Editing DNA (section)

== Interactions with proteins ==
All the functions of DNA depend on interactions with proteins. These [[protein interactions]] can be non-specific, or the protein can bind specifically to a single DNA sequence. Enzymes can also bind to DNA and of these, the polymerases that copy the DNA base sequence in transcription and DNA replication are particularly important.

=== DNA-binding proteins ===
{{further|DNA-binding protein}}
[[File:Nucleosome1.png|thumb|260px|left|Interaction of DNA (in orange) with [[histone]]s (in blue). These proteins' basic amino acids bind to the acidic phosphate groups on DNA.]]

Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called [[chromatin]]. In eukaryotes, this structure involves DNA binding to a complex of small basic proteins called [[histone]]s, while in prokaryotes multiple types of proteins are involved.<ref>{{cite journal | vauthors = Sandman K, Pereira SL, Reeve JN | s2cid = 21101836 | title = Diversity of prokaryotic chromosomal proteins and the origin of the nucleosome | journal = Cellular and Molecular Life Sciences | volume = 54 | issue = 12 | pages = 1350–64 | date = December 1998 | pmid = 9893710 | doi = 10.1007/s000180050259 | pmc = 11147202 }}</ref><ref>{{cite journal | vauthors = Dame RT | title = The role of nucleoid-associated proteins in the organization and compaction of bacterial chromatin | journal = Molecular Microbiology | volume = 56 | issue = 4 | pages = 858–70 | date = May 2005 | pmid = 15853876 | doi = 10.1111/j.1365-2958.2005.04598.x | s2cid = 26965112 | doi-access = free }}</ref> The histones form a disk-shaped complex called a [[nucleosome]], which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones, making [[ionic bond]]s to the acidic sugar-phosphate backbone of the DNA, and are thus largely independent of the base sequence.<ref>{{cite journal | vauthors = Luger K, Mäder AW, Richmond RK, Sargent DF, Richmond TJ | title = Crystal structure of the nucleosome core particle at 2.8 A resolution | journal = Nature | volume = 389 | issue = 6648 | pages = 251–60 | date = September 1997 | pmid = 9305837 | doi = 10.1038/38444 | bibcode = 1997Natur.389..251L | s2cid = 4328827 }}</ref> Chemical modifications of these basic amino acid residues include [[methylation]], [[phosphorylation]], and [[acetylation]].<ref>{{cite journal | vauthors = Jenuwein T, Allis CD | title = Translating the histone code | journal = Science | volume = 293 | issue = 5532 | pages = 1074–80 | date = August 2001 | pmid = 11498575 | doi = 10.1126/science.1063127 | s2cid = 1883924 | url = http://www.gs.washington.edu/academics/courses/braun/55104/readings/jenuwein.pdf | url-status=live | archive-url = https://web.archive.org/web/20170808142426/http://www.gs.washington.edu/academics/courses/braun/55104/readings/jenuwein.pdf | archive-date = 8 August 2017 | df = dmy-all }}</ref> These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to [[transcription factor]]s and changing the rate of transcription.<ref>{{cite book | vauthors = Ito T | title = Protein Complexes that Modify Chromatin | chapter = Nucleosome Assembly and Remodeling | series = Current Topics in Microbiology and Immunology | volume = 274 | pages = 1–22 | year = 2003 | pmid = 12596902 | doi = 10.1007/978-3-642-55747-7_1 | isbn = 978-3-540-44208-0 }}</ref> Other non-specific DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted DNA.<ref>{{cite journal | vauthors = Thomas JO | title = HMG1 and 2: architectural DNA-binding proteins | journal = Biochemical Society Transactions | volume = 29 | issue = Pt 4 | pages = 395–401 | date = August 2001 | pmid = 11497996 | doi = 10.1042/BST0290395 }}</ref> These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes.<ref>{{cite journal | vauthors = Grosschedl R, Giese K, Pagel J | title = HMG domain proteins: architectural elements in the assembly of nucleoprotein structures | journal = Trends in Genetics | volume = 10 | issue = 3 | pages = 94–100 | date = March 1994 | pmid = 8178371 | doi = 10.1016/0168-9525(94)90232-1 }}</ref>

