scholarly journals The Spacious Active Site of a Y-Family DNA Polymerase Facilitates Promiscuous Nucleotide Incorporation Opposite a Bulky Carcinogen-DNA Adduct

2004 ◽  
Vol 279 (35) ◽  
pp. 36951-36961 ◽  
Author(s):  
Rebecca A. Perlow-Poehnelt ◽  
Ilya Likhterov ◽  
David A. Scicchitano ◽  
Nicholas E. Geacintov ◽  
Suse Broyde
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Dna Adduct ◽  
Nucleotide Incorporation ◽  
Y Family

scholarly journals DNA Polymerase and dRP-lyase activities of polymorphic variants of human Pol ι

2021 ◽  
Vol 478 (7) ◽  
pp. 1399-1412
Author(s):  
Evgeniy S. Shilkin ◽  
Anastasia S. Gromova ◽  
Margarita P. Smal ◽  
Alena V. Makarova
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Altered Expression ◽  
Dna Polymerase Iota ◽  
Nucleotide Incorporation ◽  
Lyase Activity ◽  
Polymorphic Variants ◽  
Y Family

Y-family DNA polymerase iota (Pol ι) is involved in DNA damage response and tolerance. Mutations and altered expression level of POLI gene are linked to a higher incidence of cancer. We biochemically characterized five active site polymorphic variants of human Pol ι: R71G (rs3218778), P118L (rs554252419), I236M (rs3218784), E251K (rs3218783) and P365R (rs200852409). We analyzed fidelity of nucleotide incorporation on undamaged DNA, efficiency and accuracy of DNA damage bypass, as well as 5′-deoxyribophosphate lyase (dRP-lyase) activity. The I236M and P118L variants were indistinguishable from the wild-type Pol ι in activity. The E251K and P365R substitutions altered the spectrum of nucleotide incorporation opposite several undamaged DNA bases. The P365R variant also reduced the dRP-lyase activity and possessed the decreased TLS activity opposite 8-oxo-G. The R71G mutation dramatically affected the catalytic activities of Pol ι. The reduced DNA polymerase activity of the R71G variant correlated with an enhanced fidelity of nucleotide incorporation on undamaged DNA, altered lesion-bypass activity and reduced dRP-lyase activity. Therefore, this amino acid substitution likely alters Pol ι functions in vivo.

scholarly journals Deoxynucleotide Triphosphate Binding Mode Conserved in Y Family DNA Polymerases

2003 ◽  
Vol 23 (8) ◽  
pp. 3008-3012 ◽  
Author(s):  
Robert E. Johnson ◽  
José Trincao ◽  
Aneel K. Aggarwal ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Dna Polymerase ◽  
Close Contact ◽  
Dna Polymerases ◽  
Binding Mode ◽  
Nucleotide Incorporation ◽  
The Family ◽  
And Function ◽  
High Degree ◽  
Y Family ◽  
Deoxynucleotide Triphosphate

ABSTRACT Although DNA polymerase η (Polη) and other Y family polymerases differ in sequence and function from classical DNA polymerases, they all share a similar right-handed architecture with the palm, fingers, and thumb domains. Here, we examine the role in Saccharomyces cerevisiae Polη of three conserved residues, tyrosine 64, arginine 67, and lysine 279, which come into close contact with the triphosphate moiety of the incoming nucleotide, in nucleotide incorporation. We find that mutational alteration of these residues reduces the efficiency of correct nucleotide incorporation very considerably. The high degree of conservation of these residues among the various Y family DNA polymerases suggests that these residues are also crucial for nucleotide incorporation in the other members of the family. Furthermore, we note that tyrosine 64 and arginine 67 are functionally equivalent to the deoxynucleotide triphosphate binding residues arginine 518 and histidine 506 in T7 DNA polymerase, respectively.

scholarly journals Human Y-Family DNA Polymerase κ Is More Tolerant to Changes in Its Active Site Loop than Its Ortholog Escherichia coli DinB

2017 ◽  
Vol 30 (11) ◽  
pp. 2002-2012 ◽  
Author(s):  
Nicole M. Antczak ◽  
Morgan R. Packer ◽  
Xueguang Lu ◽  
Ke Zhang ◽  
Penny J. Beuning
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Active Site ◽  
Y Family

scholarly journals MsDpo4—a DinB Homolog fromMycobacterium smegmatis—Is an Error-Prone DNA Polymerase That Can Promote G:T and T:G Mismatches

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Amit Sharma ◽  
Deepak T. Nair
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Polymerase ◽  
Environmental Conditions ◽  
Mycobacterium Smegmatis ◽  
Molecular Level ◽  
Dna Polymerases ◽  
Adaptive Mutagenesis ◽  
E Coli ◽  
Nucleotide Incorporation ◽  
Y Family

Error-prone DNA synthesis in prokaryotes imparts plasticity to the genome to allow for evolution in unfavorable environmental conditions, and this phenomenon is termed adaptive mutagenesis. At a molecular level, adaptive mutagenesis is mediated by upregulating the expression of specialized error-prone DNA polymerases that generally belong to the Y-family, such as the polypeptide product of thedinBgene in case ofE. coli. However, unlikeE. coli, it has been seen that expression of the homologs ofdinBinMycobacterium tuberculosisare not upregulated under conditions of stress. These studies suggest that DinB homologs inMycobacteriamight not be able to promote mismatches and participate in adaptive mutagenesis. We show that a representative homolog fromMycobacterium smegmatis(MsDpo4) can carry out template-dependent nucleotide incorporation and therefore is a DNA polymerase. In addition, it is seen that MsDpo4 is also capable of misincorporation with a significant ability to promote G:T and T:G mismatches. The frequency of misincorporation for these two mismatches is similar to that exhibited by archaeal and prokaryotic homologs. Overall, our data show that MsDpo4 has the capacity to facilitate transition mutations and can potentially impart plasticity to the genome.

scholarly journals Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase η

2003 ◽  
Vol 23 (14) ◽  
pp. 5107-5112 ◽  
Author(s):  
M. Todd Washington ◽  
Sandra A. Helquist ◽  
Eric T. Kool ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Hydrogen Bonding ◽  
Hydrogen Bonds ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Geometric Constraints ◽  
High Fidelity ◽  
Nucleotide Incorporation

ABSTRACT Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase η (Polη) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Polη are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Polη; in this regard, Polη differs strikingly from classical high-fidelity DNA polymerases.

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