**3.4 Z-DNA and genetic instability**

Z-DNA can be seen as the high-energy conformers of B-DNA that forms *in vivo* during transcription as a result of torsion strain generated by a moving polymerase (Wang, 1984; Casasnovas & Azorin, 1987; Johnston, 1988; Hebert & Rich, 1996). It has been thoroughly studied since 1957, how a right-handed B-DNA adopting a Z-DNA *in vitro* through ''flipping" the base pairs upside down, and rotating every other purine from *anti* to *syn* conformation (Johnston, 1988; Hebert & Rich, 1996). Compared to B-DNA, Z-DNA does not have a major groove, therefore could potentially impact transcription by physically blocking RNA polymerase, or by relaxing negative supercoiling turns, or by acting as an enhancer through recruiting transacting factors.

In Z-DNA, the guanosine nucleotides are in *syn* position where the bases are found over the sugar without protection, thus more accessible to DNA damaging factors, more resistant to processing by DNA repair enzymes. For example, alkylating damage such as *N7* methylguanine, which is typically removed by a DNA glycosylase in B-DNA is not efficiently repaired when present in Z-DNA (Pfohl-Leszkowicz et al., 1983; Boiteux et al., 1985).

Further, DNA sequences with the potential to adopt Z-DNA are associated with recombination hot spots in eukaryotic cells (Wang et al., 2006). A hot spot of 1000 bp in the major histocompatibility complex (MHC) in mice, containing several copies of long GT repeats, may account for up to 2% of the recombination events occurring on the chromosome (Crouau-Roy, 1999). In *E.coli***,** the RecA molecules show a much higher binding affinity for Z-DNA than for normal B-DNA and single-stranded DNA, and show a Z-DNA structure-stimulated ATPase activity, implicating a recombination hot spot of Z-DNA in prokaryotes as well. Genetic recombination in Z-DNA can potentially induce deletion instability and/ or produce DNA double-strand breaks. For example, a CG (12) sequence forming Z-DNA induces high levels of genetic instability in both bacterial and mammalian cells (Casasnovas & Azorin, 1987).

Recently, proteins binding Z-DNA are found, including specific proteins, such as Zα domain-containing proteins ADAR1 and ESL, and fairly low specific proteins, such as HMG proteins (Suda et al., 1996; Lange et al., 2009).

#### **3.5 H-DNA and H-DNA induced DSBs and genetic instability**

H-DNA, alternatively known as triplex DNA can be classified into either pyrimidine motif or purine motif according to the orientation and composition of the third strand in a triple stranded DNA structure (Fig. 3). The third strand can form either Hoogsteen or reverse-Hoogsteen hydrogen bonds with the purine-rich strand of the duplex DNA. Therefore, the third strand can be both pyrimidine-rich and parallel to the complementary strand (Y\* R: Y) or purine-rich and antiparallel to the complementary strand (R\* R: Y), producing either pyrimidine motif or purine motif triple stranded DNA (as described previously).

Whereas (R\* R: Y) triplexes form under conditions of physiological pH, triplex of the (Y\* R: Y) composition form most readily under conditions of acidic pH. At physiological pH, triplex

Besides, some other proteins were also found to bind the structural elements in cruciforms. For example, HMG proteins, replication initiation protein RepC, cruciform binding protein CBP, and four-way junction resolvases have all been indentified to bind cruciforms (Pearson

Z-DNA can be seen as the high-energy conformers of B-DNA that forms *in vivo* during transcription as a result of torsion strain generated by a moving polymerase (Wang, 1984; Casasnovas & Azorin, 1987; Johnston, 1988; Hebert & Rich, 1996). It has been thoroughly studied since 1957, how a right-handed B-DNA adopting a Z-DNA *in vitro* through ''flipping" the base pairs upside down, and rotating every other purine from *anti* to *syn* conformation (Johnston, 1988; Hebert & Rich, 1996). Compared to B-DNA, Z-DNA does not have a major groove, therefore could potentially impact transcription by physically blocking RNA polymerase, or by relaxing negative supercoiling turns, or by acting as an enhancer

In Z-DNA, the guanosine nucleotides are in *syn* position where the bases are found over the sugar without protection, thus more accessible to DNA damaging factors, more resistant to processing by DNA repair enzymes. For example, alkylating damage such as *N7* methylguanine, which is typically removed by a DNA glycosylase in B-DNA is not efficiently repaired when present in Z-DNA (Pfohl-Leszkowicz et al., 1983; Boiteux et al.,