A distinct group of DNA-binding proteins is the DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication [[protein A]] is the best-understood member of this family and is used in processes where the double helix is separated, including DNA replication, recombination, and DNA repair.<ref>{{cite journal | vauthors = Iftode C, Daniely Y, Borowiec JA | title = Replication protein A (RPA): the eukaryotic SSB | journal = Critical Reviews in Biochemistry and Molecular Biology | volume = 34 | issue = 3 | pages = 141–80 | year = 1999 | pmid = 10473346 | doi = 10.1080/10409239991209255 }}</ref> These binding proteins seem to stabilize single-stranded DNA and protect it from forming [[stem-loop]]s or being degraded by [[nuclease]]s.

[[File:Lambda repressor 1LMB.png|thumb|upright=1.1|The lambda repressor [[helix-turn-helix]] transcription factor bound to its DNA target<ref>{{Cite web| vauthors = Beamer LJ, Pabo CO |title=RCSB PDB – 1LMB: Refined 1.8 Å crystal structure of the lambda repressor-operator complex |url=https://www.rcsb.org/structure/1LMB|access-date=2023-03-27|website=www.rcsb.org|language=en-US}}</ref>]]
In contrast, other proteins have evolved to bind to particular DNA sequences. The most intensively studied of these are the various [[transcription factor]]s, which are proteins that regulate transcription. Each transcription factor binds to one particular set of DNA sequences and activates or inhibits the transcription of genes that have these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription.<ref>{{cite journal | vauthors = Myers LC, Kornberg RD | title = Mediator of transcriptional regulation | journal = Annual Review of Biochemistry | volume = 69 | pages = 729–49 | year = 2000 | pmid = 10966474 | doi = 10.1146/annurev.biochem.69.1.729 }}</ref> Alternatively, transcription factors can bind [[enzyme]]s that modify the histones at the promoter. This changes the accessibility of the DNA template to the polymerase.<ref>{{cite journal | vauthors = Spiegelman BM, Heinrich R | title = Biological control through regulated transcriptional coactivators | journal = Cell | volume = 119 | issue = 2 | pages = 157–67 | date = October 2004 | pmid = 15479634 | doi = 10.1016/j.cell.2004.09.037 | doi-access = free }}</ref>

As these DNA targets can occur throughout an organism's genome, changes in the activity of one type of transcription factor can affect thousands of genes.<ref>{{cite journal | vauthors = Li Z, Van Calcar S, Qu C, Cavenee WK, Zhang MQ, Ren B | title = A global transcriptional regulatory role for c-Myc in Burkitt's lymphoma cells | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 14 | pages = 8164–69 | date = July 2003 | pmid = 12808131 | pmc = 166200 | doi = 10.1073/pnas.1332764100 | bibcode = 2003PNAS..100.8164L | doi-access = free }}</ref> Consequently, these proteins are often the targets of the [[signal transduction]] processes that control responses to environmental changes or [[cellular differentiation]] and development. The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to "read" the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible.<ref name="Pabo1984" />

