Editing Z-DNA (section)

==Biological significance==
A biological role for Z-DNA in the regulation of type I interferon responses has been confirmed in studies of three well-characterized rare Mendelian Diseases: Dyschromatosis Symmetrica Hereditaria (OMIM: 127400), Aicardi-Goutières syndrome (OMIM: 615010) and Bilateral Striatal Necrosis/Dystonia. Families with haploid ADAR transcriptome enabled mapping of Zα variants directly to disease, showing that genetic information is encoded in DNA by both shape and sequence.<ref name="Herbert 2019 114–117">{{cite journal|last=Herbert|first=A.|title=Mendelian disease caused by variants affecting recognition of Z-DNA and Z-RNA by the Zα domain of the double-stranded RNA editing enzyme ADAR. |journal=European Journal of Human Genetics |volume= 8|pages= 114–117|year=2019 |issue=1|pmid=31320745 |doi=10.1038/s41431-019-0458-6 |pmc=6906422}}</ref> A role in regulating type I interferon responses in cancer is also supported by findings that 40% of a panel of tumors were dependent on the ADAR enzyme for survival.<ref>{{cite journal|last=Herbert|first=A.|title=ADAR and Immune Silencing in Cancer. |journal=Trends in Cancer |volume= 5|issue=5 |pages=272–282 |year=2019 |pmid=31174840 |doi=10.1016/j.trecan.2019.03.004|s2cid=155209484 }}</ref>

In previous studies, Z-DNA  was linked to both [[Alzheimer's disease]] and [[systemic lupus erythematosus]]. To showcase this, a study was conducted on the DNA found in the hippocampus of brains that were normal, moderately affected with Alzheimer's disease, and severely affected with Alzheimer's disease. Through the use of [[circular dichroism]], this study showed the presence of Z-DNA in the DNA of those severely affected.<ref name=":2">{{Cite journal|last1=Suram|first1=Anitha|last2=Rao|first2=Jagannatha K. S.|last3=S.|first3=Latha K.|last4=A.|first4=Viswamitra M.|date=2002|title=First Evidence to Show the Topological Change of DNA from B-DNA to Z-DNA Conformation in the Hippocampus of Alzheimer's Brain|journal=NeuroMolecular Medicine|volume=2|issue=3|pages=289–298|doi=10.1385/nmm:2:3:289|pmid=12622407|s2cid=29059186|issn=1535-1084}}</ref> In this study it was also found that major portions of the moderately affected DNA was in the B-Z intermediate conformation. This is significant because from these findings it was concluded that the transition from B-DNA to Z-DNA is dependent on the progression of Alzheimer's disease.<ref name=":2" /> Additionally, Z-DNA is associated with systemic lupus erythematosus (SLE) through the presence of naturally occurring antibodies. Significant amounts of anti Z-DNA antibodies were found in SLE patients and were not present in other rheumatic diseases.<ref>{{Cite journal|last1=Lafer|first1=E M|last2=Valle|first2=R P|last3=Möller|first3=A|last4=Nordheim|first4=A|last5=Schur|first5=P H|last6=Rich|first6=A|last7=Stollar|first7=B D|date=1983-02-01|title=Z-DNA-specific antibodies in human systemic lupus erythematosus.|journal=Journal of Clinical Investigation|volume=71|issue=2|pages=314–321|doi=10.1172/jci110771|issn=0021-9738|pmc=436869|pmid=6822666}}</ref> There are two types of these antibodies. Through radioimmunoassay, it was found that one interacts with the bases exposed on the surface of Z-DNA and denatured DNA, while the other exclusively interacts with the zig-zag backbone of only Z-DNA. Similar to that found in Alzheimer's disease, the antibodies vary depending on the stage of the disease, with maximal antibodies in the most active stages of SLE.