Further, DNA sequences with the potential to adopt Z-DNA are associated with recombination hot spots in eukaryotic cells (Wang et al., 2006). A hot spot of 1000 bp in the major histocompatibility complex (MHC) in mice, containing several copies of long GT repeats, may account for up to 2% of the recombination events occurring on the chromosome (Crouau-Roy, 1999). In *E.coli***,** the RecA molecules show a much higher binding affinity for Z-DNA than for normal B-DNA and single-stranded DNA, and show a Z-DNA structure-stimulated ATPase activity, implicating a recombination hot spot of Z-DNA in prokaryotes as well. Genetic recombination in Z-DNA can potentially induce deletion instability and/ or produce DNA double-strand breaks. For example, a CG (12) sequence forming Z-DNA induces high levels of genetic instability in both bacterial and mammalian

Recently, proteins binding Z-DNA are found, including specific proteins, such as Zα domain-containing proteins ADAR1 and ESL, and fairly low specific proteins, such as HMG

H-DNA, alternatively known as triplex DNA can be classified into either pyrimidine motif or purine motif according to the orientation and composition of the third strand in a triple stranded DNA structure (Fig. 3). The third strand can form either Hoogsteen or reverse-Hoogsteen hydrogen bonds with the purine-rich strand of the duplex DNA. Therefore, the third strand can be both pyrimidine-rich and parallel to the complementary strand (Y\* R: Y) or purine-rich and antiparallel to the complementary strand (R\* R: Y), producing either

Whereas (R\* R: Y) triplexes form under conditions of physiological pH, triplex of the (Y\* R: Y) composition form most readily under conditions of acidic pH. At physiological pH, triplex

pyrimidine motif or purine motif triple stranded DNA (as described previously).

et al., 1996; Jin et al., 1997; Novac et al., 2002; Lange et al., 2009; lilley, 2010).

**3.4 Z-DNA and genetic instability** 

through recruiting transacting factors.

cells (Casasnovas & Azorin, 1987).

proteins (Suda et al., 1996; Lange et al., 2009).

**3.5 H-DNA and H-DNA induced DSBs and genetic instability** 

1985).

may be stabilized by negative supercoiling, modified with phosphorothioate groups, or polyvalent cations such as spermine and spermidine. For the R\* R: Y intramolecular triplexes and T: A\* T and C+: G\* C triplets for the Y\* R: Y intramolecular triplexes are included since these are considered the most stable triplet combinations.

Fig. 3. H-DNA (Star/ Dot marks Hoogsteen hydrogen bonded bases; colon/ line shows Watson–Crick hydrogen bonded bases)

In general, formation of a triplex DNA was a role of sequence, topology (supercoil density), ionic conditions, protein binding, methylation, carcinogen binding, and other factors. Global negative supercoil density acts in concert with local transient waves of topological changes produced by replication or transcription, and both have a critical influence on forming and stabilizing triplex DNA *in vivo*. It has been reported that a higher negative supercoiling destabilized long CTG· CAG, CCG· CGG, and GAA· TTC repeats in *Escherichia coli.* Similarly a 2.5-kb poly (R· Y) tract from the human PKD1 gene lowered the viability of the host cells (Bacolla et al., 2001; Patel et al., 2004).

Several types of DNA damages induced by H-DNA have been reported, including single and/ or double strand breaks. For example, the endogenous H-DNA forming sequences from the human c-*myc* promoter was shown to be intrinsically mutagenic in mammalian cells because of the generation of either single or double strand breaks in the H-DNA, or near the H-DNA locus. Besides, the single-stranded area, or the triplex region is also a target of various nucleases, resulting in single or DSBs formation, and the increased mutagenesis or recombination (Wang & Vasquez, 2006).

Although triplex (H-DNA) DNA occurs mainly at poly (purine·pyrimidine) ((R·Y) n) tracts, it can also be induced to form with the sequence specific DNA recognition and binding of some synthetic triplex-forming oligonucleotides (TFOs) (Casey & Glazer, 2001; Mukherjee & Vasquez, 2011). TFOs bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA to induce forming the triplex (Casey & Glazer, 2001; Mukherjee & Vasquez, 2011). During which the duplex DNA may have to undergo helical distortions on TFO binding and the distortions trigger endogenous recombination and repair mechanisms in the cell (Raghavan et al., 2004, 2005).

Indeed it has been reported that formation of TFO-induced triplex can induce sequencespecific DNA damages both in cells and in animals (Chan, et al., 1999; Kalish et al., 2005).

