Báo cáo khoa học: Structure, location and interactions of G-quadruplexes pdf

In this minireview, I describe the detailed structure of various G-quadruplexes and the computational tools that may be used to predict their formation and stability.. As an example, the

Structure, location and interactions of G-quadruplexes

Julian L Huppert

Cavendish Laboratory, University of Cambridge, UK

Introduction

Nucleic acids can form a very wide range of different

structures, aside from the well-known DNA double

helix This double helix is highly unusual in that the

structure is largely independent of the sequence For

other structures, what form they adopt, and how

sta-bly, is controlled by their sequences, and in particular

the different chemical properties of the nucleobases

It was noted in 1910 that guanine behaved

differ-ently from all other nucleobases, in that it could

spon-taneously form a gel [1] It then took over 50 years for

the structure responsible to be discovered [2] The core

consists of square arrangements of four guanines,

bound together using two hydrogen bonds for each

side of the square, and with a monovalent cation

(pref-erably K+) in the centre These squares, known as

G-tetrads, can then stack on each other to form higher

order structures called G-quadruplexes, typically with

the cations now in the interstices, each interacting with

eight guanines [3,4]

Although these structures can form from individual guanine bases, in a biological context there are few free bases around and they form from DNA (or RNA) sequences, with the bases held together by the back-bone These structures vary in their molecularity and may be tetramolecular, with one guanine in each square coming from a particular strand, bimolecular

or unimolecular In these latter two cases, there are loops connecting different runs of guanine and these loops play a very important role in controlling the details of the structure and stability of the resulting G-quadruplex [5]

For a while, G-quadruplexes were simply a struc-tural curiosity, but recently it has become clear that they play important physiological roles They are found in telomeres [6] and have been implicated in reg-ulating transcription, translation and replication [7] This interest has been reflected in the rate of publi-cation, with an exponential growth in the number of

Keywords

bioinformatics; biophysics; computational

biology; DNA; G-quadruplex; G-quartet;

G-tetraplex; genomics; RNA; transcription;

translation

Correspondence

J L Huppert, Cavendish Laboratory,

University of Cambridge, JJ Thomson Ave,

Cambridge CB3 0HE, UK

Fax: +44 1223 337000

Tel: +44 1223 337256

E-mail: jlh29@cam.ac.uk

(Received 23 February 2010, revised 23

May 2010, accepted 30 June 2010)

doi:10.1111/j.1742-4658.2010.07758.x

Four-stranded G-rich DNA structures called G-quadruplexes have been the subject of increasing interest recently Experimental and computational techniques have been used to implicate them in important biological pro-cesses such as transcription and translation In this minireview, I discuss how they form, what structures they adopt and with what stability I then discuss the computational approaches used to predict them on a genomic scale and how the information derived can be combined with experiments

to understand their biological functions Other minireviews in this series deal with G-quadruplex nucleic acids and human disease [Wu Y & Brosh

RM Jr (2010) FEBS J] and making sense of G-quadruplex and i-motif function in oncogene promoters [Brooks TA et al (2010) FEBS J]

Abbreviations

FRET, fluorescence resonance energy transfer; UTR, untranslated region.

articles mentioning the term G-quadruplex (or

G-tetra-plex, an alternative name) over the past decade

In this minireview, I describe the detailed structure

of various G-quadruplexes and the computational

tools that may be used to predict their formation and

stability I then describe where they are found in the

genomes of various organisms and how such

informa-tion allows us to predict their funcinforma-tionality

Topological variety

G-quadruplex structures may be considered to be

com-prised of a core G-rich component, consisting of

G-tetrads stacked on top of each other and zero or

more connecting loops, which may be of variable

composition

The G-rich core typically consists of two or more

stacked G-tetrads with a right-handed helical twist

The stacks are joined together by the normal sugar–

phosphate backbone The binding energy arises from

three main factors: hydrogen-bonding between the

guanines in a plane, p–p interactions between the

gua-nines in adjacent planes and charge–charge interactions

between the partially negative O6 of the guanines and

cations that typically sit in the octahedral position

between the stacks Monovalent cations, especially

K+, are particularly stabilizing Varying any of the

bases to non-G bases is highly destabilizing and such

mutant sequences are unlikely to form G-quadruplexes

in vivo[8]

This G-core can form the major part of the

struc-tures that form, as found in short sequences such as

d(TGGGGT)4, which tetramerizes to form a

four-stacked G-quadruplex with trailing ends [9] Another

example is G-wires, very long polymeric sequences of

continuously stacked G-tetrads These are currently

being investigated for their interesting electronic

prop-erties [10]

