posted 26. February 2003 00:06                   

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So now anyone who finds some cellular structure, that has no documented evolutionary history, would say "This is an example of irreducible complexity".

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Structure of the topoisomerase VI-B subunit: implications for type II topoisomerase mechanism and evolution.

Corbett KD , Berger JM

Department of Molecular and Cellular Biology, University of California, Berkeley, 327 Hildebrand Hall 3206, Berkeley, CA 94720, USA.

Type IIA and type IIB topoisomerases each possess the ability to pass one DNA duplex through another in an ATP-dependent manner. The role of ATP in the strand passage reaction is poorly understood, particularly for the type IIB (topoisomerase VI) family. We have solved the structure of the ATP-binding subunit of topoisomerase VI (topoVI-B) in two states: an unliganded monomer and a nucleotide-bound dimer. We find that topoVI-B is highly structurally homologous to the entire 40-43 kDa ATPase region of type IIA topoisomerases and MutL proteins. Nucleotide binding to topoVI-B leads to dimerization of the protein and causes dramatic conformational changes within each protomer. Our data demonstrate that type IIA and type IIB topoisomerases have descended from a common ancestor and reveal how ATP turnover generates structural signals in the reactions of both type II topoisomerase families. When combined with the structure of the A subunit to create a picture of the intact topoisomerase VI holoenzyme, the ATP-driven motions of topoVI-B reveal a simple mechanism for strand passage by the type IIB topoisomerases.

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Convergent Evolution of MutS and Topoisomerase II for Clamping DNA crossovers and stacked Holliday junctions

Abstract
This study shows that topoisomerase II and MutS proteins share a structural motif that has, in its dimeric form, a suitable geometry for clamping the two arms of either right-handed DNA crossovers or their isostructural stacked Holliday junctions. This defines a new protein family selected by convergent evolution for sensing DNA topology and binding recombination intermediates. This study also proposes that MutS binding on 2-fold right-handed crossover provides a mechanism for strand discrimination during DNA translocation.

The crystal structures of prokaryotic mismatch repair proteins MutS (1,2) complexed with mispaired substrate DNA have been solved recently (3, 4). Despite the absence of sequence homology, the overall architecture of MutS is strikingly reminiscent of type II topoisomerases (5, 6) which play essential functions in the segregation of newly replicated chromosome pairs, in chromosome condensation and in altering superhelicity (7). MutS and topoisomerase II homodimers form rings of similar size enclosing a central channel surrounded by multiple DNA binding domains (fig. 1a). In addition, MutS interacts with MutL (8) that resembles closely to the ATPase domain of type II topoisomerases (9). Experimental studies have shown that both enzymes bind recombination intermediates (10, 11) and DNA crossovers (12, 13). It is therefore likely that their common structural features reflect their common functions. The present study shows that the two enzymes share a structural motif that has, in its dimeric form, a suitable geometry for binding either DNA crossovers or Holliday junctions.

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Topoisomerase type I in the news
The complex structure of eukaryotic (non-microbial) DNA is such that it must be relaxed whenever it is needed for transcription (making of messenger RNA) or replication. Topoisomerase, a multi-subdomain enzyme, is able to accomplish this task on both positively and negatively supercoiled DNA. The 20 = (angstrom) pore provides a highly positively charged region that binds to DNA regardless of genetic sequence. The recent evidence shows that the Topoisomerase type I from eukaryotes shares no sequence or structural similarity with that from prokaryotes. Evolution would predict that eukaryotic topoisomerase type I would share some sequence homology with prokaryotic topoisomerase type I.

Rebinbo, M.R., et al. March 6, 1998. Crystal structure of human topoisomerase I in covalent and non-covalent complexes with DNA. Science 279: 477.)

