Accepted Manuscript Title: Contributions of the specialised DNA polymerases to replication of structured DNA Author: Caroline M. Wickramasinghe Hayat Arzouk Alexander Frey Ahmed Maiter Julian E. Sale PII: DOI: Reference:

S1568-7864(15)00016-6 http://dx.doi.org/doi:10.1016/j.dnarep.2015.01.004 DNAREP 2039

To appear in:

DNA Repair

Received date: Accepted date:

14-11-2014 16-1-2015

Please cite this article as: C.M. Wickramasinghe, H. Arzouk, A. Frey, A. Maiter, J.E. Sale, Contributions of the specialised DNA polymerases to replication of structured DNA, DNA Repair (2015), http://dx.doi.org/10.1016/j.dnarep.2015.01.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Contributions of the specialised DNA polymerases to replication of structured

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DNA

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Caroline M. Wickramasinghe, Hayat Arzouk, Alexander Frey, Ahmed Maiter and

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Julian E. Sale1.

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MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH,

Corresponding author, email: [email protected]; tel. +44 1223 267099

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1

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U.K.

Keywords: Replication, DNA secondary structures, DNA polymerases, translesion synthesis, G quadruplex

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Abstract It is becoming increasingly clear that processive DNA replication is threatened not only by DNA damage but also by secondary structures that can form in the DNA

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template. Failure to resolve these structures promptly leads to both genetic instability, for instance DNA breaks and rearrangements, and to epigenetic instability, in which inaccurate propagation of the parental chromatin state leads to unscheduled changes

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in gene expression. Multiple overlapping mechanisms are needed to deal with the wide range of potential DNA structural challenges to replication. This review focuses

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on the emerging mechanisms by which specialised DNA polymerases, best known for their role in the replication of damaged DNA, contribute to the replication of

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undamaged but structured DNA, particularly G quadruplexes.

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1. Introduction

DNA replication is an extraordinarily complex and rapid enzymatic reaction that leads to the duplication of gigabases of DNA with astonishing fidelity in the space of just a

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few hours. However, its delicately balanced mechanism is readily perturbed by

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problems in the DNA template. Over the years, the ways in which the replication machinery deals with damaged DNA has received much attention. However, it is

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clear that many problems encountered during replication arise from the nature of the DNA template itself. When unwound from its normal B-form duplex, DNA can form a range of secondary structures that are dependent on local sequence features, such as symmetry, repetitive tracts and G-richness and on topology, for example supercoiling (Figure 1). Many of these structures are sufficiently stable to stall DNA polymerases in vitro [1-4] although proving that a given sequence forms a particular structure in

vivo is difficult. Nonetheless both indirect and, increasingly, direct evidence is accumulating for their existence in vivo. Some DNA structures are important for

normal cellular physiology. For example, the secondary structures that form in the repeat sequences of telomeres are likely to play a crucial role in the protection of telomeres from degradation and fusion [5]. More recently, a subset of G quadruplexforming sequences has have been linked to the specification of replication origins in vertebrates [6-8]. Nonetheless, even these sequences potentially pose a threat to the processivity of replication and to genome stability. It is not surprising, therefore, that 2

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multiple mechanisms contribute to dismantling secondary structures during replication. Much attention has been paid to the roles of specialised helicases and nucleases, a topic recently reviewed in this journal [9]. The present review will focus on the emerging role of specialised DNA polymerases in processing of DNA

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2. DNA secondary structures interfering with DNA replication

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secondary structures, particularly G quadruplexes, during replication.

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There are many natural impediments to DNA replication, of which structured DNAs are just one class. For a comprehensive review of this broader area, Mirkin and

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Mirkin’s 2007 review still provides an excellent starting point [10]. Assembly of nonB DNA structures is influenced by local base composition and sequence symmetry and often arises in areas of repetitive DNA, such as inverted repeats (IR), mirror

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repeats (MR), direct repeats (DR) and regions capable of forming more complex structures such as G quadruplexes and triplex DNA (Figure 1A). While secondary structures can readily be induced to form in single stranded DNA in vitro the extent to

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which they form in vivo has been unclear, since they are often less energetically

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favourable than duplex DNA. Further, the requirement for exposure of single stranded DNA suggests that formation must occur during replication, transcription or through

