Polymerase active site and environs

Motif 2 and the Pol A motif are both critical for catalysis but contribute to catalysis by different mechanisms. Therefore, a mutation in subcluster 1D should be evaluated by the motif to which it is closest in primary sequence.

Motif 2 (residues 845-863) In PolG, motif 2 in the pol domain has been termed the "RR loop" because of two sequential arginines (R852 and R853) located on the tip of the loop, whereas family A polymerases have only a single arginine (equivalent to R853) (Euro et al, 2011). Overall, motif 2 is critical for binding correctly base-paired primer-template DNA in the minor groove, and to stabilize the template DNA backbone in the active site (Loh and Loeb, 2005). These residues form specific contacts with DNA in the pol active site and are major determinants for the catalytic efficiency and fidelity of DNA synthesis by Pol ?. Motif 2 mutations should always cause reduced pol activity but may also cause a DNA binding defect depending on the residue, such as the 5-fold defects reported for G848S and R852C (Kasiviswanathan et al, 2009). The DNA binding affinity of T851A, R853Q, Q879H, T885S may be decreased slightly, but the reduction in pol activity is much greater (Kasiviswanathan et al, 2009). Overall, it is unlikely for motif 2 mutations to present as dominant mutations because these mutant forms of Pol ? would not compete effectively with wild type Pol ? for DNA binding.

Pol A motif (residues 887-896) The Pol A motif of family A DNA polymerases forms the site of catalysis where it contacts closely the primer strand and binds the Mg2+ ion required for the chemical steps of nucleotide polymerization (D890 in PolG). Additionally, a residue of the Pol A motif discriminates against ribonucleotide incorporation (E895 in Pol ?), and mutation causes severe pol defects in addition to increased ribonucleotide incorporation (Astatke et al, 1998). Mutations in the Pol A motif at either D890 or E895 in Pol ? would likely manifest a dominant lethal phenotype, and the only patient carrying the E895G mutation to date died immediately after birth (Spinazzola et al, 2009). In contrast to these two critical residues, adjacent residues within the Pol A motif play lesser roles in pol activity and mutations in them are observed more frequently, resulting in reduced pol activity without a significant effect on DNA binding affinity, and thus they can produce a dominant phenotype. Overall, though dominant mutations are fairly rare for subcluster 1D, novel mutations affecting residues of the Pol A motif would present a moderate risk for dominant inheritance.

Upstream DNA binding channel

Partitioning loop

Distal accessory subunit interface

Putative protein-protein interactions