scholarly journals Evidence for a Watson-Crick Hydrogen Bonding Requirement in DNA Synthesis by Human DNA Polymerase κ

2005 ◽  
Vol 25 (16) ◽  
pp. 7137-7143 ◽  
Author(s):  
William T. Wolfle ◽  
M. Todd Washington ◽  
Eric T. Kool ◽  
Thomas E. Spratt ◽  
Sandra A. Helquist ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Dna Synthesis ◽  
Active Site ◽  
Base Pair ◽  
High Efficiency ◽  
Dna Lesions ◽  
Geometric Constraints ◽  
Polymerization Reaction ◽  
Nucleotide Incorporation ◽  
Low Efficiency

ABSTRACT The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Polη, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polκ, which differs from Polη in having a higher fidelity and which, unlike Polη, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Polη. We find that an isosteric thymidine analog is replicated with low efficiency by Polκ, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Polη and Polκ rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.

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.

scholarly journals A Single Domain in Human DNA Polymerase ι Mediates Interaction with PCNA: Implications for Translesion DNA Synthesis

2005 ◽  
Vol 25 (3) ◽  
pp. 1183-1190 ◽  
Author(s):  
Lajos Haracska ◽  
Narottam Acharya ◽  
Ildiko Unk ◽  
Robert E. Johnson ◽  
Jerard Hurwitz ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Tight Binding ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Binding Motifs ◽  
Translesion Dna Synthesis ◽  
Nucleotide Incorporation ◽  
Y Family ◽  
Stalled Replication Forks ◽  
Proliferating Cell

ABSTRACT DNA polymerases (Pols) of the Y family rescue stalled replication forks by promoting replication through DNA lesions. Humans have four Y family Pols, η, ι, κ, and Rev1, of which Pols η, ι, and κ have been shown to physically interact with proliferating cell nuclear antigen (PCNA) and be functionally stimulated by it. However, in sharp contrast to the large increase in processivity that PCNA binding imparts to the replicative Pol, Polδ, the processivity of Y family Pols is not enhanced upon PCNA binding. Instead, PCNA binding improves the efficiency of nucleotide incorporation via a reduction in the apparent Km for the nucleotide. Here we show that Polι interacts with PCNA via only one of its conserved PCNA binding motifs, regardless of whether PCNA is bound to DNA or not. The mode of PCNA binding by Polι is quite unlike that in Polδ, where multisite interactions with PCNA provide for a very tight binding of the replicating Pol with PCNA. We discuss the implications of these observations for the accuracy of DNA synthesis during translesion synthesis and for the process of Pol exchange at the lesion site.

scholarly journals Stimulation of DNA Synthesis Activity of Human DNA Polymerase κ by PCNA

2002 ◽  
Vol 22 (3) ◽  
pp. 784-791 ◽  
Author(s):  
Lajos Haracska ◽  
Ildiko Unk ◽  
Robert E. Johnson ◽  
Barbara B. Phillips ◽  
Jerard Hurwitz ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Protein A ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Physical Interaction ◽  
Abasic Site ◽  
Nucleotide Incorporation ◽  
Synthesis Activity ◽  
Factor C

ABSTRACT Humans have three DNA polymerases, Polη, Polκ, and Polι, which are able to promote replication through DNA lesions. However, the mechanism by which these DNA polymerases are targeted to the replication machinery stalled at a lesion site has remained unknown. Here, we provide evidence for the physical interaction of human Polκ (hPolκ) with proliferating cell nuclear antigen (PCNA) and show that PCNA, replication factor C (RFC), and replication protein A (RPA) act cooperatively to stimulate the DNA synthesis activity of hPolκ. The processivity of hPolκ, however, is not significantly increased in the presence of these protein factors. The efficiency (V max/K m ) of correct nucleotide incorporation by hPolκ is enhanced ∼50- to 200-fold in the presence of PCNA, RFC, and RPA, and this increase in efficiency is achieved by a reduction in the apparent K m for the nucleotide. Although in the presence of these protein factors, the efficiency of the insertion of an A nucleotide opposite an abasic site is increased ∼40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPolκ would bypass an abasic site by inserting a nucleotide opposite the site.

scholarly journals Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Olga Rechkoblit ◽  
Robert E. Johnson ◽  
Yogesh K. Gupta ◽  
Louise Prakash ◽  
Satya Prakash ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Thermodynamic Stability ◽  
Active Site ◽  
Base Pair ◽  
Translesion Synthesis ◽  
Human Cells ◽  
Structural Basis ◽  
Human Dna ◽  
Dna Template

AbstractPrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3′−terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3′-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.

scholarly journals Role of Hoogsteen Edge Hydrogen Bonding at Template Purines in Nucleotide Incorporation by Human DNA Polymerase ι

2006 ◽  
Vol 26 (17) ◽  
pp. 6435-6441 ◽  
Author(s):  
Robert E. Johnson ◽  
Lajos Haracska ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Hydrogen Bonding ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Human Dna ◽  
Nucleotide Incorporation ◽  
Template Specificity ◽  
Hydrogen Bonding Potential ◽  
Dna Polymerase Ι