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significant negative supercoiling, a situation that is unlikely in eukaryotic genomes [10]. Thus, secondary structure formation on the lagging strand has been favoured in many models and there is certainly evidence that lagging strand structures can pose impediments to replication and induce genetic instability. For instance, in telomeres, T-loops and lagging strand G4 motifs impede replication, can act as fragile sites and require specific factors for their safe replication [11,12]. However, during normal replication the lagging strand is coated with the single strand binding protein RPA, which is able to counteract secondary structure formation [13] and this helps prevent deletions between direct repeats by Polδ in vitro [14]. Whether this also applies to the

single stranded DNA exposed between the helicase and polymerase on the leading strand is less clear. Either way, there is strong evidence that at least G quadruplex structures are able to interrupt leading strand replication as well [15,16]. 2.1 Structures arising in repetitive DNA 3

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Direct tandem repeats are common genomic features and the most studied of these are dinucleotide and trinucleotide repeats, which simply consist of non-interrupted iterations of a core repeat unit or two or three nucleotides in size, respectively. Common structures arising in this microsatellite (repeat ≤ 9) or minisatellite (repeat ≥

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10) (MS) DNA are left handed Z-DNA and slipped DNA (S-DNA), as well as G quadruplexes (Figure 1). Z-DNA arises in sequences consisting of alternating purines

and pyrimidines, which can mis-fold to a left-handed double helical structure termed

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Z-DNA [17]. S-DNA is formed when repeats of various length, complementarily

mispair following denaturation / renaturation resulting in combinations of stretches of

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single stranded DNA and hairpin structures. MS DNA is prone to expansion and is associated with several serious inherited genetic diseases including myotonic

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dystrophy, Friedreich’s ataxia and Huntington’s disease, as well as being observed in a number of human cancers [18]. Inverted repeats (IRs) are complementary strands of DNA extending equidistantly from a symmetrical centre and have the potential to

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form cruciform structures and hairpins [19,20]. Mirror repeats are made up of identical DNA sequences arranged sequentially and can form triple helical conformations termed H-DNA. H-DNA can also be formed in supercoiled

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homopurine-homopyrimidine MRs when a third strand, either from the same DNA

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molecule (intramolecular) or from a separate DNA molecule (intermolecular) invades and binds to the groove within the double helix via Hoogsteen pairing [21].

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Remarkably regions of repetitive sequence, including those described above, make up ~50% of the human genome and thus the opportunity for them to present challenges to replication fork progression is high. In addition to the predominantly intramolecular secondary structures discussed above a series of displacement loop (D-loop) structures can present barriers to replication. D-loops are formed when one strand of nucleic acid invades a DNA duplex and pairs with one of the strands, displacing the naturally complementary DNA strand. They are physiological intermediates in homologous recombination and are found at telomeres (T-loops), where they have been proposed to contribute to telomere end protection [5]. However, they can also form with an invading RNA strand (R-loops). These structures have both physiological roles, for instance in transcription termination [22], but also pose a potential impediment to replication and genome integrity [23]. 4

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2.3 G quadruplexes It is over a hundred years since Bang first noted the propensity for guanylic acid to form gels in solution [24], but it was not until 1962 that Gellert and colleagues

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suggested that this property resulted from the formation of planar quartets of four dGs interacting via Hoogsteen base pairing (Figure 1G) [25]. Later still, the ability of these G quartets to stack to form what is now known as a G quadruplex (Figure 1G) was

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invoked as a mechanism for intermolecular pairing of telomeric sequences [26,27].

However, G quadruplexes (G4s) can also form intramolecular structures, which if

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present on the template strand of a DNA polymerase create a replication impediment [28]. The sequence requirements for G4 formation are often cited as G3-5-N1-7-G3-5-

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N1-7-G3-5-N1-7-G3-7, which gives around 376,000 potential G4-forming sequences (G4 motifs) in the human genome [29]. However, recent evidence suggests that both structures with potential for a stack of only two quartets, as well as G4s with longer

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loops of N > 7, can also create replication impediments in vivo [16,30]. Despite an abundance of biophysical evidence supporting the formation of G

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quadruplexes in vitro and evidence of their ability to stall DNA synthesis in primer

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extension assays [28], acceptance of their existence in vivo has been slow. However, numerous converging lines of evidence now support the formation of G4 structures in

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vivo and their ability to act as replication impediments. Several helicases have been shown to unwind G4 structures in vitro including PIF1, FANCJ (Dog-1 in C. elegans)