=== DNA-modifying enzymes ===

==== Nucleases and ligases ====
[[File:EcoRV 1RVA.png|thumb|left|upright=1.1|The [[restriction enzyme]] [[EcoRV]] (green) in a complex with its substrate DNA<ref>{{Cite web| vauthors = Kostrewa D, Winkler FK |title=RCSB PDB – 1RVA: Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 Å resolution |url=https://www.rcsb.org/structure/1RVA|access-date=2023-03-27|website=www.rcsb.org|language=en-US}}</ref>]]
[[Nuclease]]s are [[enzyme]]s that cut DNA strands by catalyzing the [[hydrolysis]] of the [[phosphodiester bond]]s. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called [[exonuclease]]s, while [[endonuclease]]s cut within strands. The most frequently used nucleases in [[molecular biology]] are the [[restriction enzyme|restriction endonucleases]], which cut DNA at specific sequences. For instance, the EcoRV enzyme shown to the left recognizes the 6-base sequence 5′-GATATC-3′ and makes a cut at the horizontal line. In nature, these enzymes protect [[bacteria]] against [[Bacteriophage|phage]] infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the [[restriction modification system]].<ref>{{cite journal | vauthors = Bickle TA, Krüger DH | title = Biology of DNA restriction | journal = Microbiological Reviews | volume = 57 | issue = 2 | pages = 434–50 | date = June 1993 | pmid = 8336674 | pmc = 372918 | doi = 10.1128/MMBR.57.2.434-450.1993 }}</ref> In technology, these sequence-specific nucleases are used in [[molecular cloning]] and [[Genetic fingerprinting|DNA fingerprinting]].

Enzymes called [[DNA ligase]]s can rejoin cut or broken DNA strands.<ref name=Doherty>{{cite journal | vauthors = Doherty AJ, Suh SW | title = Structural and mechanistic conservation in DNA ligases | journal = Nucleic Acids Research | volume = 28 | issue = 21 | pages = 4051–58 | date = November 2000 | pmid = 11058099 | pmc = 113121 | doi = 10.1093/nar/28.21.4051 }}</ref> Ligases are particularly important in [[Replication fork|lagging strand]] DNA replication, as they join the short segments of DNA produced at the [[replication fork]] into a complete copy of the DNA template. They are also used in [[DNA repair]] and [[genetic recombination]].<ref name=Doherty />

==== Topoisomerases and helicases ====
[[Topoisomerase]]s are enzymes with both nuclease and ligase activity. These proteins change the amount of [[DNA supercoil|supercoiling]] in DNA. Some of these enzymes work by cutting the DNA helix and allowing one section to rotate, thereby reducing its level of supercoiling; the enzyme then seals the DNA break.<ref name=Champoux /> Other types of these enzymes are capable of cutting one DNA helix and then passing a second strand of DNA through this break, before rejoining the helix.<ref>{{cite journal | vauthors = Schoeffler AJ, Berger JM | title = Recent advances in understanding structure-function relationships in the type II topoisomerase mechanism | journal = Biochemical Society Transactions | volume = 33 | issue = Pt 6 | pages = 1465–70 | date = December 2005 | pmid = 16246147 | doi = 10.1042/BST0331465 }}</ref> Topoisomerases are required for many processes involving DNA, such as DNA replication and transcription.<ref name=Wang />

[[Helicase]]s are proteins that are a type of [[molecular motor]]. They use the chemical energy in [[nucleoside triphosphate]]s, predominantly [[adenosine triphosphate]] (ATP), to break hydrogen bonds between bases and unwind the DNA double helix into single strands.<ref>{{cite journal | vauthors = Tuteja N, Tuteja R | title = Unraveling DNA helicases. Motif, structure, mechanism and function | journal = European Journal of Biochemistry | volume = 271 | issue = 10 | pages = 1849–63 | date = May 2004 | pmid = 15128295 | doi = 10.1111/j.1432-1033.2004.04094.x | url = http://repository.ias.ac.in/52775/1/40-pub.pdf | doi-access = free }}</ref> These enzymes are essential for most processes where enzymes need to access the DNA bases.

==== Polymerases ====
[[Polymerase]]s are [[enzyme]]s that synthesize polynucleotide chains from [[nucleoside triphosphate]]s. The sequence of their products is created based on existing polynucleotide chains—which are called ''templates''. These enzymes function by repeatedly adding a nucleotide to the 3′ [[hydroxyl]] group at the end of the growing polynucleotide chain. As a consequence, all polymerases work in a 5′ to 3′ direction.<ref name=Joyce>{{cite journal | vauthors = Joyce CM, Steitz TA | title = Polymerase structures and function: variations on a theme? | journal = Journal of Bacteriology | volume = 177 | issue = 22 | pages = 6321–29 | date = November 1995 | pmid = 7592405 | pmc = 177480 | doi=10.1128/jb.177.22.6321-6329.1995}}</ref> In the [[active site]] of these enzymes, the incoming nucleoside triphosphate base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their template. Polymerases are classified according to the type of template that they use.