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Previously we have characterized type IB DNA topoisomerase V (topo V) in the hyperthermophile Methanopyrus kandleri. The enzyme has a powerful topoisomerase activity and is abundant in M. kandleri. Here we report two characterizations of topo V. First, we found that its N-terminal domain has sequence homology with both eukaryotic type IB topoisomerases and the integrase family of
tyrosine recombinases. The C-terminal part of the sequence includes 12 repeats, each repeat consisting of two similar but distinct helix-hairpin-helix motifs; the same arrangement is seen in recombination protein RuvA and mammalian DNA polymerase b.

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The discovery of topo V suggests that type IB enzymes may also be ubiquitous and not confined to eukaryotes, as originally thought.

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posted 26. February 2003 02:49                    

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Please refrain from just posting a link, with no commentary or insights of your own provided. It's always best to at least give an short description of how the article you reference is relevant to the point you're making. In this case, it would be good to know precisely how [these] article[s] [relate] to the point David is making.

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posted 26. February 2003 03:17                   

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So now anyone who finds some cellular structure, that has no documented evolutionary history, would say "This is an example of irreducible complexity".

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The precise 3D arrangement of the topo II enzyme is a great example of irreducible complexity. I prefer to use the term irreducible function because a series of necessary functions is what makes whatever the level of actual complexity is important. But the level of complexity of this particular enzyme is over 1500 amino acids long.

Let me know what you think - Is this an example of irreducible complexity?

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posted 26. February 2003 05:09                   

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T Cavalier-Smith. Origins of the machinery of recombination and sex. February 2002, Volume 88, Number 2, Pages 125-141

Mutation plays the primary role in evolution that Weismann mistakenly attributed to sex. Homologous recombination, as in sex, is important for population genetics - shuffling of minor variants, but relatively insignificant for large-scale evolution. Major evolutionary innovations depend much more on illegitimate recombination, which makes novel genes by gene duplication and by gene chimaerisation - essentially mutational forces. The machinery of recombination and sex evolved in two distinct bouts of quantum evolution separated by nearly 3 Gy of stasis; I discuss their nature and causes. The dominant selective force in the evolution of recombination and sex has been selection for replicational fidelity and viability; without the recombination machinery, accurate reproduction, stasis, resistance to radical deleterious evolutionary change and preservation of evolutionary innovations would be impossible. Recombination proteins betray in their phylogeny and domain structure a key role for gene duplication and chimaerisation in their own origin. They arose about 3.8 Gy ago to enable faithful replication and segregation of the first circular DNA genomes in precellular ancestors of Gram-negative eubacteria. Then they were recruited and modified by selfish genetic parasites (viruses; transposons) to help them spread from host to host. Bacteria differ fundamentally from eukaryotes in that gene transfer between cells, whether incidental to their absorptive feeding on DNA and virus infection or directly by plasmids, involves only genomic fragments. This was radically changed by the neomuran revolution about 850 million years ago when a posibacterium evolved into the thermophilic cenancestor of eukaryotes and archaebacteria (jointly called neomurans), radically modifying or substituting its DNA-handling enzymes (those responsible for transcription as well as for replication, repair and recombination) as a coadaptive consequence of the origin of core histones to stabilise its chromosome. Substitution of glycoprotein for peptidoglycan walls in the neomuran ancestor and the evolution of an endoskeleton and endomembrane system in eukaryotes alone required the origin of nuclei, mitosis and novel cell cycle controls and enabled them to evolve cell fusion and thereby the combination of whole genomes from different cells. Meiosis evolved because of resulting selection for periodic ploidy reduction, with incidental consequences for intrapopulation genetic exchange. Little modification was needed to recombination enzymes or to the ancient bacterial catalysts of homology search by spontaneous base pairing to mediate chromosome pairing. The key innovation was the origin of meiotic cohesins delaying centromere splitting to allow two successive divisions before reversion to vegetative growth and replication, necessarily yielding two-step meiosis. Also significant was the evolution of synaptonemal complexes to stabilise bivalents and of monopolins to orient sister centromeres to one spindle pole. The primary significance of sex was not to promote evolutionary change but to limit it by facilitating ploidy cycles to balance the conflicting selective forces acting on rapidly growing phagotrophic protozoa and starved dormant cysts subject to radiation and other damage.