[31-34], WRN and BLM [35-38]. Cells mutant in these helicases exhibit genetic [3134] and epigenetic [39] instability that is focussed on G4 motifs in vivo. Further, compounds that are able to selectively bind to G4 structures can induce genetic instability or signs of DNA damage at G4 motifs. Thus, the bisquinolinium compound PhenDC3 induces rearrangements within the human CEB1 minisatellite inserted into the budding yeast genome [40], mimicking the effect of deletion of the PIF1 helicase [41]. Additionally, the highly G4-selective small molecule pyridostatin [42] induces transcription and replication-dependent damage in human cells, indicated by accumulation of γH2Ax at sites with high G quadruplex-forming potential [43]. Building on an earlier demonstration in the ciliate Stylonychia [44], G4-specific monoclonal antibodies have now been used to recognise a subset of G4 structures in mammalian cells [45,46]. Finally, recent data from our lab have demonstrated a close 5

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correlation between the ability of a G4 motif to induce replication-dependent epigenetic instability of a locus and its ability to form an intramolecular G4 structure [16]. Nonetheless, the relationship between G4 structures characterised in vitro and the structures formed by the same motif in the context of chromosomal DNA remains

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unclear. Interestingly, our recent study on G4-dependent epigenetic instability revealed a poor correlation between in vitro melting temperature of G4 structures and

the ability of the corresponding motif to cause epigenetic instability in REV1-

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deficient cells [see below and 16].

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3. Consequences of failure to process secondary structures

It is becoming evident that the apparent serene rapidity of unperturbed replication actually hides the fact that the replisome is continually dealing with rucks and tangles

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in the template DNA, but that this only becomes problematic when other factors, such as replication stress or mutations in replisome components, come into play.

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structures

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3.1 Replication slow zones and fragile sites: genetic instability at secondary Some genomic sites appear particularly prone to problems during replication.

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Identified in yeast as ‘replication slow zones’ [47] and in mammalian cells as ‘common fragile sites (CFS)’ [48], these regions are particularly prone to form replication-dependent DNA breaks under conditions of replication stress and when the intra-S phase checkpoint is disabled. For instance cytologically identifiable common fragile sites, which can extend over several hundred kilobases of DNA, are classically induced by treatment of cells with the DNA polymerase inhibitor aphidicolin. The underlying cause for such sites has been a subject of long debate. Some have been associated with sequences that could form secondary structures, but in many cases such potential is not apparent. Indeed, early genome-wide analyses of fragile sites did not reveal a clear association with any particular secondary-structure forming sequence features [49]. However, a wider analysis of over 600,000 breakpoints associated with somatic copy number variations in human cancer uncovered an association with G4 motifs [50] and a distinct class of early replicating fragile sites in B cells have been associated with repetitive elements [51]. In contrast, 6

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the Debatisse group suggested an alternative explanation for the existence of CFS. They noted that the most prominent CFS in human lymphocytes, FRA3B, resulted from a local paucity of replication initiation sites meaning that individual forks from flanking regions have to traverse large distances, about 700 kb. Since the flanking

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origins fire only in mid-S phase, fork slowing with aphidicolin resulted in a region in which replication was not completed [52]. It thus seems likely that multiple factors,

including replication stress, secondary structures and a paucity of initiation events,

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3.2 Epigenetic instability at G quadruplex structures.

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can conspire to increase the chance of replication-dependent DNA breakage.

Recently, we have shown that cells lacking a range of G4-processing enzymes,

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exhibit stochastic loss of expression of genes containing G4 motifs [16,30,39]. We have suggested that this reflects leading strand replication fork arrest, at least in the case of REV1-deficient cells, that interrupts the normal recycling of parental histones carry

post-translational

modifications

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that

responsible

for

specifying

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transcriptional state of the locus. This work highlighted an unanticipated role for the Y-family DNA polymerase REV1 in the replication of G4 DNA [30] and expanded

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on the idea that specialised polymerases, as well as helicases, were involved in

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maintaining processive replication at structured DNA.

