BioSystems, 19 (1986) 1.23-126 123 Elsevier Scientific Publishers Ireland Ltd.

DNA S T R U C T U R A L VARIABILITY AS A F A C T O R IN G E N E E X P R E S S I O N AND E V O L U T I O N

MICHAEL CONRAD", S.K BRAHMACHARI b and V. SASISEKHARAN b aDepartments o f Computer Science and Biological Sciences, Wayne State University, Detroit, MI 48202 (U.S.A.) and ~' Molecular Biophysics Unit, Indian Institute o f Science, Bangalore 560 012 (India)

(Received December 10th, 1985)

Redundant DNA can buffer sequence dependent structural deviations from an ideal double helix. Buffering serves a mechanistic function by reducing extraneous conformational effects which could interfere with readout or which would impose energetic constraints on evolution. It also serves an evolutionary function by allowing for gradual variations in conformation-dependent regulation of gene expression. Such gradualism is critical for the rate of evolution. The buffer structure concept provides a new interpretation for repetitive DNA and for exons and introns.

Keywords: Redundant DNA; Introns; DNA evolution.

A n u m b e r of exp lana t ions have been sugges ted for the h igh r e d u n d a n c y of DNA in e u k a r y o t i c cells and for the p h e n o m e n o n of exons and in t rons . One plausible idea is t ha t r e d u n d a n t DNA is basical ly pa ras i t i c (Dooli t t le and Sapienza, 1980; Orgel and Crick, 1980). In th is no te we will sugges t an a l t e rna t ive exp lana t ion in which the r edunda n t DNA serves a phys io logica l and evo lu t i ona ry buffer ing func t ion .

The model is based on the r ecogn i t i on t h a t DNA is not an ideal double helix. Right and left handed double helical DNA (e.g. B and Z DNA) can occur and reversa l s of h a n d e d n e s s are possible due to con fo rma t iona l flexibil i ty a r i s ing f r o m sugar pucker , r o t a t i o n a round phospho-d ies t e r bonds , and g lycosyl to r s ion ( S a s i s e k h a r a n and P a t t a b i r a m a n , 1978; Gup ta et al., 1980; S a s i s e k h a r a n and B r a h m a c h a r i , ].980). H a n d e d n e s s is con- trolled by base sequence , e.g. Z DNA is f avored by sequences con ta in ing a l t e rna te pu r ine -py r imid ine res idues (Wang et al., 1979). A con,fiderable a m o u n t of con- fo rma t iona l p o l y m o r p h i s m is possible with- in bo th r igh t and left handed fo rms , w i th d i f ferent base s e quences leading to d i f ferent dev ia t ions f r o m a pure helical p a t t e r n

(Sas i s ekha ran , 1983). The pa r t i cu la r con- f o r m a t i o n a s s u m e d by DNA appears to be s t rong ly mil ieu dependent , wi th f ac to r s such as superhel ica l dens i ty , ca t ion concen t r a t i on , me thy la t ion , and s t ruc tu ra l p ro te ins exe r t ing s igni f icant effects (Behe and Felsenfeld, 1981; S t i rd ivan t e t al., 1982; La tha et al., 1983; R a m e s h and B r a h m a c h a r i , 1983; La th a and B r a h m a c h a r i , 1985).

The var iabi l i ty of DNA c o n f o r m a t i o n undoub ted ly plays a va r i e ty of roles . A plausible a s s u m p t i o n is t h a t one of these roles involves gene express ion . The DNA should p re sen t a recognizable t a rg e t for a r e a d o u t enzyme. Some s tudies indicate t h a t th i s m a y be a t leas t in pa r t a shape p h e n o m e n o n (Singleton et al., 1982). DNA g e o m e t r y probably plays an i m p o r t a n t role in gene express ion .

Since the detai led connec t ions be tween the d i f ferent DNA c o n f o r m a t i o n s and func t ion have not y e t been worked out , we will p roceed on the bas is of the fol lowing simple c lass i f ica t ion of DNA base sequences . A segmen t of DNA will be called a coding sequence if it is t r an sc r i b ed to RNA. A seg m en t of DNA will be called r egu l a to ry if it specifically cont ro ls the exp res s ion of one or

0303-2647/86/$03.50 ~ 1986 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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more coding segments. Segments of DNA which are neither coding or specifically controlling will be called extrafunctional. We wish to consider whether these extra- functional regions actually have any function.

An important feature of coding segments is tha t they must be good information stores. That is, it should be possible to code for any possible protein. Particular proteins should not be precluded because the DNA which codes for them is conformationally unfavorable for transcription. If this happened the possibilities for evolution would be greatly restricted. In the classical double helical model of DNA this presented no problem. All DNA base sequences were associated with the same secondary structure. The fact that sequence can influence conformation complicates the situation. It is possible that an alteration in a coding sequence could affect DNA structure. This would be undesirable on two grounds. First it might interfere with readout enzymes acting on the coding sequence, especially if these readout enzymes were highly specific. Second it might lead to extraneous "regulatory" effects on neighboring coding sequences.

Both of these problems may be eliminated by using extrafunctional DNA as a mechanistic buffer. If a long coding sequence exhibits undesirable conformational varia- bility it can be broken up by intervening extrafunctional sequences which serve to absorb some of the conformational strain. In this case the coding pieces would correspond to exons and the extrafunctional sequences to introns. Separating coding regions by blocks of repetitive DNA would also serve to control the ramification of conformational effects. Repetitive DNA is more useful for this purpose than non-repetitive DNA since the latter would allow even greater structural variability and could itself introduce extraneous conformational strains. Re- petitive DNA, on the other hand, would allow the conformational strains to be absorbed into a highly ordered structure,

thereby reducing the effects of these strains on the overall s tructure and function of DNA to a minimum.

