Nair, Deepak T and Johnson, Mary K and Kottur, Jithesh (2019) A polar filter in DNA polymerases prevents ribonucleotide incorporation. Nucleic Acids Research, 47 (20). pp. 10693-10705. ISSN 0305-1048

Text A Polar Filter in DNA Polymerases Prevents Ribonucleotide Incorporation.pdf Restricted to Repository staff only Download (6Mb)

Abstract

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. The dPol named MsDpo4 (Mycobacterium smegmatis) naturally lacks this steric filter and hence is capable of rNTP addition. 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 (PolIV) from Escherichia coli exhibited stringent selection for deoxyribonucleotides. A comparison of MsDpo4 and PolIV led to the discovery of an additional polar filter responsible for sugar selectivity. Thr43 represents the filter in PolIV and this residue forms interactions with the incoming nucleotide to draw 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. The substitution of the equivalent residue in MsDpo4-Cys47, with Thr led to a drastic reduction in the ability of the mycobacterial enzyme to incorporate rNTPs. Overall, our studies evince that the polar filter serves to prevent ribonucleotide incorporation by dPols.

Item Type:	Article
Subjects:	Biochemical and Biophysical Sciences
Depositing User:	RCB Library
Date Deposited:	25 Jun 2020 07:43
Last Modified:	25 Jun 2020 07:43
URI:	http://rcb.sciencecentral.in/id/eprint/266

Actions (login required)

View Item