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DNA topoisomerases also must date back to the time of the first circular DNA chromosomes. Topoisomerase Ia was required for releasing strain ahead of the replication fork, while topisomerase II was essential for decatenating daughter circles after the termination of replication to allow their separation into separate daughters. Since such decatenation often produces circular dimers, additional special termination enzymes able to cut and rejoin DNA probably evolved during the origin of efficient circle replication. Since circles are the simplest way of avoiding both the end replication problem caused by the origin of separate primase and replication polymerases and avoiding digestion of ends by exonucleases (whether the organism's own or those of predators or competitors: Cavalier-Smith, 2001), it is likely that both kinds of topoisomerases and the DnaG DNA primase evolved almost simultaneously during a huge bout of enzymatic innovation of many basic DNA-handling enzymes.

Clear support for this comes from a remarkable relationship between several key DNA handling enzymes; several enzymes that interact with double-stranded DNA to catalyse apparently distinct reactions share a 100-amino acid domain called the Toprim domain because it is found in most topoisomerases and bacterial DnaG primases (Aravind et al, 1998). The Toprim domain carries the active centre that cuts and joins the DNA in topoisomerases of both the Ia and II classes; in DnaG primase this domain is flanked by longer DNA regions: an upstream one with a zinc finger domain and a downstream one with a domain for interaction with DnaB, a DNA helicase protein that helps load it onto the lagging strand. The larger and more complex topoisomerases have quite different domains associated with the Toprim domain. In topoisomerase Ia (the eukaryote homologue is confusingly called topoisomerase III) the single polypeptide chain has a Toprim domain at its N-terminal end and three C4 'little finger' DNA-binding domains near the C-terminus.

By contrast each of the two dissimilar (but evolutionarily related) polypeptides of topoisomerase II has two other domains upstream from the Toprim domain: an N-terminal MutL/Hsp90 ATPase domain is separated from the Toprim domain by an S5 domain shared with the S5 protein of the small ribosome subunit. MutL is centrally important to the universal long patch mismatch excision repair mechanism, acting as a scaffold to connect the ATPase (MutS) that recognises the mismatch to the endonuclease that nicks the DNA near the damage to allow the repair exonuclease to remove it. It also loads DNA helicase II onto the DNA so it may be unwound. This ATPase domain is essential for the active negative supercoiling of DNA by DNA gyrase, a type II topoisomerase universal in eubacteria, which also probably evolved when chromosomes first became circular, as its underwinding of DNA is essential for efficient transcription of circles and segregation of compact nucleoids in eubacteria.

A Toprim domain is also found in another ancient eubacterial recombination protein, RecR a non-enzymatic component of the minor (RecFOR) recombination pathway. In this small single polypeptide it is downstream of a C4 finger. Other kinds of proteins with Toprim domains are less universal and may be less ancient. The scattered distribution of OLD endonucleases with Toprim domains, often virally encoded, and of various phage proteins with such domains suggests that they may have arisen secondarily and been distributed by lateral gene transfer after the cenancestor (Aravind et al, 1998).

The key catalyst for homology search during recombination, RecA, is evolutionarily related to the eubacterial replicative DNA helicase, DnaB, and probably evolved from it by gene duplication and divergence prior to the cenancestor. As replication is much more basic and essential than recombination, the reverse suggestion that DnaB evolved from RecA (Leipe et al, 2000) is highly improbable and based on the widespread, but palaeontologically refuted, view that the universal tree is rooted between eubacteria and neomura and reluctance to accept the loss of DnaB by neomura (explained below).

