scholarly journals Accuracy, lesion bypass, strand displacement and translocation by DNA polymerases

2004 ◽  
Vol 359 (1441) ◽  
pp. 17-23 ◽  
Author(s):  
Thomas A. Steitz ◽  
Y. Whitney Yin
Keyword(s):  
Conformational Change ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Lesion Bypass ◽  
Dna Structures ◽  
Template Strand ◽  
Pol I ◽  
Deoxynucleoside Triphosphate ◽  
Copy Dna

The structures of DNA polymerases from different families show common features and significant differences that shed light on the ability of these enzymes to accurately copy DNA and translocate. The structure of a B family DNA polymerase from phage RB69 exhibits an active–site closing conformational change in the fingers domain upon forming a ternary complex with primer template in deoxynucleoside triphosphate. The rotation of the fingers domain α–helices by 60° upon dNTP binding is analogous to the changes seen in other families of polymerases. When the 3' terminus is bound to the editing 3' exonuclease active site, the orientation of the DNA helix axis changes by 40° and the thumb domain re–orients with the DNA. Structures of substrate and product complexes of T7 RNA polymerase, a structural homologue of T7 DNA polymerase, show that family polymerases use the rotation conformational change of the fingers domain to translocate down the DNA. The fingers opening rotation that results in translocation is powered by the release of the product pyrophosphate and also enables the Pol I family polymerases to function as a helicase in displacing the downstream non–template strand from the template strand.

scholarly journals Structure of an open conformation of T7 DNA Polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active-site

2021 ◽  
Author(s):  
Victor Juárez-Quintero ◽  
Antolin Peralta-Castro ◽  
Claudia G. G Benítez-Cardoza ◽  
Tom Ellenberger ◽  
Luis G Brieba
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Double Helix ◽  
Dna Polymerases ◽  
Structural Features ◽  
Modular Architecture ◽  
Template Strand ◽  
Helix Loop Helix ◽  
Exonuclease Domain ◽  
Dna Double Helix

The crystal structure of full-length T7 DNA polymerase in complex with its processivity factor thioredoxin and double-stranded DNA in the polymerization active site exhibits two novel structural motifs in family-A DNA polymerases: an extended b-hairpin at the fingers subdomain, that interacts with the DNA template strand downstream the primer-terminus, and a helix-loop-helix motif (insertion1) located between residues 102 to 122 in the exonuclease domain. The extended b-hairpin is involved in nucleotide incorporation on substrates with 5'-overhangs longer than 2 nucleotides, suggesting a role in stabilizing the template strand into the polymerization domain. Our biochemical data reveal that insertion1 of the exonuclease domain makes stabilizing interactions that facilitate proofreading by shuttling the primer strand into the exonuclease active site. Overall, our studies evidence conservation of the 3'-5' exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating b-hairpin guides the template-strand into the polymerization active site after the T7 primase-helicase unwinds the DNA double helix ameliorating the formation of secondary structures and decreasing the appearance of indels

scholarly journals The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 648
Author(s):  
del Prado ◽  
Santos ◽  
Lázaro ◽  
Salas ◽  
de Vega
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Structural Changes ◽  
Binding Capacity ◽  
Dna Polymerases ◽  
Crystallographic Structure ◽  
Genome Replication ◽  
Terminal Protein ◽  
Phi29 Dna Polymerase

Bacteriophage Phi29 DNA polymerase belongs to the protein-primed subgroup of family B DNA polymerases that use a terminal protein (TP) as a primer to initiate genome replication. The resolution of the crystallographic structure showed that it consists of an N-terminal domain with the exonuclease activity and a C-terminal polymerization domain. It also has two subdomains specific of the protein-primed DNA polymerases; the TP Regions 1 (TPR1) that interacts with TP and DNA, and 2 (TPR2), that couples both processivity and strand displacement to the enzyme. The superimposition of the structures of the apo polymerase and the polymerase in the polymerase/TP heterodimer shows that the structural changes are restricted almost to the TPR1 loop (residues 304–314). In order to study the role of this loop in binding the DNA and the TP, we changed the residues Arg306, Arg308, Phe309, Tyr310, and Lys311 into alanine, and also made the deletion mutant Δ6 lacking residues Arg306–Lys311. The results show a defective TP binding capacity in mutants R306A, F309A, Y310A, and Δ6. The additional impaired primer-terminus stabilization at the polymerization active site in mutants Y310A and Δ6 allows us to propose a role for the Phi29 DNA polymerase TPR1 loop in the proper positioning of the DNA and TP-priming 3’-OH termini at the preinsertion site of the polymerase to enable efficient initiation and further elongation steps during Phi29 TP-DNA replication.

