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 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 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

The Mechanism of Replication of Single-Stranded DNA. VI. Requirements for Ribonucleoside Triphosphates and DNA Polymerase III

1973 ◽  
Vol 51 (12) ◽  
pp. 1588-1597 ◽  
Author(s):  
David T. Denhardt ◽  
Makoto Iwaya ◽  
Grant McFadden ◽  
Gerald Schochetman
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase Iii ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii

Evidence is presented that in Escherichia coli made permeable to nucleotides by exposure to toluene, the synthesis of a DNA chain complementary to the infecting single-stranded DNA of bacteriophage [Formula: see text] requires ATP as well as the four deoxyribonucleoside triphosphates. This synthesis results in the formation of the parental double-stranded replicative-form (RF) molecule. The ATP is not required simply to prevent degradation of the ribonucleoside or deoxyribonucleoside triphosphates; it can be partially substituted for by other ribonucleoside triphosphates.No single one of the known E. coli DNA polymerases appears to be uniquely responsible in vivo for the formation of the parental RF. Since [Formula: see text] replicates well in strains lacking all, or almost all, of the in-vitro activities of DNA polymerases I and II, neither of these two enzymes would seem essential; and in a temperature-sensitive E. coli mutant (dnaEts) deficient in DNA polmerase-I activity and possessing a temperature-sensitive DNA polymerase III, the viral single-stranded DNA is efficiently incorporated into an RF molecule at the restrictive temperature. In contrast, both RF replication and progeny single-stranded DNA synthesis are dependent upon DNA polymerase III activity.

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.

Interaction of 5-Methoxymethyl-2′-Deoxyuridine Triphosphate with DNA Polymerases: Effects of the 5-Substituent and Comparison with the Deoxycytidine Derivative

1992 ◽  
Vol 3 (4) ◽  
pp. 243-247 ◽  
Author(s):  
P. J. Aduma ◽  
S. V. Gupta ◽  
A. L. Stuart
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Competitive Inhibitor ◽  
E Coli ◽  
The Difference ◽  
Simplex Virus ◽  
Selective Activity ◽  
Hsv 1

5-Methoxymethyl-2′-deoxyuridine (MMdUrd) is a selective anti-herpes agent that is dependent upon initial phosphorylation by Herpes simplex virus-induced deoxythymidine kinase. In order to determine its mechanism of action, MMdUrd was converted to the 5′-triphosphate (MMdUTP) and the nature of interaction of MMdUTP and dTTP with DNA polymerase of E. coli, HSV-1, and human α was investigated. The order of utilization of deoxyuridine analogues by bacterial and HSV-1 DNA polymerases for DNA synthesis was: dTTP > MMdUTP. In contrast, 5-methoxymethyl-2′-deoxycytidine-5′-triphosphate (MMdCTP) was a better substrate for HSV DNA polymerase compared to dCTP. MMdUTP is a competitive inhibitor of HSV-1 DNA polymerase with respect to dTTP incorporation (Ki = 2.9 × 10−6M). The IC50 values of MMdUTP for both HSV and human αDNA polymerases were 4.5 × 10 −6M. These data suggest that the selective activity of MMdUrd is due to its preferential phosphorylation by viral thymidine kinase and not at the DNA polymerase level. These results may also account for the difference in anti-HSV activity between MMdUrd and its deoxycytidine analogue.

scholarly journals Cloning and Characterization of Arylamine N -Acetyltransferase Genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: Increased Expression Results in Isoniazid Resistance

1999 ◽  
Vol 181 (4) ◽  
pp. 1343-1347 ◽  
Author(s):  
Mark Payton ◽  
Roy Auty ◽  
Rupika Delgoda ◽  
Martin Everett ◽  
Edith Sim
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Antitubercular Drug ◽  
Isoniazid Resistance ◽  
E Coli ◽  
Monospecific Antiserum ◽  
Many Sources ◽  
Eukaryotic Organisms

ABSTRACT Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned fromMycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli andM. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT fromM. smegmatis and cross-reacts with recombinant NAT fromM. tuberculosis. Overexpression of mycobacterialnat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatisas the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.