In biological contexts, however, multimerization is

relatively unlikely and most interest has focused on

unimolecular G-quadruplexes This requires at least

four runs of guanine to be joined together by (at least)

three loops These loops can have varying length and

sequence, and this controls the topology of the final

structures and is related to the direction of the G-rich

strands making up the core

Loops may link positions on the top (or bottom) of

the stacks, forming diagonal or lateral loops depending

on which guanines are being linked Alternatively, they

may link a guanine on the top of the stack to a

guan-ine on the bottom, resulting in a double-chain reversal

loop Further details and images are available in Phan

[11]

The nature of the loops is also related to the direc-tionality of the four G-rich runs making up the core of the structure – these may be parallel (requiring double-chain reversal loops) [12], antiparallel (with two strands running in each direction and either lateral or diagonal loops) [13] or a mixed ‘3 + 1’ hybrid, with three strands in one direction and one in the other, and a mixture of loop types [14] Which of these struc-tures is preferred depends on the sequence and length

of the loops – in general, shorter loops favour parallel structures [15]

There are also more exotic ways of arranging the loops, as found in one G-quadruplex taken from the promoter of the c-kit oncogene – its structure involves various internal loops [16]

Given this complexity, there are clearly many possi-ble structures for most G-quadruplex-forming sequences and experimental evidence suggests that in many cases they are of very similar energies Hence, many of these structures seem to exist in vitro as a set

of polymorphs and small changes in the conditions can favour one or more different detailed structures As an example, the human telomeric repeat (see below) has been shown to form parallel, antiparallel and hybrid structures under subtly different conditions [17] This

is clearly a big challenge both for structure determina-tion and also for targeting, whether pharmaceutically

or naturally

Telomeres in organisms

All organisms with linear chromosomes have to mark out the ends of their chromosomes in order to distin-guish them from unwanted double-strand breaks [18]

In all species studied to date, this is achieved with repetitive sequences of variable lengths characterized

by runs of guanines on one strand In humans, there are typically around 1000 repeats of the sequence d(GGGTTA) in duplex form, followed by a single-stranded overhang of hundreds of bases All vertebrates have the same sequence and most other organisms have very slight variants on this basic sequence, of the form d(G2–4T1–4A0–1) All of these have been shown to form stable G-quadruplexes under appropriate in vitro conditions

This remarkable property, suggesting that all linear chromosomes have G-quadruplex caps, implies strongly that there is a function to these structures Because telomeric regions are generally capped by a wide variety of proteins, it is not entirely clear exactly what form the DNA adopts in vivo [19,20] By devel-oping an antibody that bound specifically to parallel G-quadruplexes,

Plu¨ckthun and co-workers [21] demonstrated that

G-quadruplexes form in vivo in the ciliate Stylonichia

lemnae Although this was an elegant experiment, it

has not been replicated for other organisms and it is

possible that G-quadruplex formation is induced by the

presence of the antibody, although si-RNA experiments

suggest that this is not the case [19] Nonetheless, it is

hard to see how G-quadruplexes cannot play a critical

role in telomere structure and function

Predicting G-quadruplexes

An active field of research at the moment is to

inves-tigate whether G-quadruplexes can form in locations

in the genome other than just the telomeres, and

what other functions they may have In order to do

this, however, it is necessary to have a way of

pre-dicting G-quadruplex formation for other sequences

[22–24]

The simplest approach is to identify a

G-quadru-plex-like sequence in a region of interest and then to

study it in vitro This approach was taken in the classic

work by Hurley on the c-myc oncogene [25,26] The

NHE III1 domain of the c-myc promoter was known

to be a transcriptional repressor; Hurley proposed and

then demonstrated that it could form a G-quadruplex

and suggested that this could be responsible for the

repressor activity

However, such identifications do not lend themselves

to widespread target discovery and it is necessary to

develop some kind of predictive algorithm to identify

possible sequences We [27] and others [28,29] have

developed a variety of different predictive tools, which

are reviewed elsewhere [22,23] Our tool, quadparser,

is freely available online at http://www.quadruplex.org/

?view=quadparser and identifies sequences of the form

G3+N1–7G3+N1–7G3+N1–7G3+, with four runs of

gua-nines separated by variable loops [27]

This and other tools are based on a series of

biophysi-cal experiments that have been performed, studying the

thermodynamic equilibrium of G-rich oligonucleotides

between a G-quadruplex form and an unstructured

single strand These experiments are typically performed

using either ultraviolet melting [30] or fluorescence

melting [31] and have led to much information

associa-ting stability with particular sequences [32]

