Abstract
BackgroundA wide variety of DNA lesions interfere with replication and transcription, leading to mutations and cell death. DNA repair mechanisms act upon these DNA lesions present in the genomic DNA. To investigate a DNA repair mechanism elaborately, an in vitro DNA repair substrate containing DNA lesions at a specific site is required. Previously, to prepare the substrate, phagemid ssDNA and DNA lesion-harboring oligonucleotides were employed with considerable amounts of DNA polymerase and DNA ligase. However, preparing in vitro DNA repair substrate in general is difficult and labor intensive.ResultsHere, we modified the construction method of in vitro mismatch repair substrate using a nicking-endonuclease, which produces gap corresponding to the ssDNA in the plasmid DNA, and swaps DNA lesion-containing oligonucleotide upon addition of restriction enzyme and T5 exonuclease. This modified method is able to produce in vitro DNA repair substrates containing adenine:cytosine mismatch basepair, 8-oxoG, and uracil. The DNA repair enzyme, each Fpg, hOGG1 could cleave an 8-oxoG-containing DNA substrate, the mixture of UDG and APE1 could cleave a uracil-containing DNA substrate. Omitting a column purification step, DNA repair substrates were prepared by one-pot synthesis.ConclusionsWe were able to prepare in vitro DNA repair substrates using this simple method involving restriction enzymes and T5 exonuclease. It is anticipated that this method, termed as “Oligo Swapping Method”, will be valuable for understanding the DNA repair machinery.
Highlights
A wide variety of DNA lesions interfere with replication and transcription, leading to mutations and cell death
4 pp),; base excision repair (BER), which is used for non-bulky and non-helix-distorting DNA modifications induced by alkylation, oxidation (e.g. 8-oxoG), and deamination; and mismatch repair (MMR), which mends the mismatch base pairings during DNA replication
We have modified the method by addition of a restriction enzyme and T5 exonuclease that is capable of removing linear Double strand DNA (dsDNA) and nicked plasmid DNA
Summary
A wide variety of DNA lesions interfere with replication and transcription, leading to mutations and cell death. To investigate a DNA repair mechanism elaborately, an in vitro DNA repair substrate containing DNA lesions at a specific site is required. The induced DNA lesions interfere with DNA and RNA synthesis during replication and transcription, respectively, leading to mutations and cell death [1]. As a result, these genomic mutations and subsequently, the disruption of cellular processes may cause cancer, congenital diseases, and aging. To analyze DNA repair mechanisms in vitro, we required DNA repair substrates containing a DNA lesion at specific site. For the in vitro BER assay for short patch repair, chemically synthesized DNA lesion bearing oligonucleotides annealed with complementary strands were employed as DNA lesion substrates [4, 5]. In case of in vitro NER assay [6, 7], at least ~ 120-bp long DNA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