In DNA replication, DNA-dependent [[DNA polymerase]]s make copies of DNA polynucleotide chains. To preserve biological information, it is essential that the sequence of bases in each copy are precisely complementary to the sequence of bases in the template strand. Many DNA polymerases have a [[Proofreading (biology)|proofreading]] activity. Here, the polymerase recognizes the occasional mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. If a mismatch is detected, a 3′ to 5′ [[exonuclease]] activity is activated and the incorrect base removed.<ref>{{cite journal | vauthors = Hubscher U, Maga G, Spadari S | s2cid = 26171993 | title = Eukaryotic DNA polymerases | journal = Annual Review of Biochemistry | volume = 71 | pages = 133–63 | year = 2002 | pmid = 12045093 | doi = 10.1146/annurev.biochem.71.090501.150041 | url = http://pdfs.semanticscholar.org/e941/98efed7eb8fa606b87d9a44c118c235a62e9.pdf | archive-url = https://web.archive.org/web/20210126170051/http://pdfs.semanticscholar.org/e941/98efed7eb8fa606b87d9a44c118c235a62e9.pdf | url-status = dead | archive-date = 26 January 2021 }}</ref> In most organisms, DNA polymerases function in a large complex called the [[replisome]] that contains multiple accessory subunits, such as the [[DNA clamp]] or [[helicase]]s.<ref>{{cite journal | vauthors = Johnson A, O'Donnell M | title = Cellular DNA replicases: components and dynamics at the replication fork | journal = Annual Review of Biochemistry | volume = 74 | pages = 283–315 | year = 2005 | pmid = 15952889 | doi = 10.1146/annurev.biochem.73.011303.073859 }}</ref>

RNA-dependent DNA polymerases are a specialized class of polymerases that copy the sequence of an RNA strand into DNA. They include [[reverse transcriptase]], which is a [[virus|viral]] enzyme involved in the infection of cells by [[retrovirus]]es, and [[telomerase]], which is required for the replication of telomeres.<ref name=Greider /><ref name=Tarrago-Litvak1994>{{cite journal | vauthors = Tarrago-Litvak L, Andréola ML, Nevinsky GA, Sarih-Cottin L, Litvak S | title = The reverse transcriptase of HIV-1: from enzymology to therapeutic intervention | journal = FASEB Journal | volume = 8 | issue = 8 | pages = 497–503 | date = May 1994 | pmid = 7514143 | doi = 10.1096/fasebj.8.8.7514143 | doi-access = free | s2cid = 39614573 }}</ref> For example, HIV reverse transcriptase is an enzyme for AIDS virus replication.<ref name=Tarrago-Litvak1994 /> Telomerase is an unusual polymerase because it contains its own RNA template as part of its structure. It synthesizes [[telomeres]] at the ends of chromosomes. Telomeres prevent fusion of the ends of neighboring chromosomes and protect chromosome ends from damage.<ref name=Nugent />

Transcription is carried out by a DNA-dependent [[RNA polymerase]] that copies the sequence of a DNA strand into RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of DNA called a promoter and separates the DNA strands. It then copies the gene sequence into a [[messenger RNA]] transcript until it reaches a region of DNA called the [[terminator (genetics)|terminator]], where it halts and detaches from the DNA. As with human DNA-dependent DNA polymerases, [[RNA polymerase II]], the enzyme that transcribes most of the genes in the human genome, operates as part of a large [[protein complex]] with multiple regulatory and accessory subunits.<ref>{{cite journal | vauthors = Martinez E | s2cid = 24946189 | title = Multi-protein complexes in eukaryotic gene transcription | journal = Plant Molecular Biology | volume = 50 | issue = 6 | pages = 925–47 | date = December 2002 | pmid = 12516863 | doi = 10.1023/A:1021258713850 }}</ref>