4. The specialised DNA polymerases and the replication of damaged DNA When the replication fork encounters a damaged DNA template, the replicative polymerase stall as they are generally not able to incorporate nucleotides opposite damaged bases. However, if the cells attempt to employ excision repair of the damage at this point, when it is exposed in single stranded DNA, they will collapse the replication fork, creating a double strand break, a far more deleterious lesion. To circumvent this problem they employ DNA damage tolerance pathways to get the lesion back into duplex DNA to allow its safe repair. There are two main mechanisms of DNA damage tolerance [53]. Template switching involves the stalled nascent strand switching to use an undamaged template, most commonly the newly synthesised daughter strand on the sister chromatid. Alternatively, the stalled replicative polymerase is replaced with one or more specialised polymerases that are 7

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able to incorporate a base opposite the lesion and to extend from the resulting mismatch. This process is known as translesion synthesis. In vertebrates there are now at least eleven polymerases, in addition to the trio of

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replicative polymerases (Polε, Polδ and Polα), involved in various aspects of DNA damage tolerance and repair [reviewed in 54]. At the heart of replicative DNA damage tolerance mechanisms are the Y-family polymerases Polη, Polι, Polκ and

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REV1 and the B-family polymerase, Polζ. Access of these polymerases to DNA is under tight control by a series of context-determined interactions with, among others,

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ubiquitinated PCNA and the C terminus of REV1 itself [54,reviewed in 55]. TLS polymerases can bypass a diverse range of DNA lesions and, while they are

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inherently mutagenic, often replicate particular lesions with a high degree of accuracy. For example Polη is highly accurate when bypassing UV-induced cis-syn cyclobutane T-T dimers, while Polκ is proficient at TLS past bulky lesions, such as

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benzo(a)pyrene adducts [56,57]. However, TLS polymerases usually work in concert, with Polζ functioning to extend from the mismatch created following insertion opposite a lesion by one of the Y-family polymerases [58]. In this context REV1

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appears to serve as an adaptor between PCNA, the other Y-family 'inserter'

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polymerases and the 'extender' polymerase, Polζ potentially aiding polymerase switching [reviewed in 55,59,60]. The structural and biochemical features underlying

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lesion bypass by TLS polymerases may also apply to resolution of secondary structures at the replication fork [reviewed in 48].

5. Resolution of secondary structures: the contribution of the specialised polymerases

5.1 The TLS polymerases in replication of undamaged DNA: what’s the problem? In addition to their role in replicating damaged DNA, it is increasingly evident that several of the TLS DNA polymerases play a role in the replication of undamaged templates. The key questions is, what are the problems being tackled by these enzymes?

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Polζ is required for the majority of damage-induced mutagenesis in budding yeast, but is also responsible for at least half the spontaneous mutagenesis that arises in the absence of exogenously applied damaging agents. Importantly, Polζ–dependent mutagenesis is increased when replication is stressed, and this results from it copying

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undamaged DNA rather than from bypass of DNA lesions [61]. Under these conditions, Polζ, together with REV1, is required for replicating ‘difficult’ sequences that form, for instance, small hairpin structures [62]. This often results in complex

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mutations, a long documented feature of Polζ-deficiency [63], that result from template switching primed by the dC transferase activity of REV1 [62]. A recent

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study in human cells has suggested that Polζ is also required for the replication and stability of common fragile sites in G2/M in human cells [64], although curiously this

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appears to involve the catalytic REV3 subunit acting independently of REV7, a ‘regulatory’ subunit normally required for Polζ function.

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There is incomplete evidence implicating the Y-family polymerases, Polη and Polκ in replication of potentially structured or repetitive DNA, particularly G-rich tracts. C. elegans lacking the FANCJ helicase homologue Dog-1, and depleted of Polη and

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Polκ, by RNAi have been reported to exhibit an increase in deletions at G-tracts,

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suggesting that both polymerases participate in the deletion-free replication of these sequences [65]. However, a more recent and extensive study using knock-out alleles

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did not find any impact of Polη or Polκ status on G-tract stability in either wild type

or Dog-1 deficient worms [66]. In human cells it has been reported that Polη and Polκ,

but not Polι, are needed to prevent breaks in cells harbouring multiple copies of the G4-forming sequence from the human c-MYC gene [67] and, further, Polη helps

prevent CFS expression during an otherwise unperturbed S-phase [68]. These observations might well reflect defective replication of 'difficult' sequences rather than an increase in replication stress per se since absence of Polη does not affect

global replication fork rates [68,69]. Consistent with this idea, the need for Polη in

replicating CFS becomes more pronounced under conditions of mild replication stress [70]. It is clear that further work is needed to clarify the role of Polη and Polκ during replication of potentially structured DNA sequences, in human cells, and to identify the circumstances under which they may be deployed.

