Research Update
An Unexpected Discovery in DNA Repair Pathway
The mechanisms nature developed to prevent or correct damage at the cellular and molecular levels are extensive and largely still a mystery. Researchers from the Stockholm University shed light on one of the enzymes critical to the prevention of DNA damage by reactive oxygen radicals produced as a side product of metabolism. The MutT Homolog 1 (MTH1) protein catalyzes the hydrolysis of 8-oxo-dGTP, a common oxygen radical, to relatively inert 8-oxo-dGMP.
The researchers elucidated the crystal structure of MTH1 with 8-oxo-dGMP by crystallizing the protein in the presence of 8-oxo-dGTP purchased from TriLink (Cat# N-2034).
As expected, the crystal structure showed that the nucleotide in the complex was in the monophosphate form; the hydrolysis reaction occurs too quickly to allow capture of a complex with the triphosphate analog. However, what was completely unexpected was that the 8-oxo-2′-deoxygaunosine was in the anti configuration, with the base extended as normally depicted in drawings of 2′-deoxyguanosine. Previous nucleotide analog studies suggested the nucleobase was rotated into the syn configuration for MTH1, which was confirmed to be the case by crystal structure studies of the E. coli version of the MTH protein, MutT. This suggests that although the proteins have the same catalytic activities, they derive from different evolutionary paths. On the other hand, the substrate hydrolysis domain appears to be largely conserved, so the paths certainly have a common ancestry at some point.
The authors suggest that the binding of the nucleotide in the anti configuration by MTH1 explains the broader substrate specificity the protein exhibits compared to MutT.
Another rather surprising finding was that although MTH1 is very specific for 8-oxo purine nucleotide substrates (it also recognizes 8-OH-dATP, 2-OH-dATP and 2-OH-rATP), there doesn’t appear to be any specific interaction of the protein with the 8-oxo position. The authors suggest the 8-oxo may actually stabilize the 6-enol-8-keto tautomer which may be the key to substrate binding.
TriLink offers the following products for DNA damage/repair mechanism research:
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Q. Can N-8001 (1-thio-dATP) and N-8002 (1-thio-dCTP) be used by Taq polymerase in PCR?
- Chun-Nan Chen, Single Cell Technology
A. 1-thio-2′-deoxyadenosine-5′-triphosphate (N-8001) and 1-thio-2′-deoxycytidine-5′-triphosphate (N-8002) can be incorporated by Taq in PCR. However, not all analogs will incorporate with the same efficiency in PCR; some optimization may be needed. We recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions for modified nucleotide incorporation with robust amplicon yield.
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