DNA Repair Enzymes: Cleaving modified bases using novel molecular biology tools from NEB

DNA Repair Enzymes: Cleaving modified bases using novel molecular biology tools from NEB. In today's webinar, we will cover common types of modified bases, activities of DNA repair enzymes, which enzyme to choose, and review workflows.

DNA is susceptible to many types of damage resulting from exposure to a variety of chemical or environmental reagents, manipulation, or simply aging. Common types of DNA modification that often give rise to mutagenic effects include oxidation and deamination of DNA bases. Many types of base modifications are created by oxidation, but the conversion of guanine to 8-oxoguanine is one of the most common. 8-oxoguanine can base pair with adenine and is therefore mutagenic.

Deamination of bases is a common type of DNA damage that occurs when an amino group is removed from a nitrogenous base. Deamination can occur through various means, including spontaneous hydrolysis, deamination by deaminase enzymes, or environmental factors. Deamination of bases can lead to the formation of mutagenic lesions such as hypoxanthine or inosine, which is formed when adenine loses an amine group, uracil which is formed when cytosine loses an amine group, or xanthine which is formed when guanine loses an amine group.
While modified bases are introduced naturally into DNA sequences through biological processes such as DNA replication, direct enzymatic modification, or spontaneous DNA damage, these modified bases can also be intentionally engineered into DNA sequences and used with their cognitive DNA repair enzymes such as DNA glycosylases and DNA endonucleases as useful molecular biology tools.

NEB currently offers two enzymes that will act on 8-oxoguanine. Both FPG and thermostable OGG enzymes are bifunctional DNA glycosylases and AP lyases that will recognize and remove the damaged purine base. First, the glycosylase activity of each enzyme will recognize the 8-oxoguanine in a DNA duplex and remove the damaged base. The AP lyase activity of these enzymes will then cleave the DNA at the generated Abasic or AP site to create a one nucleotide gap at the site of the 8-oxoguanine. The products remaining from the cleavage events, however, are different depending on the enzyme. FPG leaves a phosphate at both the three-prime and five-prime end, whereas thermostable OGG leaves a three-prime phospho-alpha-beta unsaturated aldehyde and a five-prime phosphate at the location of the generated one nucleotide gap.

One key difference between FPG and thermostable OGG enzymes is in their specificity for modified bases. As shown in the left panel, FPG is more promiscuous and exhibits activity on a number of modified bases, including but not limited to 8-oxoguanine. Whereas thermostable OGG is specific for 8-oxoguanine modified base. Another key difference to note between FPG and thermostable OGG includes the temperature dependence of the enzymes. FPG is optimally active at 37 degrees Celsius and can be heat inactivated at 60 degrees Celsius. Thermostable OGG, on the other hand, is a thermostable enzyme that is active over a wide range of temperatures anywhere between 16 to 80 degrees Celsius. However, it is optimally active between 60 to 80 degrees and cannot be heat inactivated.

Deamination of deoxyadenosine can give rise to deoxyinosine in DNA. Endonuclease V is an enzyme that recognizes DNA containing deoxyinosines on both double-stranded and single-stranded DNA and cleaves the second phosphodiester bond three-prime to the deoxyinosine, leaving a Nick with a three-prime hydroxyl and five-prime phosphate. It is important to note that endonuclease V does not remove the damaged deoxyinosine base, but rather cuts the DNA downstream of the base modification, keeping the modification intact. Also, the Nick generated has a three-prime hydroxyl and five-prime phosphate, meaning that it can be religated by a DNA ligase. In addition to deoxyinosine, endo V exhibits activity, albeit to a lesser degree, on DNA containing Abasic or AP sites, urea, base mismatches, insertion-deletion mismatches, hairpin or unpaired loops, flaps, and pseudo-Y structures.

NEB also cells hAAG or human alkaladenine DNA glycosylase, which will remove the inosine base and leave an Abasic or AP site. As hAAG is only a glycosylase and does not contain an AP lyase activity, an additional step of base hydrolysis or cleavage of the AP site by an endonuclease would then be required to cleave the DNA backbone.

As a constituent nucleobase in RNA, the incorporation and cleavage at uracil is a widely used molecular biology tool. For cleavage at deoxyuracils, NEB offers a suite of USER enzymes or uracil-specific excision reagent. These USER enzymes are actually a combination of two enzymes. The first enzyme, a uracil DNA glycosylase or UDG catalyzes the excision of the uracil base forming an Abasic or AP site while leaving the phosphodiester backbone intact. The lyase activity of the second enzyme, an endonuclease, and in this case for USER enzyme, endonuclease VIII, breaks the phosphodiester backbone at the three-prime and five-prime sides of the Abasic site so that a one nucleotide gap is created. In addition to USER enzyme, NEB also offers a thermolabile version of USER, Thermolabile USER II Enzyme. This is a mixture of Antarctic Thermolabile UDG and endonuclease III. This mixture has the same functionality of USER enzyme where the Antarctic Thermolabile UDG excises the uracil base leaving an AP site that is then cut by endo III, leaving in one nucleotide gap.