=== Z-DNA in transcription ===
Z-DNA is commonly believed to provide [[Strain (chemistry)#Torsional strain|torsional strain]] relief during [[DNA transcription|transcription]], and it is associated with [[DNA supercoiling|negative supercoiling]].<ref name="Ha2005" /><ref name="Rich2003">{{cite journal  |last1=Rich |first1=A |last2=Zhang |first2=S |year=2003 |title=Timeline: Z-DNA: the long road to biological function  |journal=Nature Reviews Genetics |volume=4 |issue=7 |pages=566–572 |pmid=12838348 |doi=10.1038/nrg1115|s2cid=835548 }}</ref> However, while supercoiling is associated with both DNA transcription and replication, Z-DNA formation is primarily linked to the rate of [[transcription (biology)|transcription]].<ref>{{cite journal|last1=Wittig|first1=B.|last2=Dorbic|first2=T.|last3=Rich|first3=A.|title=Transcription is associated with Z-DNA formation in metabolically active permeabilized mammalian cell nuclei |journal=Proceedings of the National Academy of Sciences |volume=88 |issue=6 |pages=2259–2263 |year=1991 |pmid=2006166 |doi=10.1073/pnas.88.6.2259|pmc=51210|bibcode=1991PNAS...88.2259W|doi-access=free}}</ref>

A study of [[Chromosome 22 (human)|human chromosome 22]] showed a correlation between Z-DNA forming regions and promoter regions for [[nuclear factor I]].  This suggests that transcription in some human genes may be regulated by Z-DNA formation and nuclear factor I activation.<ref name=Champ2004/>

Z-DNA sequences upstream of promoter regions have been shown to stimulate transcription.  The greatest increase in activity is observed when the Z-DNA sequence is placed three helical turns after the [[Promoter (genetics)|promoter sequence]]. Furthermore, using micrococcal nuclease-crosslinking technique,<ref name=":5" /> Z-DNA is unlikely to form [[nucleosome]]s, which are often located before and/or after a Z-DNA forming sequence.  Because of this property, Z-DNA is hypothesized to code for the boundary in nucleosome positioning.  Since the placement of nucleosomes influences the binding of [[transcription factor]]s, Z-DNA is thought to regulate the rate of transcription.<ref name=":5">{{cite journal |last1=Wong|first1=B.|last2=Chen|first2=S.|last3=Kwon|first3=J.-A.|last4=Rich|first4=A.|title=Characterization of Z-DNA as a nucleosome-boundary element in yeast ''Saccharomyces cerevisiae''|journal=Proceedings of the National Academy of Sciences |volume=104 |issue=7 |pages=2229–2234 |year=2007 |pmid=17284586 |doi=10.1073/pnas.0611447104|pmc=1892989|bibcode=2007PNAS..104.2229W|doi-access=free}}</ref>

Developed behind the pathway of [[RNA polymerase]] through negative supercoiling, Z-DNA formed via active transcription has been shown to increase genetic instability, creating a propensity towards [[mutagenesis]] near promoters.<ref name="Wang2006">{{cite journal |last1=Wang|first1=G.|last2=Christensen|first2=L. A.|last3=Vasquez|first3=K. M.|title=Z-DNA-forming sequences generate large-scale deletions in mammalian cells |journal=Proceedings of the National Academy of Sciences |volume=108 |issue=8 |pages=2677–2682 |year=2006 |pmid=16473937 |doi=10.1073/pnas.0511084103|pmc=1413824|bibcode=2006PNAS..103.2677W|doi-access=free}}</ref> A study on ''[[Escherichia coli]]'' found that gene [[Deletion (genetics)|deletions]] spontaneously occur in [[plasmid]] regions containing Z-DNA-forming sequences.<ref name="Freund1989">{{cite journal |last1=Freund |first1=A. M.|last2=Bichara|first2=M.|last3=Fuchs|first3=R. P.|title=Z-DNA-forming sequences are spontaneous deletion hot spots |journal=Proceedings of the National Academy of Sciences |volume=86 |issue=19 |pages=7465–7469 |year=1989 |pmid=2552445|doi=10.1073/pnas.86.19.7465 |pmc=298085|bibcode=1989PNAS...86.7465F|doi-access=free}}</ref> In mammalian cells, the presence of such sequences was found to produce large genomic fragment deletions due to chromosomal [[DNA repair|double-strand breaks]]. Both of these genetic modifications have been linked to the [[Chromosomal translocation|gene translocations]] found in cancers such as [[leukemia]] and [[lymphoma]], since breakage regions in [[tumor cell]]s have been plotted around Z-DNA-forming sequences.<ref name="Wang2006" /> However, the smaller deletions in bacterial plasmids have been associated with [[replication slippage]], while the larger deletions associated with mammalian cells are caused by [[non-homologous end-joining]] repair, which is known to be prone to error.<ref name="Wang2006" /><ref name="Freund1989" />

