A polar filter in DNA polymerases prevents ribonucleotide incorporation

The presence of ribonucleotides in DNA can lead to genomic instability and cellular lethality. To prevent adventitious rNTP incorporation, the majority of the DNA polymerases (dPols) possess a steric filter in the form of a bulky residue that stacks against the ribose sugar and clashes with 2’ -OH of incoming ribonucleotide. MsDpo4 from Mycobacterium smegmatis, naturally lack this steric filter and hence is capable of rNTP addition. We observed that the introduction of the steric filter in MsDpo4 did not result in complete abrogation of the ability of this enzyme to incorporate ribonucleotides. In comparison, DNA polymerase IV from Escherichia coli (PolIV) exhibited stringent selection for deoxyribonucleotides during DNA synthesis. A rigorous comparison of MsDpo4 and E. coli PolIV led to the discovery of an additional filter that contributes substantially towards sugar selectivity. This filter is represented by a polar residue that forms interactions with the incoming nucleotide and draws it closer to the enzyme surface. As a result, the 2’ –OH in rNTPs will clash with the enzyme surface, and therefore ribonucleotides cannot be accommodated in the active site in a conformation compatible with productive catalysis. A comprehensive analysis of members of different families of dPols suggested that the polar filter may be a conserved feature of Y- and B- family dPols. Studies conducted on a representative member of the B-family, DNA polymerase II from E. coli showed that members of this family also possess a polar filterto aid ribonucleotide exclusion.Overall, our studies show that a polar and steric filter act in tandem to reduce rNTP incorporation by DNA polymerases.

For full article: https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkz792/5572565