ABSTRACT Human DNA polymerase ι (Pol ι) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol ι with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol ι active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N6 of an A or the O6 of a G, here we provide evidence that whereas hydrogen bonding at N6 is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O6 is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O6 and N7 hydrogen bonding to DNA synthesis by Pol ι, we have examined its proficiency for replicating through the 6 O-methyl guanine and 8-oxoguanine lesions, which affect the O6 and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O6 is an imperative. The dispensability of N6 hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol ι with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N6 positions of templating A.

scholarly journals A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol

2020 ◽  
Author(s):  
Kazutoshi Kasho ◽  
Gorazd Stojkovič ◽  
Cristina Velázquez-Ruiz ◽  
Maria Isabel Martínez-Jiménez ◽  
Timothée Laurent ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Dna Lesions ◽  
Replication Forks ◽  
Flexible Loop ◽  
Nucleotide Incorporation ◽  
Dntp Binding ◽  
Incorporation Efficiency

ABSTRACTReplication forks often stall at damaged DNA. Resumption of DNA synthesis can occur by replacement of the replicative DNA polymerase with specialized, error-prone translesion DNA polymerases (TLS), that have higher tolerance for damaged substrates. Several of these polymerases (Polλ, Polη and PrimPol) are stimulated in DNA synthesis through interaction with PolDIP2, however the mechanism of this PolDIP2-dependent stimulation is still unclear. Here we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol’s enhanced processivity. PolDIP2 increases PrimPol’s primer-template and dNTP binding affinity, which concomitantly enhances PrimPol’s nucleotide incorporation efficiency. This activity is dependent on a unique arginine cluster in PolDIP2 and could be essential for PrimPol to function in vivo, since the polymerase activity of PrimPol alone is very limited. This mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, could be common to all other PolDIP2-interacting TLS polymerases, i.e. Polλ, Polη, Polζ and REV1, and might be critical for their in vivo function of tolerating DNA lesions at physiological nucleotide concentrations.

scholarly journals MARKER DISCRIMINATION AND MUTAGEN-INDUCED ALTERATIONS IN PNEUMOCOCCAL TRANSFORMATION

Genetics ◽  
1974 ◽  
Vol 77 (3) ◽  
pp. 449-458
Author(s):  
Jean-Gérard Tiraby ◽  
Maurice S Fox
Keyword(s):  
Biological Activity ◽  
Base Pair ◽  
Fusidic Acid ◽  
Nitrous Acid ◽  
High Efficiency ◽  
Chemical Modifications ◽  
Substantial Fraction ◽  
Resistant Mutants ◽  
Low Efficiency

ABSTRACT Nitrous acid-induced and hydroxylamine-induced chemical alterations in transforming DNA result in the loss of biological activity and in mutagenesis. The function responsible for the discrimination between high efficiency and low efficiency markers in pneumococcal transformation, and for the elimination of a substantial fraction of spontaneously occurring mutational events, does not appear to act on integrated DNA carrying these chemical alterations. The chemical modifications result in mutations that are evident among bacteria transformed with the treated DNA. Fusidic acid-resistant mutants isolated in this way have been shown to be predominantly of the low efficiency class. We have previously reported that most mutations of spontaneous origin occurring in this locus are of the high efficiency class, and have suggested that discrimination results from the elimination of specific classes of base pair mismatches that occur as intermediates in transformation. It would appear that the base pair mismatches most effectively eliminated in the discriminating strain of pneumococcus are those of the A:C and G:T type and that the immediate product of transformation with mutagenized DNA involves intermediates that are not recognized as A:C or G:T by the discrimination system.

scholarly journals Non-bulky Lesions in Human DNA: The Ways of Formation, Repair, and Replication

Acta Naturae ◽  
2017 ◽  
Vol 9 (3) ◽  
pp. 12-26 ◽  
Author(s):  
А. V. Ignatov ◽  
K. A. Bondarenko ◽  
A. V. Makarova
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
High Efficiency ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Translesion Dna Synthesis ◽  
Human Dna ◽  
Mechanisms Of Formation ◽  
Bulky Dna Lesions

DNA damage is a major cause of replication interruption, mutations, and cell death. DNA damage is removed by several types of repair processes. The involvement of specialized DNA polymerases in replication provides an important mechanism that helps tolerate persistent DNA damage. Specialized DNA polymerases incorporate nucleotides opposite lesions with high efficiency but demonstrate low accuracy of DNA synthesis. In this review, we summarize the types and mechanisms of formation and repair of non-bulky DNA lesions, and we provide an overview of the role of specialized DNA polymerases in translesion DNA synthesis.

scholarly journals A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol

2021 ◽  
Author(s):  
Kazutoshi Kasho ◽  
Gorazd Stojkovič ◽  
Cristina Velázquez-Ruiz ◽  
Maria Isabel Martínez-Jiménez ◽  
Mara Doimo ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Mammalian Cells ◽  
Polymerase Activity ◽  
Dna Lesions ◽  
Binding Affinities ◽  
Dna Lesion ◽  
Nucleotide Incorporation ◽  
Timely Fashion ◽  
Dntp Binding

Abstract Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.

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