The toxic effect of [[ethidium bromide]] (EtBr) on [[trypanosoma]]s is caused by shift of their [[kinetoplast]]id DNA to Z-form. The shift is caused by [[intercalation (biochemistry)|intercalation]] of EtBr and subsequent loosening of DNA structure that leads to unwinding of DNA, shift to Z-form and inhibition of DNA replication.<ref name="pmid21187912">{{cite journal |last1= Roy Chowdhury |first1 = A. |last2=Bakshi |first2=R.|last3=Wang |first3=J. |last4=Yıldırır |first4=G. |last5=Liu |first5=B. |last6=Pappas-Brown |first6=V. |last7=Tolun |first7=G. |last8=Griffith |first8=J. D. |last9=Shapiro |first9=T. A. |last10=Jensen |first10=R. E. |last11=Englund |first11=P. T. | title = The killing of African trypanosomes by ethidium bromide | journal = PLOS Pathogens | volume = 6 | issue = 12 | pages = e1001226 | date = Dec 2010 | pmid = 21187912 | pmc = 3002999 | doi = 10.1371/journal.ppat.1001226 |doi-access = free }}</ref>

=== Discovery of the Zα domain ===
The first domain to bind Z-DNA with high affinity was discovered in [[ADAR|ADAR1]] using an approach developed by Alan Herbert.<ref name="Herbert1993">{{cite journal |last1=Herbert |first1=A. |last2=Rich |first2=A. |year=1993 |title=A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide |journal=Nucleic Acids Research |volume=21 |issue=11 |pages=2669–2672 |pmid=8332463 |pmc= 309597 |doi=10.1093/nar/21.11.2669}}</ref><ref name="Herbert1997">{{cite journal |last1=Herbert |first1=A. |last2=Alfken |first2=J. |last3=Kim |first3=Y. G. |last4=Mian |first4=I. S. |last5=Nishikura |first5=K. |last6=Rich |first6=A. |year=1997 |title=A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase. |journal=Proceedings of the National Academy of Sciences |volume=94 |issue=16 |pages=8421–8426 |pmid=9237992 |pmc=22942 |doi=10.1073/pnas.94.16.8421 |bibcode=1997PNAS...94.8421H|doi-access=free }}</ref> [[Crystallography|Crystallographic]] and [[nuclear magnetic resonance|NMR]] studies confirmed the biochemical findings that this domain bound Z-DNA in a non-sequence-specific manner.<ref name="Herbert1998">{{cite journal|last1=Herbert |first1=A. |last2=Schade |first2=M. |last3=Lowenhaupt |first3=K. |last4=Alfken |first4=J |last5=Schwartz |first5=T. |last6=Shlyakhtenko |first6=L. S. |last7=Lyubchenko |first7=Y. L. |last8=Rich |first8=A. |year=1998 |title=The Zα domain from human ADAR1 binds to the Z-DNA conformer of many different sequences |journal=Nucleic Acids Research |volume=26 |issue=15 |pages=2669–2672 |pmid=9671809 |pmc=147729 |doi=10.1093/nar/26.15.3486}}</ref><ref name="Schwartz1999">{{cite journal |last1=Schwartz |first1=T. |last2=Rould |first2=M. A. |last3=Lowenhaupt |first3=K. |last4=Herbert |first4=A. |last5=Rich |first5=A. |year=1999 |title=Crystal structure of the Zα domain of the human editing enzyme ADAR1 bound to left-handed Z-DNA |journal=Science |volume=284 |issue=5421 |pages=1841–1845 |pmid=10364558 |doi=10.1126/science.284.5421.1841}}</ref><ref name="Schade1999">{{cite journal|last1=Schade |first1=M. |last2=Turner |first2=C. J. |last3=Kühne |first3=R. |last4=Schmieder |first4=P. |last5=Lowenhaupt |first5=K. |last6=Herbert |first6=A. |last7=Rich |first7=A. |last8=Oschkinat |first8=H |year=1999 |title=The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA |journal=Proceedings of the National Academy of Sciences |volume=96 |issue=22 |pages=2465–2470 |pmid=10535945 |pmc=22950 |doi=10.1073/pnas.96.22.12465 |bibcode=1999PNAS...9612465S|doi-access=free }}</ref> Related domains were identified in a number of other proteins through [[sequence homology]].<ref name="Herbert1997" /> The identification of the Zα domain provided a tool for other crystallographic studies that lead to the characterization of Z-RNA and the B–Z junction. Biological studies suggested that the Z-DNA binding domain of ADAR1 may localize this enzyme that modifies the sequence of the newly formed RNA to sites of active transcription.<ref name="Herbert2001">{{cite journal|last1=Herbert |first1=A. |last2=Rich |first2=A. |year=2001 |title=The role of binding domains for dsRNA and Z-DNA in the ''in vivo'' editing of minimal substrates by ADAR1 |journal=Proceedings of the National Academy of Sciences |volume=98 |issue=21 |pages=12132–12137 |pmid=11593027 |doi=10.1073/pnas.211419898 |bibcode=2001PNAS...9812132H |pmc=59780|doi-access=free }}</ref><ref name="Halber1999">{{cite web |title=Scientists observe biological activities of 'left-handed' DNA  |last=Halber |first=D. |date=1999-09-11 |publisher=MIT News Office |url=http://web.mit.edu/newsoffice/1999/zdna-0911.html |access-date=2008-09-29}}</ref> A role for Zα, Z-DNA and Z-RNA in defense of the genome against the invasion of Alu retro-elements in humans has evolved into a mechanism for the regulation of innate immune responses to dsRNA. Mutations in Zα are causal for human interferonopathies such as the Mendelian Aicardi-Goutières Syndrome.<ref name="Herbert2019">{{cite journal |last1=Herbert |first1=A. |year=2019 |title= Z-DNA and Z-RNA in human disease|journal=Communications Biology |volume=2  |page=7 |doi=10.1038/s42003-018-0237-x|pmid=30729177 |pmc=6323056 }}</ref><ref name="Herbert 2019 114–117"/>Additionally, Zα domains are demonstrated to localize at the stress granules because of their innate ability in binding nucleic acid. Furthermore, different Zα domains bind to the Z conformation of nucleic acid differently providing important avenues for specific targeting in drug discovery.

