A nick is a discontinuity in a double stranded DNA molecule where there is no

phosphodiester bond In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is ...

between adjacent nucleotides of one

strand Strand may refer to: Topography *The flat area of land bordering a body of water, a: ** Beach ** Shoreline * Strand swamp, a type of swamp habitat in Florida Places Africa *Strand, Western Cape, a seaside town in South Africa * Strand Street, ...

typically through damage or enzyme action. Nicks allow DNA strands to untwist during replication, and are also thought to play a role in the

DNA mismatch repair DNA mismatch repair (MMR) is a system for recognizing and repairing erroneous insertion, deletion, and mis-incorporation of bases that can arise during DNA replication and recombination, as well as repairing some forms of DNA damage. Mismatch ...

mechanisms that fix errors on both the leading and lagging daughter strands.Williams JS, Kunkel TA. Ribonucleotides in DNA: Origins, repair and consequences. DNA repair. 2014;19:27-37. doi:10.1016/j.dnarep.2014.03.029

Formation of nicks

The diagram shows the effects of nicks on intersecting DNA in a twisted plasmid. Nicking can be used to dissipate the energy held up by intersecting states. The nicks allow the DNA to take on a circular shape.Chao Ji, Lingyun Zhang, and Pengye Wang,
Configuration Transitions of Free Circular DNA System Induced by Nicks
" Journal of Nanomaterials, vol. 2015, Article ID 546851, 7 pages, 2015. doi:10.1155/2015/546851 Effects of nicks on intersecting DNA forms

Effects of nicks on intersecting DNA forms

Nicked DNA can be the result of DNA damage or purposeful, regulated biomolecular reactions carried out in the cell. During processing, DNA can be nicked by physical shearing, over-drying or enzymes. Excessive rough handling in pipetting or vortexing creates physical stress that can lead to breaks and nicks in DNA. Overdrying of DNA can also break the phosphodiester bond in DNA and result in nicks. Nicking endonuclease enzymes can assist with this process. A single-stranded break (nick) in DNA can be formed by the hydrolysis and subsequent removal of a phosphate group within the helical backbone. This leads to a different DNA conformation, where a hydrogen bond forms in place of the missing piece of the DNA backbone in order to preserve the structure.

Repair of nicks

Ligases are versatile and ubiquitous enzymes that join the 3’ hydroxyl and 5’ phosphate ends to form a phosphodiester bond, making them essential in nicked DNA repair, and ultimately genome fidelity. This biological role has also been extremely valuable in sealing the

sticky ends DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends, one strand is longer than the ...

of plasmids in molecular cloning. Their importance is attested by the fact most organisms have multiple ligases dedicated to specific pathways of repairing DNA. In eubacteria these ligases are powered by NAD+ rather than ATP. Each nick site requires 1 ATP or 1 NAD+ to power the ligase repair. Ligase_nick_repair_mecanism

In order to join these fragments, the ligase progresses through three steps: # Addition of an

adenosine monophosphate Adenosine monophosphate (AMP), also known as 5'-adenylic acid, is a nucleotide. AMP consists of a phosphate group, the sugar ribose, and the nucleobase adenine; it is an ester of phosphoric acid and the nucleoside adenosine. As a substituent it t ...

(AMP) group to the enzyme, referred to as adenylylation, # Adenosine monophosphate transfer to the DNA and # Nick sealing, or phosphodiester bond formation. One particular example of a ligase catalyzing nick closure is the E. coli NAD+ dependent DNA ligase, LigA. LigA is a relevant example as it is structurally similar to a clade of enzymes found across all types of bacteria. Ligases have a metal binding site which is capable of recognizing nicks in DNA. The ligase forms a DNA-adenylate complex, assisting recognition. With human DNA ligase, this forms a crystallized complex. The complex, which has a DNA–adenylate intermediate, allows

DNA ligase I DNA ligase 1 is an enzyme that in humans is encoded by the ''LIG1'' gene. DNA ligase I is the only known eukaryotic DNA ligase involved in both DNA replication and repair, making it the most studied of the ligases. Discovery It was known that ...

to institute a conformational change in the DNA for the isolation and subsequent repair of the DNA nick.

Biological implications

Role in mismatch repair

Single-stranded nicks act as recognizable markers to help the repair machinery distinguish the newly synthesized strand (daughter strand) from the template strand (parental strand).

(MMR) is an important DNA repair system that helps maintain genome plasticity by correcting mismatches, or non Watson-Crick base pairs in the a DNA duplex. Some sources of mismatched base pairs include replication errors and deamination of 5-methylcytosine DNA to form thymine. MMR in most bacteria and eukaryotes is directed to the erroneous strand of the mismatched duplex through recognition of strand discontinuities, while MMR in E. coli and closely related bacteria is directed to the strand on the basis of the absence of

methylation In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These t ...