Large conformational deviations from the average DNA conformation in the coding sequence are more likely to have deleterious effects on gene expression if the read- out enzymes are highly specific. If the enzymes are on the less specific side the extrafunctional sequences become unnecessary. The sequences are absent in prokaryotes. The alternative, but in a sense equivalent s ta tement is, if an organism cannot afford to maintain extrafunctional sequences it cannot support highly specific readout and regulatory machinery. This is consistent with the well known fact tha t microorganisms with extraneous DNA are evolutionarily unstable and outcompeted by variants which dispense with these extraneous pieces (Zamenhof and Eichhorn, 1967). As the number of genes becomes larger the problem of finding an optimum genetic s tructure by the Darwinian mechanism of variation and selection, or indeed by any mechanism, becomes more difficult. The presence of parasitic DNA becomes inevitable. But this so-called parasitic DNA is extrafunctional DNA which can serve to mechanistically buffer the effects of sequence variations in coding and regulatory regions. As genomes become larger they automatically accumulate the regulatory mechanisms which allow them to develop further complexity by regulatory evolution.

Extrafunctional DNA has an even more far-reaching effect as an evolutionary buffer which modulates shape change in the regulatory sequences. It is important that regulation be gradually tunable. Single genetic changes should be able to give rise to useful regulatory changes, otherwise evolution through regulatory changes would be too slow. This is due to the fact tha t the rate of evolution scales a s pn, where p is the probability of a genetic event and n is the number of simultaneous genetic events

required to produce an improved or at least an acceptable form (see Conrad, 1983 for detailed calculations). The best way to organize a DNA molecule to achieve this is to introduce repetitive blocks of DNA which can serve to buffer the effects of base sequence change on the geometry of the regulatory segments. In addition such intervening segments would isolate regulatory segments , allowing for independent ad jus tmen t of different regulatory controls. A DNA molecule organized in this way would support a larger range of accessible regulatory variations.

It might be thought tha t extrafunctional DNA serving as an evolutionary buffer could not arise in evolution since it is a cost to the individual organism. However, there are at least two mechanisms by which such extrafunctional buffering material could a-rise. The first is that extrafunctional sequences which serve a mechanist ic buffering role would also serve an evolution- buffering role. The second is tha t extrafunctional sequences would hitchhike along with the evolutionary advances which they facilitated. Hitchhiking has been proposed as a mechanism for the evolution of high mutat ion rates (Cox and Gibson, 1974) and as a mechanism for the evolution of redundancies which buffer the effects of muta t ion on protein s t ructure and funct ion (Conrad, 1979, 1982). The accumulat ion of parasi t ic DNA is also a hitchhiking phenomenon, but one which is unaided by any sys temat ic evolutionary advantage. Repetit ive DNA is most likely to arise through specific duplicative mechanisms and to be propagated by either direct selection on the individual (in mechanist ic buffering) or by hitchhiking selection.

A buffering effect has recently been demonstra ted in supercoiled plasmids containing d(CG), inserts near the recognition sequence (G-G-A-T-C-C) for the restr ict ion endonu.clease BamH1 (Azorin et al., 1984). Formation of the left handed Z- DNA conformation in the d(CG), insert

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resulted in a B-Z junct ion between opposing helical forms that inhibited cleavage by BamH1 when its recognition sequence was located immediately adjacent to the insert or four base pairs away from it. Cleavage by BamHl appears to be highly sensitive to DNA conformation. No inhibition of cleavage was found when the BamH1 site was eight base pairs away. The implication is that the structural perturbation of the recognition sequence by the B-Z junction is buffered by the introduction of intervening base pairs. Depending on the context, such intervening base pairs could serve primarily as a readout buffer or primarily as an evolutionary buffer; in general they are likely to contribute in some degree to both types of buffering. DNA which accumulates buffering sequences will exhibit an enormous amount of neutral or quasi-neutral conformational polymorphism. This fact should be recognized in any interpretation of the functional significance of DNA conformation.

Buffering of regulatory evolution by extrafunctional DNA has profound im- plications for evolutionary theory. The most prominent evolutionary mechanisms in prokaryotes involve mutation and other genetic operations involving the sequence variability of DNA. The differences within wide taxonomic categories of metazoans are often largely regulatory. For example, the differences among the primates may be more a matter of regulation than of base sequence differences in the coding regions (King and Wilson, 1975). The major adaptive radiat ions among these forms are likely to have been mediated by regulatory evolution. For this type of evolution the s tructural variability of DNA is much more important than the pure sequence variability. So-called redundant DNA, rather than being an epiphenomenon of evolution, serves to facilitate the changes in DNA geometry which underlie regulatory evolution. With such facilitation DNA structural changes are capable of playing as important a role in the diversification of metazoan organizations as DNA sequence

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changes played in the diversification of proteins and of microorganisms.

Acknowledgements

This paper was written during a sabbatical visit by M.C. to the Molecular Biophysics Unit, Indian Institute of Science. Support by National Science Foundation grant INT-83- 11410 and by the Indian Institute of Science is gratefully acknowledged. S.B. and V.S. acknowledge support from the Department of Science and Technology, New Delhi.

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