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A nick is a discontinuity in a double stranded DNA molecule where there is no phosphodiester bond between adjacent nucleotides of one strand typically through damage or enzyme action. Nicks allow for the much-needed release of torsion in the strand during DNA replication. Nicks are also thought to play a role in the DNA mismatch repair mechanisms that fix errors on both the leading and lagging daughter strands.[1] The nicks act as recognizable markers to help the repair machinery distinguish the newly synthesized strand (daughter strand) from the template strand (parental strand).[1] However, the mechanism between the leading and lagging strand differs. On the lagging strand, nicks exist between Okazaki fragments and are easily recognizable by the DNA mismatch repair machinery prior to ligation. Due to the continuous replication that occurs on the leading strand, the mechanism there is slightly more complex. During replication, ribonucleotidesare added by replication enzymes and these ribonucleotides are nicked by an enzyme called RNase H2.[1] Together, the presence of a nick and a ribonucleotide make the leading strand easily recognizable to the DNA mismatch repair machinery.
Role In Mismatch Repair
[edit]Nicks can serve as signals that are recognized by mismatch repair proteins.[1] During DNA replication, nicks are found between Okazaki fragments, and provide a recognition site for mismatch proofreading proteins to make sure the newly synthesized strands do not contain mismatched nucleotides. Nicks are also used as recognition sites when random mismatches in the DNA occur. When a mismatch occurs, the nucleotides do not compliment each other and will not exist in the same conformation as two complimentary nucleotides. This failure to assume a normal conformation can cause nicks that cannot be sealed by nick ligation.[2]
Mismatch proteins recognize the nick and can begin mismatch repair on the erroneous base pair. If a nick does not occur naturally in this way when a mismatch has occurred, mismatch repair proteins create a nick in order to proceed with the excision and repair. The mismatch proteins MutS, MutH, and MutL have been identified in E.coli and have been observed to create and utilize nicks in repair.[3] MutS binds to DNA and acts as the activator for MutH and MutL. Once activated, MutH generates the nick at the 5' or 3' end, depending on the location of the mismatch, to begin the excision process. MutL recruits helicase II, which unwinds the DNA at the nick toward the mismatch.[3] A directional specific exonuclease is recruited and excises the strand containing the nick from the nick site. Finally, the single-stranded gap is repaired and synthesized by DNA polymerase, and ultimately ligated by DNA ligase.[3]
Nicks also serve a role in the mismatch repair of ribonucleotides. Ribonucleotide excision repair begins when the enzyme RNase H2 creates a nick at the ribonucleotide site in the DNA. FEN1 then cleaves the ribonucleotide at the nick site, excising it from the strand. DNA ligase seals the nick to complete the repair process.[4]
Nick Ligation
[edit]Single-stranded and double stranded nicks are repaired by DNA ligase, an enzyme with similar structural elements to DNA polymerase.[2] The role of DNA ligase's interaction with nicks is primarily to catalyze the the joining of the two ends of the nick.[5] During DNA replication, DNA ligase creates a complex with the nicked DNA between Okazaki fragments. The complex lets DNA ligase momentarily unwind part of the double-helix to expose the nick for ligation, and the DNA binding domain of the ligase stabilizes the structure and positions the nick on the enzyme's catalytic site.[6] Without this stabilization, torque can occur on the DNA double helix, making nick repair difficult.[7]
DNA nicks are repaired via several steps. First, DNA ligase is activated by reaction with ATP molecules (or NAD+ molecules in eubacteria) to form a ligase-adenylate intermediate, which is the addition of AMP to a lysine residue in the active site of DNA ligase.[7] This reaction converts the ATP into AMP. Next, AMP is transferred from the ligase to the 5' phosphate end of the nick to form the DNA-adenylate intermediate. Finally, DNA ligase catalyses the formation of the the phosphodiester bond by connecting the 3' hydroxyl group of the nick to the DNA-adenylate intermediate, and AMP is released.[7] In single-stranded nicks, DNA ligase uses the nucleotides on the complementary strand as a template to close the nick.[6]
- ^ Constantin, Nicoleta; Dzantiev, Leonid; Kadyrov, Farid A.; Modrich, Paul (2005-12-02). "Human Mismatch Repair RECONSTITUTION OF A NICK-DIRECTED BIDIRECTIONAL REACTION". Journal of Biological Chemistry. 280 (48): 39752–39761. doi:10.1074/jbc.M509701200. ISSN 0021-9258. PMC 1435381. PMID 16188885.
{{cite journal}}: CS1 maint: unflagged free DOI (link) - ^ a b Cherepanov, Alexei V.; de Vries, Simon (11 December 2002). "Dynamic mechanism of nick recognition by DNA ligase". European Journal of Biochemistry. 269 (24): 5993–5999.
- ^ a b c Li, Guo-Min (2008-01-01). "Mechanisms and functions of DNA mismatch repair". Cell Research. 18 (1): 85–98. doi:10.1038/cr.2007.115. ISSN 1001-0602.
- ^ Williams, Jessica S.; Kunkel, Thomas A. (2014-07-01). "Ribonucleotides in DNA: origins, repair and consequences". DNA repair. 19: 27–37. doi:10.1016/j.dnarep.2014.03.029. ISSN 1568-7856. PMC 4065383. PMID 24794402.
- ^ Timson, David J; Singleton, Martin R; Wigley, Dale B (2000-08-30). "DNA ligases in the repair and replication of DNA". Mutation Research/DNA Repair. 460 (3–4): 301–318. doi:10.1016/S0921-8777(00)00033-1.
- ^ a b Pascal, John M.; O'Brien, Patrick J.; Tomkinson, Alan E.; Ellenberger, Tom (25 November 2004). "Human DNA ligase I completely encircles and partially unwinds nicked DNA". Nature. 432: 473–478.
- ^ a b c Crut, Aurélien; Nair, Pravin A.; Koster, Daniel A.; Shuman, Stewart; Dekker, Nynke H. (2008-05-13). "Dynamics of phosphodiester synthesis by DNA ligase". Proceedings of the National Academy of Sciences. 105 (19): 6894–6899. doi:10.1073/pnas.0800113105. ISSN 0027-8424. PMC 2383972. PMID 18458338.