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Both Polκ and REV1 have also been implicated in maintaining the stability of repetitive sequences. In vitro experiments have shown that Polκ may play an important role in both the accurate replication and evolution of microsatellite repeats. On dinucleotide repeats, Polκ is as, if not more, accurate than the replicative Polδ and

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can facilitate the completion of synthesis over a repeat by Polδ [71]. However, when faced with a mononucleotide [T]11 repeat, Polκ frequently inserted dG or dC thereby breaking up the repeat, [72] potentially facilitating subsequent replication rounds. In

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yeast, REV1 has been implicated in maintaining stability of CAG.CTG trinucleotide repeats, a role that is independent of the catalytic dC transferase activity of the

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enzyme [73]. Recent work has also implicated REV1 in efficient, error-free replication of G4-forming sequences [30,74] and it is the potential mechanisms by

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which REV1 may facilitate replication of these structures that we consider next. 5.2 The role of REV1 in the replication of G4 DNA

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REV1 is an unusual member of the Y-family of DNA polymerases as its catalytic activity is restricted to deoxycytidyl transfer [75]. It is proficient at dC incorporation opposite template G, abasic sites and some bulky minor groove adducts [76-78]. More

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bizarre is its catalytic mechanism. The enzyme flips the template base out of the

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active site and actually incorporates dC by pairing with its own Arg 324 [79]. However, REV1 also plays a central role in the coordination of TLS through

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interactions with its C-terminus. This region can bind PCNA, ubiquitin, the other Yfamily polymerases and Polζ [reviewed in 55]. REV1 plays an important role in

maintaining replication fork progression in the face of a damaged DNA template [69,80], but surprisingly is also required for the prompt replication of G4 DNA [30]. Cells lacking REV1 are deficient in maintaining a replicating plasmid containing a leading, but not lagging, strand G4 motif and efficient G4 replication requires the Cterminus and, to some extent, the catalytic activity of the enzyme [30]. The data from these plasmid replication experiments is mirrored by an assay for epigenetic instability of the BU-1 locus. Stochastic loss of high level expression is dependent on replication stalling at a single G4 motif and can be monitored by loss of surface expression (Figure 2A) [16,39]. Using this approach we have shown that the Cterminus of REV1 plays an essential role in maintaining epigenetic stability [39]. This would be consistent with the known role of this domain interacting with the other Yfamily polymerases and Polζ [60]. These observations led us to propose a speculative 10

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model in which replication of the G-rich quadruplex structure might be facilitated by the dC transferase activity of REV1 which would be able to provide a 'bait' to compete for the dG bases at the start of the quadruplex [30]. Once catalysis across the first run of dGs had been completed, we proposed that the C terminus of REV1 would

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facilitate hand off to another Y-family polymerases, as suggested by the involvement of Polη and Polκ in G4 replication [67], or to Polζ. This handoff model predicts that

the epigenetic instability of rev1 mutants would be similar to mutants lacking the

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other Y family polymerases and Polζ. Interestingly, this is not the case (Figure 2B). While minor instability is observed in polκ and polζ cells, even a triple mutant lacking

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Polη, Polκ and REV3 does not exhibit instability comparable to the rev1 mutant. This suggests that REV1 is playing another role in the context of G4 replication that is

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independent of its ability to interact with other TLS polymerases. Interestingly, a recent study has shown that the catalytic core of REV1 is also able to directly destabilise G4s, independently of its catalytic activity and prevent them refolding [74].

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This function may be related to the potent single strand binding activity of REV1 [81] and suggests that REV1 might be able to passively dissolve G4 structures similar to the mechanism that has been proposed for RPA [13]. Potentially consistent with this

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idea, we have observed that epigenetic instability of the Bu-1a reporter in REV1-

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long loops [16].

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deficient DT40 cells was most pronounced when the locus contained a G4 motif with

Thus, REV1 is likely to play multiple roles in the replication of G4 structures in vivo.