=== Consequences of Z-DNA binding to vaccinia E3L protein ===
As Z-DNA has been researched more thoroughly, it has been discovered that the structure of Z-DNA can bind to Z-DNA binding proteins through [[Van der Waals force|van der Waal forces]] and [[hydrogen bonding]].<ref name=":1" /> One example of a Z-DNA binding protein is the [[vaccinia]] E3L protein, which is a product of the E3L gene and mimics a mammalian protein that binds Z-DNA.<ref name=":4">{{Cite journal|last1=Kim|first1=Y.-G.|last2=Muralinath|first2=M.|last3=Brandt|first3=T.|last4=Pearcy|first4=M.|last5=Hauns|first5=K.|last6=Lowenhaupt|first6=K.|last7=Jacobs|first7=B. L.|last8=Rich|first8=A.|date=2003-05-30|title=A role for Z-DNA binding in vaccinia virus pathogenesis|journal=Proceedings of the National Academy of Sciences|volume=100|issue=12|pages=6974–6979|doi=10.1073/pnas.0431131100|pmid=12777633|issn=0027-8424|pmc=165815|bibcode=2003PNAS..100.6974K |doi-access=free}}</ref><ref>{{Cite journal|last1=Kim|first1=Y.-G.|last2=Lowenhaupt|first2=K.|last3=Oh|first3=D.-B.|last4=Kim|first4=K. K.|last5=Rich|first5=A.|date=2004-02-02|title=Evidence that vaccinia virulence factor E3L binds to Z-DNA in vivo: Implications for development of a therapy for poxvirus infection|journal=Proceedings of the National Academy of Sciences|volume=101|issue=6|pages=1514–1518|doi=10.1073/pnas.0308260100|issn=0027-8424|pmid=14757814|pmc=341766|bibcode=2004PNAS..101.1514K |doi-access=free}}</ref> Not only does the E3L protein have affinity to Z-DNA, it has also been found to play a role in the level of severity of virulence in mice caused by vaccinia virus, a type of [[poxvirus]]. Two critical components to the E3L protein that determine virulence are the [[N-terminus]] and the [[C-terminus]]. The N-terminus is made of up a sequence similar to that of the Zα domain, also called [[Adenosine deaminase z-alpha domain]], while the C-terminus is composed of a double stranded RNA binding motif.<ref name=":4" /> Through research done by Kim, Y. et al. at the Massachusetts Institute of Technology, it was shown that replacing the N-terminus of the E3L protein with a Zα domain sequence, containing 14 Z-DNA binding residues similar to E3L, had little to no effect on pathogenicity of the virus in mice.<ref name=":4" /> In Contrast, Kim, Y. et al. also found that deleting all 83 residues of the E3L N-terminus resulted in decreased virulence. This supports their claim that the N-terminus containing the Z-DNA binding residues is necessary for virulence.<ref name=":4" /> Overall, these findings show that the similar Z-DNA binding residues within the N-terminus of the E3L protein and the Zα domain are the most important structural factors determining virulence caused by the vaccinia virus, while amino acid residues not involved in Z-DNA binding have little to no effect. A future implication of these findings includes reducing Z-DNA binding of E3L in vaccines containing the vaccinia virus so negative reactions to the virus can be minimized in humans.<ref name=":4" />

Furthermore, Alexander Rich and Jin-Ah Kwon found that E3L acts as a [[transactivator]] for human IL-6, NF-AT, and p53 genes. Their results show that [[HeLa]] cells containing E3L had increased expression of human IL-6, NF-AT, and p53 genes and point mutations or deletions of certain Z-DNA binding amino acid residues decreased that expression.<ref name=":1">{{Cite journal|last1=Kwon|first1=J.-A.|last2=Rich|first2=A.|date=2005-08-26|title=Biological function of the vaccinia virus Z-DNA-binding protein E3L: Gene transactivation and antiapoptotic activity in HeLa cells|journal=Proceedings of the National Academy of Sciences|volume=102|issue=36|pages=12759–12764|doi=10.1073/pnas.0506011102|pmid=16126896|pmc=1200295|bibcode=2005PNAS..10212759K |issn=0027-8424|doi-access=free}}</ref> Specifically, mutations in Tyr 48 and Pro 63 were found to reduce transactivation of the previously mentioned genes, as a result of loss of hydrogen bonding and london dispersion forces between E3L and the Z-DNA.<ref name=":1" /> Overall, these results show that decreasing the bonds and interactions between Z-DNA and Z-DNA binding proteins decreases both virulence and gene expression, hence showing the importance of having bonds between Z-DNA and the E3L binding protein.