How a polymerase bypasses the rules of Watson-Crick base-pairing

March 20, 2014 § 3 Comments

The half-sized ASFV DNA polymerase X does not follow the dogma of binding DNA first followed by MgdNTP. It binds synthetic dGTP prior to DNA in order to catalyze dG:dGTP incorporation. Image by Wen-Jin Wu.

The half-sized ASFV DNA polymerase X does not follow the dogma of binding DNA first followed by MgdNTP. It binds synthetic dGTP prior to DNA in order to catalyze dG:dGTP incorporation. Image by Wen-Jin Wu.

A pairs with T; G pairs with C. This is the golden rule in DNA base pairing, known as Watson-Crick base pairing. But some polymerases thumb their noses at this golden rule and can get the bases to pair with different partners. In a paper recently published in the Journal of the American Chemical Society, researchers demonstrate how a polymerase found in the African swine fever virus bypasses the golden base-pairing rule by tackling the process of making DNA in a different way.

DNA polymerases are the enzymes involved in fixing and making DNA. Ming-Daw Tsai’s laboratory at Academia Sinica in Taiwan has a longstanding interest in DNA polymerase X. This polymerase, known as Pol X, is found in the African swine fever virus. The polymerase is unusual because it can get G to pair with itself; it also can carry out canonical base pairing. So, how does the polymerase accomplish the noncanonical base pairing?

“Kinetic studies suggested that Pol X does not follow the established mechanistic paradigm that DNA polymerases bind DNA” before binding to a nucleotide complexed with a magnesium ion, explains Tsai. “However, structural studies took us a long time since Pol X does not crystallize.”

The investigators were left with studying the enzyme in solution by nuclear magnetic resonance spectroscopy. “Even though the structure of the free enzyme in 2001 was solved by NMR, using NMR to solve the structures of complexes with MgdGTP, in the absence and presence of DNA, was a lot harder,” he says.

After spending 10 years on the problem, Tsai’s group now has an answer. DNA polymerases that strictly stick to Watson-Crick base pairing, known as hgh-fidelity polymerases, first bind to the DNA and then to the necessary nucleotide complexed with a magnesium ion. The investigators found that Pol X binds to a nucleotide-magnesium ion complex first and then to the DNA. Whatever nucleotide this low-fidelity polymerase is carrying could get inserted into the DNA strand.

“This result is contrary to the thought that maybe low-fidelity polymerases are just poor polymerases that cannot achieve high fidelity,” explains Tsai. In fact, Pol X “is highly specific and seems to know what it is doing.”

He says the data show that there are two competing factors in a DNA polymerase’s function: The DNA may impose the rules of Watson-Crick base pairing on the polymerase, but the enzyme may control the DNA by prebinding its preferred nucleotide-magnesium ion complex.

Tsai emphasizes that the work by his group specifically applies to Pol X. “There has been an explosion in the study of the structure and mechanism of low-fidelity and translesion synthesis DNA polymerases in the past decade. Many beautiful structural studies have been published, and it appears that each polymerase has some of its own interesting features,” he says.

Indeed, his group will test other low-fidelity polymerases to see how they work. Also, he notes, “there could be some clinical implication as well, though it is speculative at this point.” For making new drugs against the African swine fever virus, Tsai says it’s possible “we can target Pol X alone instead of Pol X-DNA complex. The same concept can be applied to some disease-relevant DNA polymerase mutants in human, which may have developed the ability to pre-bind dNTP.”

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