. Nicking endonucleases introduce the strand discontinuities, or DNA nicks, for both respective systems. Mut L homologues from eukaryotes and most bacteria incise the discontinuous strand to introduce the entry or termination point for the excision reaction. Similarly, in E. coli, Mut H nicks the unmethylated strand of the duplex to introduce the entry point of excision. For eukaryotes specifically, the mechanism of DNA replication elongation between the leading and lagging strand differs. On the lagging strand, nicks exist between

Okazaki fragments Okazaki fragments are short sequences of DNA nucleotides (approximately 150 to 200 base pairs long in eukaryotes) which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA r ...

and are easily recognizable by the DNA mismatch repair machinery prior to

ligation Ligation may refer to: * Ligation (molecular biology), the covalent linking of two ends of DNA or RNA molecules * In medicine, the making of a ligature (tie) * Chemical ligation, the production of peptides from amino acids * Tubal ligation, a meth ...

. Due to the continuous replication that occurs on the leading strand, the mechanism there is slightly more complex. During replication, ribonucleotides are added by replication enzymes and these ribonucleotides are nicked by an enzyme called RNase H2. Together, the presence of a nick and a ribonucleotide make the leading strand easily recognizable to the DNA mismatch repair machinery. Nick translation is a biological process in which a single-stranded DNA nick serves as the marker for

DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...

to excise and replace possibly damaged nucleotides. At the end of the segment that DNA polymerase acts on, DNA ligase must repair the final segment of DNA backbone in order to complete the repair process. In a lab setting, this can be used to introduce

fluorescent Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...

or other tagged nucleotides by purposefully inducing site-specific, single-stranded nicks in DNA in vitro and then adding the nicked DNA to an environment rich in DNA polymerase and tagged nucleotide. The DNA polymerase then replaces the DNA nucleotides with the tagged ones, starting at the site of the single-stranded nick.

Role in replication and transcription

Nicked DNA plays an important role in many biological functions. For instance, single-stranded nicks in DNA may serve as purposeful biological markers for the enzyme

topoisomerase DNA topoisomerases (or topoisomerases) are enzymes that catalyze changes in the topological state of DNA, interconverting relaxed and supercoiled forms, linked (catenated) and unlinked species, and knotted and unknotted DNA. Topological issues i ...

that unwinds packed DNA and is critical to DNA replication and transcription. In these instances, nicked DNA is not the result of unwanted cell damage. Topoisomerase-1 preferentially acts at nicks in DNA to cleave adjacent to the nick and then winds or unwinds the complex topologies associated with packed DNA. Here, the nick in the DNA serves as a marker for single strand breakage and subsequent unwinding. It is possible that this is not a highly conserved process. Topoisomerase may cause short deletions when it cleaves bonds, because both full-length DNA products and short deletion strands are seen as products of topoisomerase cleavage while inactive mutants only produced full-length DNA strands. Nicks in DNA also give rise to different structural properties, can be involved in repairing damages caused by ultraviolet radiation, and are used in the primary steps that allow for genetic recombination. Nick idling is a biological process in which DNA polymerase may slow or stop its activity of adding bases to a new daughter strand during DNA replication at a nick site. This is particularly relevant to

in lagging strand in double stranded DNA replication because the direction of replication is opposite to the direction of

, therefore nick idling plays a role in stalling the complex as it replicates in the reverse direction in small fragments (Okazaki fragments) and has to stop and reposition itself in between each and every fragment length of DNA. DNA structure changes when a single-stranded nick is introduced. Stability is decreased as a break in the phosphodiester backbone allows DNA to unwind, as the built up stress from twisting and packing is not being resisted as strongly anymore. Nicked DNA is more susceptible to degradation due to this reduced stability.

In bacteria

The ''nic'' site or nick region is found within the origin of transfer (''oriT'') site and is a key in starting

bacterial conjugation Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual mode of reproduction in bacter ...

. A single strand of DNA, called the T-strand, is cut at ''nic'' by an enzyme called relaxase.Lanka Erich, Wilkins Brian M (1995). “DNA Processing Reactions in Bacterial Conjugation.” Annu. Rev. Biochem. 64: 141-69 This single strand is eventually transferred to the recipient cell during the process of

. Before this cleavage can occur, however, it is necessary for a group of proteins to attach to the ''oriT'' site. This group of proteins is called the relaxosome. It is thought that portions of the ''oriT'' site are bent in a way that creates interaction between the relaxosome proteins and the ''nic'' site. Cleaving the T-strand involves relaxase cutting a

at the nic site. The cleaved strand is left with a hydroxyl group at the 3' end, which may allow for the strand to form a circular plasmid after moving into the recipient cell.Grohmann E, Muth G, Espinosa M (2003). "Conjugative Plasmid Transfer in Gram-positive Bacteria." Microbiol Mol Biol Rev. 67(2):277-301.

References

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