The properties of steric gate mutants reveal different constraints within the active sites of Y-family and A-family DNA polymerases.

Abstract:

Y-family (lesion-bypass) DNA polymerases show the same overall structural features seen in other members of the polymerase superfamily, yet their active sites are more open, with fewer contacts to the DNA and nucleotide substrates. This raises the question of whether analogous active-site side chains play equivalent roles in the bypass polymerases and their classical DNA polymerase counterparts. In Klenow fragment, an A-family DNA polymerase, the steric gate side chain (Glu710) not only prevents ribonucleotide incorporation but also plays an important role in discrimination against purine-pyrimidine mispairs. In this work we show that the steric gate (Phe12) of the Y-family polymerase Dbh plays a very minor role in fidelity, despite its analogous role in sugar selection. Using ribonucleotide discrimination to report on the positioning of a mispaired dNTP, we found that the pyrimidine of a Pu-dPyTP nascent mispair occupies a similar position to that of a correctly paired dNTP in the Dbh active site, whereas in Klenow fragment the mispaired dNTP sits higher in the active site pocket. If purine-pyrimidine mispairs adopt the expected wobble geometry, the difference between the two polymerases can be attributed to the binding of the templating base, with the looser binding site of Dbh permitting a variety of template conformations with only minimal adjustment at the incoming dNTP. In Klenow fragment the templating base is more rigidly held, so that changes in base pair geometry would affect the dNTP position, allowing the Glu710 side chain to serve as a sensor of nascent mispairs.

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