scholarly journals Efficient and Error-Free Replication past a Minor-Groove N2-Guanine Adduct by the Sequential Action of Yeast Rev1 and DNA Polymerase ζ

2004 ◽  
Vol 24 (16) ◽  
pp. 6900-6906 ◽  
Author(s):  
M. Todd Washington ◽  
Irina G. Minko ◽  
Robert E. Johnson ◽  
Lajos Haracska ◽  
Thomas M. Harris ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Close Contact ◽  
Dna Polymerases ◽  
Minor Groove ◽  
Synthetic Activity ◽  
Oxidation Reactions ◽  
Lesion Bypass ◽  
Dna Minor Groove

ABSTRACT Rev1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase ζ (Polζ). Rev1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While Rev1 plays an indispensable structural role in Polζ-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, Rev1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N 2-adducted guanines in DNA. To test this idea, we examined whether Rev1 could incorporate a C opposite the γ-hydroxy-1,N 2-propano-2′deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N 2 of guanine. Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that Rev1 efficiently incorporates a C opposite this adduct from which Polζ subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the Rev1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N 2-guanine DNA minor-groove adducts that form in DNA.

scholarly journals Escherichia coli DNA Polymerase III Can Replicate Efficiently past a T-T cis-syn Cyclobutane Dimer if DNA Polymerase V and the 3′ to 5′ Exonuclease Proofreading Function Encoded by dnaQ Are Inactivated

2002 ◽  
Vol 184 (10) ◽  
pp. 2674-2681 ◽  
Author(s):  
Angela Borden ◽  
Paul I. O'Grady ◽  
Dominique Vandewiele ◽  
Antonio R. Fernández de Henestrosa ◽  
Christopher W. Lawrence ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Wild Type ◽  
Lesion Bypass ◽  
Base Pairs ◽  
Pol V ◽  
Pol I ◽  
Cyclobutane Dimer ◽  
Dna Polymerase V ◽  
Pol Iii

ABSTRACT Although very little replication past a T-T cis-syn cyclobutane dimer normally takes place in Escherichia coli in the absence of DNA polymerase V (Pol V), we previously observed as much as half of the wild-type bypass frequency in Pol V-deficient (ΔumuDC) strains if the 3′ to 5′ exonuclease proofreading activity of the Pol III ε subunit was also disabled by mutD5. This observation might be explained in at least two ways. In the absence of Pol V, wild-type Pol III might bind preferentially to the blocked primer terminus but be incapable of bypass, whereas the proofreading-deficient enzyme might dissociate more readily, providing access to bypass polymerases. Alternatively, even though wild-type Pol III is generally regarded as being incapable of lesion bypass, proofreading-impaired Pol III might itself perform this function. We have investigated this issue by examining dimer bypass frequencies in ΔumuDC mutD5 strains that were also deficient for Pol I, Pol II, and Pol IV, both singly and in all combinations. Dimer bypass frequencies were not decreased in any of these strains and indeed in some were increased to levels approaching those found in strains containing Pol V. Efficient dimer bypass was, however, entirely dependent on the proofreading deficiency imparted by mutD5, indicating the surprising conclusion that bypass was probably performed by the mutD5 Pol III enzyme itself. This mutant polymerase does not replicate past the much more distorted T-T (6-4) photoadduct, however, suggesting that it may only replicate past lesions, like the T-T dimer, that form base pairs normally.

Incorporation of Selected Nucleoside Phosphonates and Anti-Human Immunodeficiency Virus Nucleotide Analogues into DNA by Human DNA Polymerases α, β and γ

1997 ◽  
Vol 8 (3) ◽  
pp. 187-195 ◽  
Author(s):  
T Cihlar ◽  
MS Chen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Immunodeficiency Virus ◽  
Deoxynucleoside Triphosphate ◽  
Template Dna ◽  
Dna Polymerase Γ ◽  
Incorporation Efficiency