Thus common protein domains are found in a remarkably wide variety of DNA-handling proteins, which must have been formed during precellular evolution by duplication and shuffling by illegitimate recombination of a fairly small number of domains. Note that contrary to some assumptions (Gilbert, 1987) there is no good reason to think that introns were involved in this early domain shuffling. Nor is there any reason to think that it occurred in an RNA world - indeed the very existence of an RNA world is doubtful (Cavalier-Smith, 2001). There was however very likely to have been either a temporary RNA-protein world before the evolution of DNA replication or a NA/protein world with mixed nucleotides. Probably most of the basic DNA handling proteins evolved by gene duplication and gene chimaerisation by domain shuffling of those that interacted originally with RNA or which did not discriminate strongly between the two types of nucleotides. Distant similarities can even be detected between DNA and RNA polymerases and reverse transcriptase (Joyce and Steitz, 1994).

[...]

The recombination events that shuffled the domains making up the more complex enzymes such as topoisomerase II must themselves have depended on the prior evolution of DNA ligase at least, so DNA ligase may have been almost the earliest DNA handling enzyme - after the origin of the first DNA polymerase itself. The first DNA ligase was probably the universal ATP-dependent type. I have argued that it must have evolved in precellular evolution (Cavalier-Smith, 1987b, 2001). The NAD-dependent type restricted to eubacteria and viruses probably evolved only after the evolution of the first protocell allowed the origin of secondary metabolism and the biosynthesis of more complex nucleotide cofactors like NAD (Cavalier-Smith, 2001). Possibly it was first adopted by a DNA virus to help its replication and/or was only later recruited by the host cell as a secondary enzyme (before the cenancestor); it was probably lost by the neomuran cenancestor prior to the divergence of eukaryotes and archaebacteria.

When the first endonuclease evolved is less clear. Because DNA is made in pieces and because hydrolysis of misincorporated ribonucleotides and accidental mechanical breakage may have been quite frequent before high fidelity replication and repair, they might initially not have been needed; uncontrolled they would have been more a hazard than a benefit. The distinction between a nuclease and a DNA topoisomerase can be evolutionarily slight. Thus the restriction endonuclease NaeI can be converted into a topoisomerase II (unrelated to the natural ones) by a single amino acid substitution (Huai et al, 2000). The fact that early eukaryotes evolved a DNA topoisomerase I entirely unrelated in sequence or 3D structure (hence called Ib) to topoisomerases Ia and II also indicates that it is mechanistically relatively easy to evolve a topoisomerase from other enzymes: topoisomerase Ib is related to the site-specific integrase (sometimes called recombinase) of phage (Aravind et al, 1998). As it is found also in eukaryotic viruses like vaccinia it was probably secondarily acquired by an early eukaryote from an infecting virus - a nice example of lateral gene transfer. In keeping with its independent origin, the integrase/topoisomerase Ib family cuts the DNA differently from topoisomerase Ia and II, generating a 5'0H free end.

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Aravind L, Leipe DD, Koonin EV. Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins. Nucleic Acids Res. 1998 Sep 15;26(18):4205-13.

Iterative profile searches and structural modeling show that bacterial DnaG-type primases, small primase-like proteins from bacteria and archaea, type IA and type II topoisomerases, bacterial and archaeal nucleases of the OLD family and bacterial DNA repair proteins of the RecR/M family contain a common domain, designated Toprim (topoisomerase-primase) domain. The domain consists of approximately 100 amino acids and has two conserved motifs, one of which centers at a conserved glutamate and the other one at two conserved aspartates (DxD). Examination of the structure of Topo IA and Topo II and modeling of the Toprim domains of the primases reveal a compact beta/alpha fold, with the conserved negatively charged residues juxtaposed, and inserts seen in Topo IA and Topo II. The conserved glutamate may act as a general base in nucleotide polymerization by primases and in strand rejoining by topoisomerases and as a general acid in strand cleavage by topoisomerases and nucleases. The role of this glutamate in catalysis is supported by site-directed mutagenesis data on primases and Topo IA. The DxD motif may coordinate Mg2+that is required for the activity of all Toprim-containing enzymes. The common ancestor of all life forms could encode a prototype Toprim enzyme that might have had both nucleotidyl transferase and polynucleotide cleaving activity.

[...]

