar may have happened at the origin of Judaism, as there is a fascinating thread of historical scholarship and speculation that suggests that Moses was Egyptian, and that much of the religion of Judaism was generated as a mixture of inheritances from, or inversions of, the religion and cults of Egypt.

While the Bible is full of Moses stories, no other historical, let alone archeological, attestation exists. Thus the many authors who have striven to unearth the truth of what happened have had to be creative. The whole thing may be an out and out myth, or unrecognizably reworked. Freud wrote "Moses and Monotheism" as an exercise in retro-psychoanalysis, cutting his totemic father figure down in an imagined Oedipal paroxysm of murder, followed by remorse. Jan Assmann more recently, in "Moses the Egyptian", wrote an eliptical tale of cultural hints and suppressed memories and trauma continually expressed and re-interpreted over time in the "othering", adoption, and inversion of cultural patterns. Manetho, a third century BCE Egyptian priest, wrote a history that puts Moses, originally an Egyptian priest named Osarseph, at the head of a renegade (and leper, for good measure) army which terrorized Egypt sometime in the 1300-1500 BCE period, ending in their expulsion and exile. 

The Hyksos, a semitic people, had a distinctive look, in Egypt of ~1900 BC.

Ever since the reign of Akhenaten was unearthed in the late 1800's, it has been tempting to tie his monotheistic revolution (1353-1336 BCE) to that of Judaism, which was putatively founded in the same general time period, when Egypt was at its height of power and regional influence. In both cases, monotheism was a tough sell, and created antagonism that characterized both episodes. The Amarna period ended with complete reversal- Akhanaten being erased from his monuments and records, and Egypt returning to its traditional ways. Judaism, according to its own documents, and despite Moses's teaching, endured a lengthy period of conflict and consolidation before the monotheistic faction gained ascendence in the post-Babylonian exile period. It also generated the enduring enmity of neighboring polytheists, ultimately resulting in the military defeat and dispersal of the Jewish nation.

So what is the evidence? Moses is an Egyptian name. Like Tutmosis, Ahmose, Ramses, and many others, it means "is born", or "is child of". While there are both Hebrew and Egyptian etymologies possible, Moses is also described as practiced in all the arts of Egypt, including various forms of magic and the secret symbols, i.e. hieroglyphs. Egyptians practiced circumcision, which Judaism obviously adopted with gusto. Egyptians worshipped the ram (representing the leading god, Amun) and the bull (Osiris), which the Jews turned around and sacrificed in their rituals. Cooking meat in milk was an Egyptian practice, which may have been the source of the contrary interdiction in Kosher law. Judaism was anti-iconic, completely contrary to the abundant icons of, frankly, all the other polytheistic religions, though new icons have been snuck in, in the form of the ark of the covenant, the Torah scrolls, wailing wall, etc. The Thummim is a judicial badge and device for divination, taken from the Egyptians. It is indeed likely that originators of Judaism were assimilated Egyptians who left, whether by choice or not. The historian Tacitus noted the inverted character of Judaism vs the Egyptian religion. And Maimonides argued that the laws were a form of treatment for withdrawal from idol-addiction, in his case against the "Sabians", which in reality were the Egyptians, if they were any actual culture at all. But it served other purposes as well, such as cultural glue, which continues to be functional even when all other reasons have become irrelevant and many of the less convenient laws have been cast aside.

Whichever pharoh was the one described in Exodus, its Egyptological details, though accurate, come from a substantially later time, the 600's BC, when it was written, not from the time of the events. And Assmann argues that Manetho, for one, conflated several historical episodes to come up with his account. One was a Hyksos colony of semitic peoples that occupied northern (lower) Egypt through the second intermediate period (~1800 BC) to their defeat, about 1540 BC, by Kamose and his successor Ahmose, who were based in the south. There may have, however, been other incursions of semitic peoples from time to time, especially as records through the less organized periods of Egyptian history are sparse. A second episode was the Amarna period, which was officially suppressed, but which Assmann argues remained vivid as a traumatic memory of religious and existential revolution, informing an Egyptian official's view of "pollution" of the Egyptian culture by outsiders. 

Similarly, one can imagine that the idea of monotheism, so suddenly sprung upon the Egyptians, is something that was knocking around for longer periods of time, both before and since. Assmann goes through a long argument by Ralph Cudworth (who wrote long before the hieroglyphs were deciphered) about a possible "esoteric" theology of the Egyptians, which was monotheistic, while the cult for public consumption was polytheistic. That makes little sense, as all royal tombs and decorations hew (religiously!) to the standard story, and so clearly embody a full cast of characters, and their belief in the Osiris story and hope of continued life in the land of the un-dead. Nevertheless, even without such an esoteric/demotic split, it is natural to wonder about origins, such as where this family of gods arose from, which in turn would send thoughts in the direction of possible monotheism. Perhaps the incredible conservatism of Egyptian culture caused such thoughts to be ruthlessly stamped out, but also prone to occasional eruption in incovenient forms. We in our own time are experiencing the thrill of normative inversion, when a subculture decides that black is white, that all norms should be trampled, and a new god worshipped. 

