Abstract
The molecular structure of DNA polymerase I or family A polymerases is made up of three major domains that consist of a single polymerase domain with two extra exonuclease domains. When the N-terminal was deleted, the enzyme was still able to perform basic polymerase activity with additional traits that used isothermal amplification. However, the 3′-5′ exonuclease domain that carries a proofreading activity was disabled. Yet, the structure remained attached to the 5′-3′ polymerization domain without affecting its ability. The purpose of this non-functional domain still remains scarce. It either gives negative effects or provides structural support to the DNA polymerase. Here, we compared the effect of deleting each domain against the polymerase activity. The recombinant wild type and its variants were successfully purified and characterized. Interestingly, SK72-Exo (a large fragment excluding the 5′-3′ exonuclease domain) exhibited better catalytic activity than the native SK72 (with all three domains) at similar optimum temperature and pH profile, and it showed longer stability at 70 °C. Meanwhile, SK72-Exo2 (polymerization domain without both the 5′-3′ and 3′-5′ exonuclease domain) displayed the lowest activity with an optimum at 40 °C and favored a more neutral environment. It was also the least stable among the variants, with almost no activity at 50 °C for the first 10 min. In conclusion, cutting both exonuclease domains in DNA polymerase I has a detrimental effect on the polymerization activity and structural stability.
Highlights
Deoxyribonucleic acid (DNA) polymerase plays a key role in maintaining the genetics information of an organism from one generation to another through the catalysis of double-stranded DNA [1].This enzyme plays a significant role in the DNA repair pathway to secure the integrity of genetic materials by proofreading and recorrecting any mismatched nucleotide [2,3]
DNA polymerase enzymes have been categorized into seven different families termed as A, B, C, D, X, Y, and RT according to their similarity in amino acid sequences as well as elucidated structure analyses [4,5]
The SK72 DNA polymerase I gene was encoded for 878 polypeptides with an approximate molecular weight Domain and isoelectric pointAnalyses of 100 kDa and 5.53, respectively
Summary
Deoxyribonucleic acid (DNA) polymerase plays a key role in maintaining the genetics information of an organism from one generation to another through the catalysis of double-stranded DNA [1]. This enzyme plays a significant role in the DNA repair pathway to secure the integrity of genetic materials by proofreading and recorrecting any mismatched nucleotide [2,3]. DNA polymerase enzymes have been categorized into seven different families termed as A, B, C, D, X, Y, and RT according to their similarity in amino acid sequences as well as elucidated structure analyses [4,5]. The 50 -30 exonuclease domain serves as filling for the gaps present in the Okazaki fragment at the lagging strand, and the 30 -50 exonuclease domain plays a role in DNA excision repair processes [9,10,11]
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