Here we show that Topo IA and Topo II share a structurally conserved domain involved in DNA strand breakage and rejoining not only with one another, but also with the DnaG-type primases, a family of ATP-dependent nucleases and a family of DNA repair proteins. These observations suggest a previously unsuspected, deep mechanistic analogy between such superficially different processes as primer formation, DNA breakage and rejoining by topoisomerase and DNA cleavage by certain nucleases. We hypothesize that at a very early stage of evolution, topoisomerases and primases could have evolved from a single ancestral enzyme that might have had multiple functions in replication and repair.

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The distinction between a nuclease and a DNA topoisomerase can be evolutionarily slight. Thus the restriction endonuclease NaeI can be converted into a topoisomerase II (unrelated to the natural ones) by a single amino acid substitution (Huai et al, 2000).

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EMBO J 2000 Jun 15;19(12):3110-8

Crystal structure of NaeI-an evolutionary bridge between DNA endonuclease and topoisomerase.

Huai Q, Colandene JD, Chen Y, Luo F, Zhao Y, Topal MD, Ke H.

NAE:I is transformed from DNA endonuclease to DNA topoisomerase and recombinase by a single amino acid substitution. The crystal structure of NAE:I was solved at 2.3 A resolution and shows that NAE:I is a dimeric molecule with two domains per monomer. Each domain contains one potential DNA recognition motif corresponding to either endonuclease or topoisomerase activity. The N-terminal domain core folds like the other type II restriction endonucleases as well as lambda-exonuclease and the DNA repair enzymes MutH and Vsr, implying a common evolutionary origin and catalytic mechanism. The C-terminal domain contains a catabolite activator protein (CAP) motif present in many DNA-binding proteins, including the type IA and type II topoisomerases. Thus, the NAE:I structure implies that DNA processing enzymes evolved from a few common ancestors. NAE:I may be an evolutionary bridge between endonuclease and DNA processing enzymes.

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posted 26. February 2003 12:58                    

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By irreducibly complex I mean a single system composed of several well-matched, interacting parts that contribute to the basic function, wherein the removal of any one of the parts causes the system to effectively cease functioning.
Michael Behe, Darwin’s Black Box pg 39

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posted 26. February 2003 13:07                    

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[...]My point is that none of the articles you posted made any headway to suggesting a Darwinian scenario for how topoisomerase systems evolve; thus, they do not provide any sort of counter-argument to David's suggestion that these systems are irreducibly complex.[...]

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[...]I think that IC is demonstrated not so much in the actual topo II enzyme itself but rather the neccessary function of that enzyme (whether it is a topo II or not).[...]

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posted 26. February 2003 14:13                    

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... the defect [in my definition of IC] can be repaired easily enough by inserting a word to define IC as: a single system which is necessarily composed of several well-matched, interacting parts that contribute to the basic function, and where the removal of any one of the parts causes the system to effectively cease functioning.

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After defining the term [IC] in DBB, I went on to argue that IC systems are obstacles for Darwinian explanations.

"An IC system cannot be produced directly (that is, by continuously improving the initial function, which continues to work by the same mechanism) by slight successive modifications of a precursor system, because any precursor to an IC system that is missing a part is by definition nonfunctional DBB pg 39"

However, commentary by Robert Pennock and other has made me realize that there is a weakness in that view of IC. The current definition puts the focus on removing a part from an already-functioning system. Thus, seeking a counterexample to IC, in Tower of Bable Pennock writes about a part in a sophisticated chronometer, whose origin is simply assumed, which breaks to give a system that he posits can nonetheless work in a simpler watch in a less demanding environment. The difficult task facing Darwinian evolution, however, would not be to remove parts from sophisticated pre-existing systems; it would be to bring together components to make a new system in the first place. Thus there is an asymmetry between my current definition of IC and the task facing natural selection. I hope to repair this defect in future work. [bold mine]

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"An irreducibly complex evolutionary pathway is one that contains one or more unselected steps (that is, one or more necessary-but-unselected mutations). The degree of irreducible complexity is the number of unselected steps in the pathway."

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