Thermolabile USER II Enzyme provides the added benefit as compared to USER enzyme that it can be heat inactivated after 10 minutes at 65 degrees Celsius. Additionally, NEB offers Thermostable USER III Enzyme. This is an enzyme cocktail comprised of Afu UDG, a thermostable UDG, and endonuclease IV. Again, this mixture has the same functionality of USER enzyme and Thermolabile USER II Enzyme where the Afu UDG excises the uracil base leaving an AP site that is then cut by endonuclease IV, leaving a one nucleotide gap. However, while USER and Thermolabile USER II Enzymes are both active at 37 degrees Celsius, Thermostable USER III Enzyme is active at higher temperatures between 50 to 75 degrees Celsius.

While all three USER enzymes accomplish the same result, they each do so by different endonucleases. This is important because that means each variation of USER enzyme leaves different ends at the site of cleavage. USER enzyme contains endonuclease VIII and leaves a three-prime and five-prime phosphate after cleavage. Thermolabile USER II Enzyme contains endonuclease III and leaves a three-prime phospho-alpha-beta unsaturated aldehyde and a five-prime phosphate after cleavage. Thermostable USER III Enzyme contains endonuclease IV and leaves a three-prime hydroxyl and five-prime deoxyribose phosphate. It is important to note that none of the ends generated by any USER enzyme are capable of being religated as this would require a three-prime hydroxyl and five-prime phosphate to remain after cleavage.

USER cloning was one of the first methods that allowed simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. Using this approach, target DNA molecules and cloning vector are generated by PCR with 6 to 10 bases of homology between the neighboring fragments. PCR fragments contain a single deoxyuracil residue flanking the three-prime end of the hemology region and can be designed to accommodate nucleotide substitutions, insertions, and/or deletions.

The primers are then used to amplify the vector and target DNA in discrete overlapping fragments that incorporate a DU at each end. The subsequent treatment of PCR fragments with USER enzyme creates a single nucleotide gap at the location of each DU, resulting in PCR fragments flanked with single stranded extensions that allow seamless and directional assembly of customized DNA molecules into a linearized vector. In this way, multi-fragment assemblies as well as various mutagenic changes can all be accomplished in a single format experiment.

USER enzymes can also be used to open up the NEBNext adapter as part of multiplex library preparation for next generation sequencing on the Illumina platform. NEBNext Illumina Library Prep Kits contain the NEBNEXT loop-shaped adapter, which contains a single uracil base. In order for the adapter ligated molecules to be available for PCR, the loop must first be cleaved by a USER enzyme before PCR amplification.

USER enzymes are also used in directional RNA library preparation, the workflow of which is shown here. In the NEBNext directional RNA-seq workflow, uracil is incorporated specifically during the second strand synthesis. The DU containing second strand is selectively removed using USER. At the same time, if NEBNext adapters are used, the DU in the center of the NEBNext hairpin loop adapter is removed, opening the molecule and making it available for PCR amplification.

In addition to the three already commercially available USER formats, including USER enzyme, Thermolabile USER II Enzyme, and Thermostable USER III Enzyme, NEB offers the potential to mix and match any of our four commercially available UDGs and a selection of endonuclease enzymes. UDG enzymes include E. coli UDG, Antarctic Thermolabile UDG, the thermostable Afu UDG, and a WarmStart version of Afu UDG. Available endonucleases that can be mixed with UDGs include endonuclease VIII, endonuclease III, or endonuclease IV. The endonuclease selection can be tailored to suit your needs depending on what ends are required in your workflow at the cleavage site. Please contact NEB's customized solutions team if you are interested in a custom USER formulation.

Thermostable Endonuclease Q or EndoQ is an archaeal DNA endonuclease that cleaves the phosphate backbone of a DNA substrate five-prime to the position of modified nucleobasis of deoxyxanthosine, deoxyinosine, deoxyuracil, and an AP site. EndoQ functions optimally at 65 to 85 degrees Celsius. However, Thermostable Endonuclease Q will perform at temperatures between 37 to 90 degrees Celsius. It is important to note that like endonuclease V, Thermostable EndoQ does not remove the damaged base, but rather cuts the DNA upstream of the base modification, keeping the modification intact. Interestingly, the result of cleavage by Thermostable EndoQ is different than any available USER enzyme. The Nick generated by Thermostable EndoQ leaves a three-prime hydroxyl and a five-prime phosphate, meaning that it can be religated by a DNA ligase.