Incorporation of selected diphosphates of nucleoside phosphonates and triphosphates of currently approved anti-human immunodeficiency virus nucleoside analogues into DNA by human DNA polymerases α, β and γ was studied. All three polymerases were able to incorporate diphosphates of 9-(2-phosphonomethoxyethyl)adenine (PMEApp), 9-(2-phosphonomethoxyethyl)guanine (PMEGpp), ( R)-9-(2-phosphonomethoxypropyl)adenine (PMPApp), ( R)-9-(2-phosphononomethoxypropyl)-2,6-diaminopurine (PMPDAPpp) and ( 2R,5R)-9-[2,5-dihydro-5-(phosphonomethoxy)-2-furanyl]adenine (D4APpp) into primer/template DNA of defined sequence. After incorporation, these nucleoside phosphonates acted as terminators of primer extension. Kinetic constants of their incorporation were determined and compared with those for incorporation of ddATP, ddCTP, (-)-2′-deoxy-3′-thiacytidine triphosphate (3TC-TP), 2′,3′-didehydro-3′-deoxythymidine triphosphate (d4T-TP) and 3′-azido-3′-deoxythymidine triphosphate (AZT-TP). Relative efficiencies of incorporation (percentage of the incorporation efficiency for the corresponding natural deoxynucleoside triphosphate) by DNA polymerase a ranged from 0.05% for 3TC-TP to 51% for PMEGpp. DNA polymerase β catalysed the incorporation with relative efficiencies ranging from 0.014% for AZT-TP to 125% for ddCTP, and efficiencies of incorporation by DNA polymerase γ varied between 0.13% for 3TC-TP and 25% for ddCTP. Generally, the lowest incorporation efficiencies with all three polymerases were found for PMPApp (0.06–1.4%) and PMPDAPpp (0.075–2.2%).

scholarly journals Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site

1991 ◽  
Vol 11 (9) ◽  
pp. 4786-4795
Author(s):  
J S Gibbs ◽  
K Weisshart ◽  
P Digard ◽  
A deBruynKops ◽  
D M Knipe ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Cell Nuclei ◽  
Dna Polymerase I ◽  
Viral Dna ◽  
Gene Encoding ◽  
Simplex Virus ◽  
Polymerase I

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.

scholarly journals Mammalian DNA Polymerase Kappa Activity and Specificity

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2805 ◽  
Author(s):  
Hannah R. Stern ◽  
Jana Sefcikova ◽  
Victoria E. Chaparro ◽  
Penny J. Beuning
Keyword(s):  
Dna Polymerase ◽  
Genome Instability ◽  
Fragile Sites ◽  
Chemotherapy Response ◽  
Nucleotide Polymorphisms ◽  
Lesion Bypass ◽  
Base Pairs ◽  
Domains Of Life ◽  
Copy Dna ◽  
Y Family

DNA polymerase (pol) kappa is a Y-family translesion DNA polymerase conserved throughout all domains of life. Pol kappa is special6 ized for the ability to copy DNA containing minor groove DNA adducts, especially N2-dG adducts, as well as to extend primer termini containing DNA damage or mismatched base pairs. Pol kappa generally cannot copy DNA containing major groove modifications or UV-induced photoproducts. Pol kappa can also copy structured or non-B-form DNA, such as microsatellite DNA, common fragile sites, and DNA containing G quadruplexes. Thus, pol kappa has roles both in maintaining and compromising genomic integrity. The expression of pol kappa is altered in several different cancer types, which can lead to genome instability. In addition, many cancer-associated single-nucleotide polymorphisms have been reported in the POLK gene, some of which are associated with poor survival and altered chemotherapy response. Because of this, identifying inhibitors of pol kappa is an active area of research. This review will address these activities of pol kappa, with a focus on lesion bypass and cellular mutagenesis.

scholarly journals Two Family B DNA Polymerases from Aeropyrum pernix, an Aerobic Hyperthermophilic Crenarchaeote

1999 ◽  
Vol 181 (19) ◽  
pp. 5984-5992 ◽  
Author(s):  
Isaac K. O. Cann ◽  
Sonoko Ishino ◽  
Norimichi Nomura ◽  
Yoshihiko Sako ◽  
Yoshizumi Ishino
Keyword(s):  
Amino Acid ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Amino Acid Sequences ◽  
Pol Ii ◽  
Aeropyrum Pernix ◽  
Heat Stable ◽  
Pol I ◽  
Group I ◽  
Fraction I

ABSTRACT DNA polymerase activities in fractionated cell extract ofAeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3′→5′ exonuclease and polymerase activities. The product ofpolA gene (Pol I) was aphidicolin resistant and heat stable up to 80°C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95°C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II ofPyrodictium occultum. The deduced amino acid sequence ofA. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomainCrenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.

scholarly journals Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site.

1991 ◽  
Vol 11 (9) ◽  
pp. 4786-4795 ◽  
Author(s):  
J S Gibbs ◽  
K Weisshart ◽  
P Digard ◽  
A deBruynKops ◽  
D M Knipe ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Cell Nuclei ◽  
Dna Polymerase I ◽  
Viral Dna ◽  
Gene Encoding ◽  
Simplex Virus ◽  
Polymerase I

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.

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.

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