Participation of Distal RRRY Motif in Strand Displacement DNA synthesis by A‐Family DNA Polymerases

Strand displacement synthesis of DNA (SDSD) is an essential activity during Okazaki fragment maturation process. We have previously shown that residues in fingers subdomain and RRRY motif of E.coli DNA polymerase I participates in SDSD and 3′ – 5′ exonuclease activity, respectively ( J. Biol. Chem. 2008, 283: 17979–17990). We further investigated the function of the conserved RRRY motif in the function of DNA polymerase I enzymes with or without associated 3′‐5′ exonuclease activity. Using DNA polymerase I from M. tuberculosis , which lacks 3′ – 5′ exonuclease activity and E.coli DNA polymerase I, which contains the 3′ – 5′ exonuclease activity, we now show that the RRRY motif is essential for SDSD. A detailed biochemical properties of individual arginine to alanine mutant enzymes showed that the first and the third arginine (R796 and R798) in M. tuberculosis polymerase I are essential for the SDSD, while, the 2 nd arginine (R797) and 4 th tyrosine (Y799) are not required. Similar properties were displayed by the mutant derivatives of equivalent residues in E.coli polymerase I. The loss of strand displacement DNA synthesis activity resulted from the significant reduction in the DNA binding affinity by mutant enzymes. Two most affected mutant enzyme species were further found to be defective in the formation of the enzyme‐DNA‐dNTP ternary complexes. We further find that displaced strand cleavage is mediated by 3′ – 5′ exonuclease activity of E.coli DNA polymerase I, in RRRY‐dependent manner. Our data, for the first time, demonstrate dual function of a conserved motif despite its distal location from the active sites. Support or Funding Information None