Although hAAG has been noted in literature to cleave xanthosine modified bases, NEB has confirmed that the activity of this enzyme on DX is inefficient. On the other hand, Thermostable EndoQ will recognize and efficiently cleave xanthosine modified bases. These specificities have been noted on our DNA repair enzymes on damaged and non-standard basis chart available on NEB.com.
Thermostable EndoQ exhibits different substrate preferences than USER enzymes. USER enzymes are known to have approximately three to four-fold lower activity on uracil in single-stranded DNA versus double-stranded DNA. Conversely, EndoQ actually prefers single-stranded DNA as a substrate and is fourfold more active on a uracil in single-stranded DNA than a uracil in double-stranded DNA. Thermostable EndoQ has the same activity toward inosine, regardless of single-stranded or double-stranded DNA context, and is actually more active toward xanthosine in a double-stranded DNA context rather than single-stranded DNA context.

One potential application of both USER enzymes and Thermostable EndoQ is cleavage of DNA oligos, whether double or single-stranded from a solid surface. In anticipation of applications for cleaving synthesized DNA from solid supports, we tested how close to the DNA ends EndoQ and users can cleave a uracil. With double-stranded DNA, all user enzymes are capable of cleaving a uracil, only three nucleotides from either the three-prime or five-prime end. Thermostable EndoQ, on the other hand, struggled slightly with cleavage of a uracil three nucleotides away from the five-prime end of double-stranded DNA. Conversely, with single-stranded DNA, USER enzymes struggled with cleavage of a uracil, especially at the five-prime end of the DNA, and required at least 9 to 12 nucleotides for efficient cleavage from the five-prime end, and at least six nucleotides for cleavage from the three-prime end of single-stranded DNA. Thermostable EndoQ, on the other hand, can cleave a uracil that is only six nucleotides away from the five-prime end of single-stranded DNA, and only three nucleotides from the three-prime end of single-stranded DNA, making Thermostable EndoQ the preferred enzyme for the cleavage of single-stranded DNA from a solid support.

To summarize the differences between NEB's USER enzymes and Thermostable EndoQ, USER enzymes are a two-enzyme mixture, whereas Thermostable EndoQ is a single enzyme. USER enzymes cleave only at uracils while Thermostable EndoQ can cleave at a uracil, inosine or xanthosine. USER enzymes are available in either a thermolabile or thermostable version, whereas Thermostable EndoQ is only thermostable and cannot be heat inactivated. No USER enzyme formulation leaves ligatable ends, whereas Thermostable Endonuclease Q leaves a three-prime hydroxyl and a 5-prime phosphate, which can be religated. USER enzymes are three to fourfold less active on single-stranded DNA than double-stranded DNA, whereas Thermostable EndoQ prefers single-stranded DNA over double-stranded DNA for a DU substrate. USER enzymes require at least six to nine nucleotides from the end of single-stranded DNA for cleavage to occur, whereas Thermostable EndoQ can cleave single-stranded DNA as close as three nucleotides from the end of DNA.

In summary, NEB offers a wide variety of DNA repair enzymes, many of which we have had time to discuss here today. In addition to our NEB catalog, which contains detailed information on all of the enzymes NEB sells, NEB.com is also a great resource for determining which DNA repair enzyme fits best into your workflow. On NEB.com, you will find a number of tables, including the activities of DNA repair enzymes and structure-specific endonucleases. I really like this table because it collates all of the schematics for all of our DNA repair enzymes in one location. If you are unsure of which enzyme you may require in a workflow, this is a great way to visualize the activities of all of our DNA repair enzyme offerings.

The substrate specificity of these DNA repair enzymes can also be found in the DNA repair enzymes on damaged and non-standard bases table. This is helpful if you know which modified base you wish to remove or cleave and will help you identify the best enzyme for your workflow.

Lastly, our properties of DNA repair enzymes and structure-specific endonucleases is a great resource and provides detailed information, not only on substrates recognized by each DNA repair enzyme, but also information regarding the cleavage site, what products are formed, and what ends are left at both the five-prime and three-prime end after cleavage. This can be very helpful information if you have further downstream processing steps in your workflow, and the termini left after cleavage are important. All of these resources are available in the NEB catalog and at NEB.com, where they can be found under the resources section under selection charts.