scholarly journals Pre-Steady-State Kinetic Analysis of the Incorporation of Anti-HIV Nucleotide Analogs Catalyzed by Human X- and Y-Family DNA Polymerases

2010 ◽  
Vol 55 (1) ◽  
pp. 276-283 ◽  
Author(s):  
Jessica A. Brown ◽  
Lindsey R. Pack ◽  
Jason D. Fowler ◽  
Zucai Suo
Keyword(s):  
Mitochondrial Dna ◽  
Steady State ◽  
Substrate Specificity ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Steady State Kinetic ◽  
Y Family ◽  
State Kinetic

ABSTRACTNucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human mitochondrial DNA polymerase has been established bothin vitroandin vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X- and Y-family DNA polymerases, which participate in DNA repair and DNA lesion bypassin vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active, 5′-phosphorylated forms of tenofovir, lamivudine, emtricitabine, and zidovudine. For the six enzymes, all of the drug analogs were incorporated less efficiently (40- to >110,000-fold) than the corresponding natural nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming nucleotide analog. In general, the 5′-triphosphate forms of lamivudine and zidovudine were better substrates than emtricitabine and tenofovir for the six human enzymes, although the substrate specificity profile depended on the DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X- and Y-family DNA polymerases is a potential mechanism of NRTI drug toxicity, and we have established a structure-function relationship for designing improved NRTIs.

scholarly journals Identification and Analysis of “Extended −10” Promoters from Mycobacteria

1998 ◽  
Vol 180 (9) ◽  
pp. 2568-2573 ◽  
Author(s):  
Murali D. Bashyam ◽  
Anil K. Tyagi
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Sigma Factor ◽  
Mycobacterium Smegmatis ◽  
Important Determinant ◽  
Comparative Assessment ◽  
Binding Domain ◽  
Site Specific ◽  
E Coli

ABSTRACT Earlier studies from our laboratory on randomly isolated transcriptional signals of mycobacteria had revealed that the −10 region of mycobacterial promoters and the corresponding binding domain in the major sigma factor are highly similar to their Escherichia coli counterparts. In contrast, the sequences in −35 regions of mycobacterial promoters and the corresponding binding domain in the major sigma factor are vastly different from their E. colicounterparts (M. D. Bashyam, D. Kaushal, S. K. Dasgupta, and A. K. Tyagi, J. Bacteriol. 178:4847–4853, 1996). We have now analyzed the role of the TGN motif present immediately upstream of the −10 region of mycobacterial promoters. Sequence analysis and site-specific mutagenesis of a Mycobacterium tuberculosispromoter and a Mycobacterium smegmatis promoter reveal that the TGN motif is an important determinant of transcriptional strength in mycobacteria. We show that mutation in the TGN motif can drastically reduce the transcriptional strength of a mycobacterial promoter. The influence of the TGN motif on transcriptional strength is also modulated by the sequences in the −35 region. Comparative assessment of these extended −10 promoters in mycobacteria and E. coli suggests that functioning of the TGN motif in promoters of these two species is similar.

scholarly journals Amino Acid Architecture That Influences dNTP Insertion Efficiency in Y-Family DNA Polymerase V of E. coli

2009 ◽  
Vol 392 (2) ◽  
pp. 270-282 ◽  
Author(s):  
Kwang Young Seo ◽  
Jun Yin ◽  
Prashant Donthamsetti ◽  
Sushil Chandani ◽  
Chui Hong Lee ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Polymerase ◽  
E Coli ◽  
Dna Polymerase V ◽  
Y Family

scholarly journals A polar filter in DNA polymerases prevents ribonucleotide incorporation

2019 ◽  
Vol 47 (20) ◽  
pp. 10693-10705 ◽  
Author(s):  
Mary K Johnson ◽  
Jithesh Kottur ◽  
Deepak T Nair
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Dna Polymerases ◽  
Drastic Reduction ◽  
Equivalent Residue ◽  
Stringent Selection ◽  
Selection For ◽  
Dna Polymerase Iv

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.

Sign in / Sign up

Export Citation Format

Share Document