However, there are a number of problems with the

results so far First, our ability to accurately predict

structure or stability for a novel sequence is very

lim-ited We have recently taken a Bayesian inference

approach to come up with the first evidence-based

pre-dictor for stability This method uses Bayesian

calcul-cations to learn from experimentally determined data

and allows predictions of thermal stability for new input sequences under various conditions However, further data are required for it to be more accurate and it currently only predicts the melting temperature rather than more detailed thermodynamic parameters [33] It is freely available for use online at http:// www.quadruplex.org/?view=quadpredict

Second, the experiments largely describe only a sin-gle-strand folding and say little about the effects of having the complementary strand present Experiments that have been done show that, as expected, the com-plementary strand tends to favour the formation of duplex DNA [34], but it is not clear for which sequences G-quadruplexes will still form and with what stability compared with the duplex Elegant experiments using FRET in plasmids show that G-quadruplexes can exist in a duplex context, but it is not yet possible to generalize this result [35] Interest-ingly, the complementary strand of a G-quadruplex may form an alternative C-rich structure called an i-motif, which contains hemi-protonated C” C+ base pairs [36]

Lastly, all the experiments are performed in vitro, and so omit many factors that would be present

in vivo These factors include the many proteins that interact with G-quadruplexes, stabilizing, destabilizing (e.g acting as helicases) or cleaving them [37] It also neglects the presence of nucleosomes [38], which stably bind duplex DNA Supercoiling is also neglected, although experimental evidence for both c-kit and c-myc show that it can play a significant role in promoting G-quadruplex activity [39] Also, in vitro experiments are generally performed in dilute aqueous solution, although it has been repeatedly shown that molecular crowding (as found in vivo) can induce G-quadruplex formation [40,41]

Despite these limitations, much work has been per-formed developing and refining these predictors, which allows broad-scale studies of the presence of G-quad-ruplex-forming units in many organisms’ genomes In humans, for example, there are  375 000 possible quadruplex-forming sequences, although it is unlikely that all of these would in fact form in vivo [27,28] Nonetheless, it is interesting to note that this is signifi-cantly below the number that would be expected by chance, suggesting an evolutionary pressure to reduce the number

Genomic locations

Predicted G-quadruplexes are not located randomly throughout the genomes and tend to cluster together

in particular regions Coupled with the depletion

across the whole genome, this is highly suggestive of

evolved functionality

Aside from telomeres, one of the first regions

con-sidered for the presence of G-quadruplexes was gene

promoters Hurley’s work on c-myc established the

principle that such sequences could regulate gene

tran-scription [25,26] and other examples, such as c-kit

[42,43], were then found [44] These are discussed in

more detail in one of the other minireviews in this

series [45]

Computational analyses of the entire human genome

showed that G-quadruplexes were in fact very likely to

be found just upstream of gene TSS positions Indeed,

almost half of all known genes have a putative

G-quadruplex in their promoter in a position where it

could be involved in gene regulation These

G-quadru-plexes tend to be more thermodynamically stable than

typical [46]

These genes are not random in terms of their

func-tions Oncogenes are more likely, and tumour

supres-sors less likely, to contain G-rich sequences [29]

Detailed GO code analysis showed in general that

genes that are involved in regulation (e.g transcription

factors) are more likely to have promoter

G-quadru-plexes, whereas ‘housekeeping’ genes (e.g those

involved in protein biosynthesis) tend to be depleted in

G-quadruplexes [46] Interestingly, genes involved with

olfaction are extremely depleted, raising questions

as to their evolutionary history and how they are

regulated

Similar results have been found in a wide variety of

other organisms, yielding similar results for other

ver-tebrates and comparable results for other eukaryotes

and prokaryotes

RNA is also capable of forming G-quadruplex

struc-tures and indeed forms them even more stably than does

DNA In addition, whereas DNA in biological systems

is typically double-stranded, RNA is typically

single-stranded, and so G-quadruplex formation does not have

to compete with duplex formation and is hence more

likely However, a wide range of alternative structures is

possible in addition to G-quadruplexes [47]

The question therefore arises as to whether RNA

G-quadruplexes could play a role in translation

regula-tion, in a manner analogous to DNA G-quadruplexes

and transcription We identified a predicted

G-quadru-plex in the 5¢-UTR of the N-ras oncogene, and

demon-strated that it could form a G-quadruplex in vitro, and

that the formation of the G-quadruplex led to a

signifi-cant (fourfold) reduction in the amount of protein

pro-duced for a given amount of mRNA [48] This work

has since been developed by others, who have

investi-gated how the positioning affects the effect [48] and

demonstrated that this mechanism applies in vivo in prokaryotes and eukaryotes [49,50]