However, it is very unlikely that REV1 works alone but rather in concert with DNA helicases that can help unwind the structure. While this suggestion awaits biochemical proof, we have shown that the pattern of gene dysregulation in REV1-deficient cells overlaps significantly with that seen in cells lacking the 5'-3' helicase FANCJ, but not with cells lacking the 3'-5' helicases BLM and WRN suggesting that REV1 and FANCJ might cooperate by 'attacking' the G4 structure from opposite ends [39]. 5.4 Picking up the pieces: the role of Polθ in end joining of breaks created at G quadruplexes In C. elegans the DNA helicase Dog-1 (Deletion of Guanines) plays an important role in replicating genomic G4 structures by suppressing deletions at G4 motifs [33,34]. The human FANCJ helicase, the orthologue of dog-1 [82], which is mutated in 11

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Fanconi’s anaemia [83], appears to play similar role [31,32]. Consistent with its in vivo role, purified FANCJ is able to unwind G4 structures in vitro with a 5’ – 3’ polarity [31,32]. The deletions seen in dog-1 worms are generally short (< 300 bp) with their 3’ end close to the G4 motif [33]. Interestingly, they have a characteristic

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feature, which is a significantly higher than chance likelihood of a single base pair of homology at the end of each flank [66]. It turns out that this results from an unusual property of DNA polymerase θ, which is able to mediate end joining of breaks by

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stabilising a single base pair overlap and extending to create an insertion templated on flanking DNA [66]. In worms lacking both Dog-1 and Polθ, extremely large deletions

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are observed. Thus, Polθ acts as a backup for the failure to unwind G4 motifs and while this pathway is potentially mutagenic it avoids much more deleterious kilobase-

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scale deletions.

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6. Outlook and conclusions

Even an apparently unperturbed replication fork is continually having to deal with

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challenges arising from the sequence and conformation of the DNA template and

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requires mechanisms operating constitutively to suppress and resolve secondary structures. There is much yet to learn about how the specialised helicases,

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polymerases and single strand binding activities collaborate. We especially need more mechanistic information on how the specialised translesion polymerases contribute to the replication of structured DNA. Finally, it is worth noting that the multiple overlapping pathways for maintaining processive replication at 'difficult' structured DNA sequences offer many potential opportunities for therapeutic interventions aimed at causing lethal levels of replication disruption in cancer cells in which replication is already stressed. Acknowledgements The laboratory of JES is funded by a core grant to LMB by the Medical Research Council (U105178808). Additionally, HA is funded by a post-doctoral fellowship (11-0514) from Worldwide Cancer Research (formerly Association for International Cancer Research) and AF by a studentship from the Jürgen Manchot Stiftung. We 12

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thank Sasa Svikovic and Cristina Rada for comments on the manuscript and Shunichi Takeda, Kyoto University, for sharing polymerase deficient DT40 lines.

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Figure Legends

Figure 1. Non-B DNA structures A. Table displaying typical sequences leading to

formation of various types of structured DNA. B. Left handed Z-DNA, with right handed B-DNA for comparison. C. H-DNA (triplex DNA). An intermolecular interaction is shown. D. Hairpin DNA. E. Cruciform DNA. F. S-DNA (slipped-strand DNA). G. The canonical G quadruplex motif is show (left) and G-quartet (centre) and an intramolecular G quadruplex (right). The groups of dG bases are colour coded to show how they form into the quartet and quadruplex structures. Figure 2. Epigenetic instability at the BU-1 locus of DT40 cells in mutants of the

Y-family polymerases and Polζ. A. Epigenetic instability of BU-1 is caused by leading strand replication arrest at a G4 located 3.5 kb from the transcription start site 18

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(TSS) [16]. In mutants defective in G4 processing, the resulting post-replicative gap causes loss of a tract of parental histone marks due to interruption of histone recycling which can extend to the promoter region resulting in stochastic switching from a Bu1high to a Bu-1low state. The orange boxes represent the six exons of the BU-1 locus. Diagram not to scale. B. Fluctuation analysis for instability of Bu-1 expression. Each

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point in the scatter plot represents an individual Bu-1high cellular clone expanded for

three weeks, at which point the percentage of cells that have become Bu-1low is

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each mutant and the error bars the interquartile range.

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Wickramasinghe Figure 1 A Type of repeat

Example sequences

Potential structures

5’ - TGCGATACTCATCGCA - 3’

Inverted Repeats (IR)

Hairpins Cruciforms

3’ - ACGCTATGAGTAGCGT - 5’

CACGATT - 3’ GTGCTAA - 5’

3’ - AATCGTG

H-DNA (Triplex DNA)

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5’ - TTAGCAC

Mirror Repeats (MR)

Dinucleotide repeat: (AT)n n10nt: minisatellite

Z-DNA (left handed helix) S-DNA G Quadruplex

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Direct Repeats (DR)

Common Trinucleotide repeats (TNRs): (CAG)n / (GAA)n / (CTG)n / (CGG)n

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Wickramasinghe Figure 2

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