The design, construction, overexpression, and purification of a Klenow sub-fragment lacking the 30–50 exonuclease domain is presented here. In particular, a synthetic gene coding for the residues 515–928 of Escherichia coli DNA polymerase I was constructed. To improve the solubility and stability of the corresponding protein, the synthetic gene was designed to contain 11 site-specific substitutions. The gene was inserted into the pBADHis expression vector, generating 2 identical Klenow sub-fragments, bearing or not a hexahistidine tag. Both these Klenow sub-fragments, denominated HoLaMa and HoLaMaHis, were purified, and their catalytic properties were compared to those of Klenow enzyme. When DNA polymerase activity was assayed under processive conditions, the Klenow enzyme performed much better than HoLaMa and HoLaMaHis. However, when DNA polymerase activity was assayed under distributive conditions, the initial velocity of the reaction catalyzed by HoLaMa was comparable to that observed in the presence of Klenow enzyme. In particular, under distributive conditions HoLaMa was found to strongly prefer dsDNAs bearing a short template overhang, to the length of which the Klenow enzyme was relatively insensitive. Overall, our observations indicate that the exonuclease domain of the Klenow enzyme, besides its proofreading activity, does significantly contribute to the catalytic efficiency of DNA elongation.
Martina, C.E., Lapenta, F., Monton Silva, A., Hochkoeppler, A. (2015). HoLaMa: A Klenow sub-fragment lacking the 3′-5′ exonuclease domain. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 575, 46-53 [10.1016/j.abb.2015.04.003].
HoLaMa: A Klenow sub-fragment lacking the 3′-5′ exonuclease domain
LAPENTA, FABIO;MONTÒN SILVA, ALEJANDRO;HOCHKOEPPLER, ALEJANDRO
2015
Abstract
The design, construction, overexpression, and purification of a Klenow sub-fragment lacking the 30–50 exonuclease domain is presented here. In particular, a synthetic gene coding for the residues 515–928 of Escherichia coli DNA polymerase I was constructed. To improve the solubility and stability of the corresponding protein, the synthetic gene was designed to contain 11 site-specific substitutions. The gene was inserted into the pBADHis expression vector, generating 2 identical Klenow sub-fragments, bearing or not a hexahistidine tag. Both these Klenow sub-fragments, denominated HoLaMa and HoLaMaHis, were purified, and their catalytic properties were compared to those of Klenow enzyme. When DNA polymerase activity was assayed under processive conditions, the Klenow enzyme performed much better than HoLaMa and HoLaMaHis. However, when DNA polymerase activity was assayed under distributive conditions, the initial velocity of the reaction catalyzed by HoLaMa was comparable to that observed in the presence of Klenow enzyme. In particular, under distributive conditions HoLaMa was found to strongly prefer dsDNAs bearing a short template overhang, to the length of which the Klenow enzyme was relatively insensitive. Overall, our observations indicate that the exonuclease domain of the Klenow enzyme, besides its proofreading activity, does significantly contribute to the catalytic efficiency of DNA elongation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