Even if the Amarna period did not directly foster Moses and the Jewish form of monotheism, the latter owes a great deal to Egyptian culture, likely including some glimmer of the monotheistic idea. Within Judaism, it took a second (and certainly real) exile, in Babylon, to bring the monotheistic idea to fuller fruition, as the last set of prophets called for purification and repentance, the Torah was written down, and the second temple built.

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Mate Choice and the Origin of Species

The definition of a species is somewhat murky, because speciation is a continuous process. Which says something about race and racism as well.

Santa Claus brought me a delightful book about bird evolution, over the holidays. The writing is workman-like, and the treatment thorough. It covers the diversity, origins, and current status of birds, with an emphasis on how they inform evolutionary theory generally. One issue that is still live in evolutionary biology is the definition of a species. It is clear enough that birds and bees are different species, even different phyla. But are the six species of herring gull separate, or just sub-species? This is not just a headache for birders filling out their lists, but very much for biologists as well, in conservation, taxonomy, and evolutionary theory. How different do species have to be? Does any interbreeding / hybridization disqualify two populations from being different species? If not, how much does? And more importantly, at which point do such populations behave like different species, avoiding mating with each other, and maintaining distinct traits? This remains a difficult question, and is generally relevant, including for our own increasingly ramified family tree of ancestors.

One point that the author (Douglas Futuyma) makes, which I had not appreciated fully before, is that in the process of speciation, genetic barriers to successful mating between two species (or nascent species) come very late. In contrast, behavioral, ecological, or geographical forms of separation come early and are the real drivers. The examples are all around us as hybrid forms that can occur at range overlaps, not only among birds, but mammals as well. Polar bears can interbreed with other bears, though they don't want to! Species are recognized as separate by traits or molecular isolation long before they differ enough that they can't make viable hybrid offspring.

"So far, there doesn't seem to be any detailed evidence about how specific genes interact to cause incompatibility in hybrids, and why the genes diverged. But genetic incompatibility is seldom the cause of speciation in birds simply because premating isolation almost always evolves long before incompatibility does. Hybrids seem to be fully fit in about half of 254 different crosses between closely related species, many of which diverged as long as 5 million years ago. We can learn more about the causes of speciation if we focus on premating isolation, especially behavior. What causes behavioral isolation to occur?" p.169

This has some significant implications. Speciation turns out to arise largely from mate choice decisions, which we know in birds are highly discriminating. Why do they have all the plumage colors, song singing, and other mating behaviors, but to carry out the most careful evaluation of mates? The ideal mate is not very closely related. There are instinctive barriers to sibling mating, for instance. On the other hand, the ideal mate is also not very distantly related- not part of a differently colored sub-population or allied species. The plumage colors of birds are usually the clearest marks of speciation, both to us, and evidently to themselves- a way to keep straight who is who. On top of all that, the ideal mate has other properties that it may display during the courtship period, like vigor, courage to sing from the top-most branches, the ability to bring gifts of food, or to to construct an intricate bower.

The typical differentiation of nascent species happens due to physical separation, such as a founder finch flying to Hawaii, and starting an amazing adaptive radiation of honeycreepers and other birds. But sometimes, (as must have happened within the Hawaiian radiation of these birds), separation can be by habit or specialization within the same location- which is called sympatric speciation, or speciation in place. While difficult to demonstrate and reconstruct, this has been documented to occur, and again must be attributed to some kind of behavioral specialization and mating preference that overrides the heretofore mixed mating system of a local population.

A happy pair of horned puffins- not to be confused with tufted puffins!

It is evident that mate choice is a deeply significant and influential force. It causes all the decorative features typically displayed by males, but more deeply embodies and enforces the very concept of species as experienced by the species themselves. When a tufted puffin declines to mate with a horned puffin, it may not know whether offspring would have been possible or infertile, but it just knows that something isn't quite right about that prospective mate, and finds someone more suitable. And this is naturally relevant in humans as well. We are always making fine distinctions among ourselves in status and innumerable other qualities. Therefore it should come as little surprise that we have racist impulses, despite the fact that humans are, due to our rapid evolution and rampant internecine warfare, one of the least diverse species in existance, with no trace of genetic differentiation to support any kind of genetically based sub-speciation, races, etc.

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