Although the algorithms used to predict G-quadru-plexes have been created based on data from DNA experiments, it is still approximately possible to modify them to predict RNA G-quadruplexes, although con-siderable further work is needed to bring the accuracy

of such predictions up the same level as for DNA Nonetheless, the tools available can give a clear indica-tion of the locaindica-tion of G-quadruplexes in RNA form-ing sequences [51]

The first thing that is clear is that there is distinct asymmetry between the coding strand and the template strand – there are relative few G-quadruplex motifs in the coding strand, suggesting that they have been dis-favoured in general However, those that still exist do cluster at the 5¢-end of the 5¢-UTR of many thousand genes [51] As with promoter G-quadruplexes, there is

a clear selectivity in terms of gene functions Interest-ingly, there is a localized concentration of G-quadru-plexes immediately after the transcription end site of some genes, particularly those with additional genes immediately downstream These are regions that should never be transcribed, and have been associated with regulation of transcription termination via a paus-ing mechanism Studies uspaus-ing an antibody evolved to specifically bind G-quadruplexes showed that gene expression changed significantly for genes with G-quadruplexes in their promoters, in the 5¢-UTR or around the 3¢-UTR, suggesting a widespread range of functions and a complex response [52]

Duplex DNA in cells is not naked, but is stored as nucleosomes, wrapped around histone proteins These structures would be expected to have the effect of sta-bilizing the duplex form and preventing the formation

of G-quadruplexes or any other secondary structures However, nucleosomes are not present along the entire genome and there are gaps between nucleosomes, of a size large enough to allow G-quadruplex formation [38] Notably, the promoter region immediatetely upstream of genes, where G-quadruplexes are espe-cially common, are denuded on nucleosomes This inverse association holds true across the human gen-ome, with stable G-quadruplex sequences located in general in the gaps between nucleosomes This is con-sistent with them having a function derived from their structure, either as a result of evolutionary pressure to move them into gaps, or because the formation of G-quadruplex structures prevents nucleosomes from forming in those locations [53]

As previously discussed, the presence of a comple-mentary strand of DNA disfavours G-quadruplex formation and so we may expect that

G-quadruplex-based functionality may be more likely when DNA is

single-stranded Such conditions are found as standard

in telomeres, where there is a single-stranded overhang,

and during replication, when the strands must be

sepa-rated; G-quadruplex formation has been proposed for

both of these One other, rarer, occasion when DNA is

single-stranded is a G-loop [54], the product of

transcription of certain AG-rich sequences where the

stability of the normally transient RNA⁄ DNA hybrid

outweighs the DNA duplex, resulting in the formation

of a loop with the complementary DNA strand being

unbound [55] These structures have been shown to

form in immunoglobulin class switch regions, and

may occur elsewhere G-loop formation in plasmids

has been shown directly and indirectly to lead to

G-quadruplex formation and this may form part of

the switching mechanism [54,56]

Conclusions

G-quadruplex-forming sequences have been proposed

to play a wide range of physiological roles, including

in all the processes of the Central Dogma

Experimen-tal studies have confirmed these functions in a number

of specific genes and computational methods have

given statistical evidence that these structures may be

widespread in the genomes of many organisms

How-ever, further evidence is required to demonstrate

clearly exactly how many of these possible

G-quadru-plexes do actually form in vivo and to clarify many of

the details of the mechanisms involved

In general, it seems that G-quadruplexes are

gener-ally a bad thing and most organisms are depleted in

sequences that could form them However, some

spe-cific locations and spespe-cific G-quadruplexes appear to

be highly amplified, presumably as a result of

evolu-tionary pressures Given the stability of the

G-quadru-plex compared with the duG-quadru-plex, it seems unlikely that

they form permanently (except at telomeres or in

RNA), but are likely to be a relatively minor

compo-nent at equilibrium They therefore function as

switches, forming in response to stimuli such as

super-coiling changes Once formed, they are sufficiently

metastable to be long-lived, especially with protein

sta-bilization, and can therefore control other processes,

whether by acting as steric blocks, occluding other

active DNA sites or recruiting proteins that bind them

Once the trigger is removed, helicase activity (for

example) can then return them to their original duplex

state, ready for retriggering At any given time, the

number of folded G-quadruplexes in a given cell may

be extremely low, although many may form during the

lifetime of the cell

This field is still relatively new, but has come a long way in terms of establishing hypotheses and providing proof of concept and statistical evidence for many of them Over the next few years, it will become essential

to investigate more of the details of the field, and establish exactly how important they are in vivo I am sure the field will rise to the challenge and continue to

be an exciting place to work

Acknowledgements

JLH is a Research Councils UK Academic Fellow and Member of Parliament for Cambridge Caroline Wright is thanked for helpful discussions and Jaime Gomez Marquez is thanked for extreme patience

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