The Gratuitous Repair on Undamaged DNA Misfold 409

Unwound DNA is known to be formed by A+T -rich sequences (Fig. 5). Since A· T base pairs contain two hydrogen bonds and C· G base pairs contain three, A· T-rich tracts are less thermally stable than C· G -rich tracts in DNA. In the presence of superhelical energy, A+T rich regions can unwind and remain unwound under conditions normally found in the cell. Such sites often provide places for DNA replication proteins to enter DNA to begin the chromosome duplication. Unwound DNA can therefore be alternatively called as DNA unwinding elements (DUEs) that have been identified in both prokaryotic and eukaryotic DNA sequences. DUEs are AT-rich sequences about 30-100 bp long. They share little sequence similarity except for being AT-rich. Under torsion stress, unwinding of the double helix occurs first in AT-rich sequences; therefore, DUEs can be maintained as unpaired DNA regions in the presence of negative supercoiling. The single-stranded area of the unwound structure may be target of nuclease activity resulting in single or DSBs, leading to enhanced

Normally, curved DNA is often seen in DNA segment containing runs of three or four bases of A in one strand and a similar run of T in the other and spaced at 10-base pair intervals. Interestingly we have recently found that trinucleotide repeats AAT can also adopt curved DNA in *E.coli*, which can be repressed by H-NS and its stimulated IS1E transposition (Pan et

Apart from the roles in DNA replication, transcription and gene regulation, non-B DNA may also lead to gene instability, including chromosomal translocation, deletion and amplification in cancer and other human diseases ( Bacolla & Wells, 2009; Chen et al., 2010). Since non-B DNA abnormal folds have been addressed with generating DNA breaks, including both single and double strand DNA breaks. Non-B DNA structures recruit DNA repair machinery to the breaking sites, which then make gene mutations and chromosomal

Some regions of DNA forming non-B DNA structures in replication or transcription, which may turn to affect the DNA transactions (Van Holde & Zlatanova, 1994; Samadashwily et

**4. Biological significance of DNA abnormal folding** 

**4.1 Effects of non-B DNA structures on DNA replication / transcription** 

al., 1997; Krasilnikova et al., 2004; Lin et al., 2006; Mirkin & Mirkin, 2007)

**3.7 Unwound DNA** 

mutagenesis or recombination.

Fig. 5. Unwound DNA

rearrangements during repair.

**3.8 Curved DNA** 

al., 2010)

However, mismatch repair proteins are not involved in this TFO-induced mutagenesis. Several reports have now shown that cells that are deficient in the MutS and MutL homologues MSH2, MLH1, MSH3, or MSH6, do not show any change in TFO-induced mutagenesis. In contrasts, NER factors can recognize the intermolecular triplex at least in part. Therefore NER was involved in the triplex-induced mutagenesis and recombination in cells. For example, in *E.coli*, NER proteins, such as UvrB and UvrC, were necessary for H-DNA-induced cell growth retardation and cell lysis, similarly, recombination induced by TFOs depends also on the NER pathway (Faruqi et al., 2000).

#### **3.6 G-tetraduplex and genetic instability**

G-quadruplexes are higher-order DNA or RNA structures formed from G-rich DNA or RNA sequences that are built around tetrads of hydrogen-bonded guanine bases (Lipps & Rhodes, 2009; Sannohe & Sugiyama, 2010). Despite the wide prevalence of genomic sequences that have G-rich property and that can potentially fold into tetraplex / quadruplexes structures, a direct demonstration of their existence *in vivo* proved to be a difficult undertaking. Only recently has there evidence started to increase for their presence and role *in vivo* (Lipps & Rhodes, 2009), since most of the tetraplex/ quadruplexes forming sequences are fairly short and quadruplexes are likely to be transiently formed. G-quadruplexes (tetraduplex) may have several isomers which can be formed intramolecularly and intermolecularly (Fig. 4).

Recent progress of the related studies revealed that G-quadruple could provide a nucleic acid based mechanism, such as regulating telomere maintenance, transcription, replication as well as translation. In the same time, various G-quadruplexes binding proteins, such as, a G4 quadruplex and purine Motif triplex nucleic acid-binding protein have also been characterized (Dyke et al.,2004), many others have been summarized in the reference (Fry, 2007).

The existence of cellular proteins that preferentially interact with tetraplex DNA provides a strong argument for the existence of quadruplex formations in genomic DNA.
