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

Wolfle, William T.; Washington, M. Todd; Kool, Eric T.; Spratt, Thomas E.; Helquist, Sandra A.; Prakash, Louise; Prakash, Satya

doi:10.1128/mcb.25.16.7137-7143.2005

Cited by 54 publications

(69 citation statements)

References 49 publications

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“…Moreover, there is a significant asymmetry in the efficiency of the extension reaction, an ϳ30-fold reduction for insertion of dATP opposite template DFT and ϳ900-fold reduction for insertion of d(DFT)TP opposite template A (relative to the corresponding processes with T and dTTP, respectively (5)). The latter loss is comparable with the effects on insertion reactions involving the DFT analog seen with Y-class polymerases (5,(7)(8)(9). Together, these observations appear inconsistent with the conclusion that A-and Y-class polymerases use different mechanisms of replication and that the former may rely chiefly on shape for efficient and correct nucleotide insertion (1,2).…”

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confidence: 44%

“…Conversely, their more open active sites may render Y-class polymerases (6 -10) less sensitive to changes in base pair dimensions but more dependent on formation of hydrogen bonds between nucleotide pairs at the replicative position (6 -9). A-class DNA polymerases whose activities were assessed using apolar T analogs to date include Escherichia coli pol I (3)(4)(5) and the polymerase from phage T7 (6), and the tested Y-class DNA polymerases consisting of yeast pol (7), human pol (8), and the Dbh (DinB homolog (5)) and Dpo4 (9) polymerases from Sulfolobus acidocaldarius and Sulfolobus solfataricus, respectively.…”

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confidence: 99%

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Structure and Activity of Y-class DNA Polymerase DPO4 from Sulfolobus solfataricus with Templates Containing the Hydrophobic Thymine Analog 2,4-Difluorotoluene

Irimia

Eoff

Pallan

et al. 2007

Journal of Biological Chemistry

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The 2,4-difluorotoluene (DFT) analog of thymine has been used extensively to probe the relative importance of shape and hydrogen bonding for correct nucleotide insertion by DNA polymerases. As far as high fidelity (A-class) polymerases are concerned, shape is considered by some as key to incorporation of A(T) opposite T(A) and G(C) opposite C(G). We have carried out a detailed kinetic analysis of in vitro primer extension opposite DFT-containing templates by the trans-lesion (Y-class) DNA polymerase Dpo4 from Sulfolobus solfataricus. Although full-length product formation was observed, steady-state kinetic data show that dATP insertion opposite DFT is greatly inhibited relative to insertion opposite T (ϳ5,000-fold). No products were observed in the pre-steady-state. Furthermore, it is noteworthy that Dpo4 strongly prefers dATP opposite DFT over dGTP (ϳ200-fold) and that the polymerase is able to extend an A:DFT but not a G:DFT pair. We present crystal structures of Dpo4 in complex with DNA duplexes containing the DFT analog, the first for any DNA polymerase. In the structures, template-DFT is either positioned opposite primer-A or -G at the ؊1 site or is unopposed by a primer base and followed by a dGTP:A mismatch pair at the active site, representative of a ؊1 frameshift. The three structures provide insight into the discrimination by Dpo4 between dATP and dGTP opposite DFT and its inability to extend beyond a G:DFT pair. Although hydrogen bonding is clearly important for error-free replication by this Y-class DNA polymerase, our work demonstrates that Dpo4 also relies on shape and electrostatics to distinguish between correct and incorrect incoming nucleotide.Recent research on in vitro primer extension reactions catalyzed by a range of DNA polymerases and using the hydrophobic T isostere 2,4-difluorotoluene (DFT) 3 (Fig. 1) and other analogs with substituents of increasing size at the 2-and 4-positions of the aromatic moiety appear to support different mechanisms of nucleotide insertion by high fidelity (A-class) and trans-lesion (Y-class) DNA polymerases (reviewed in Refs. 1 and 2). Thus, accurate replication by A-class polymerases may be more dependent on a close steric match between the active site and the shape of a Watson-Crick base pair ("active site tightness") than on the formation of complementary hydrogen bonds between incoming nucleotide base and template residue (3-6). Conversely, their more open active sites may render Y-class polymerases (6 -10) less sensitive to changes in base pair dimensions but more dependent on formation of hydrogen bonds between nucleotide pairs at the replicative position (6 -9). A-class DNA polymerases whose activities were assessed using apolar T analogs to date include Escherichia coli pol I (3-5) and the polymerase from phage T7 (6), and the tested Y-class DNA polymerases consisting of yeast pol (7), human pol (8), and the Dbh (DinB homolog (5)) and Dpo4 (9) polymerases from Sulfolobus acidocaldarius and Sulfolobus solfataricus, respectively.Careful review of the accu...

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contrasting

confidence: 44%

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confidence: 99%

Structure and Activity of Y-class DNA Polymerase DPO4 from Sulfolobus solfataricus with Templates Containing the Hydrophobic Thymine Analog 2,4-Difluorotoluene

Irimia

Eoff

Pallan

et al. 2007

Journal of Biological Chemistry

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“…A crystal structure of the HIV-1 RT-DNA-PMPA-DP ternary complex revealed that PMPA-DP forms a noncanonical Watson-Crick base pair (51). Thus, this suboptimal base-pairing scheme for PMPA-DP and dT may contribute to the lower catalytic efficiency for the human enzymes, since hydrogen bonding has been shown to be important for incorporation catalyzed by Pol ␥ (27), Pol (7), Saccharomyces cerevisiae Pol (54), Pol (56), and Rev1 (6). Overall, PMPA-DP appears to be a superior drug analog because it requires only two phosphorylation events to become activated, it is a better substrate for HIV-1 RT than the seven human enzymes examined thus far (Table 3) (23,50), and it has a favorable resistance profile characterized by activity against most NRTI-resistant viruses and a low propensity for resistance development (30,34).…”

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confidence: 99%

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

Brown

Pack

Fowler

et al. 2011

Antimicrob Agents Chemother

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Nucleoside 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 both in vitro and in 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 bypass in vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases ␤, , , , , 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.More than 30 million people worldwide are infected with the human immunodeficiency virus (HIV), which is the causative agent of AIDS. To manage the life-threatening effects of HIV replication, nucleoside reverse transcriptase inhibitors (NRTIs) have been a mainstay in effective combination antiretroviral therapy. NRTIs undergo phosphorylation by host cell kinases to be converted into their active di-or triphosphate (DP or TP) forms, which can serve as nucleotide substrates for HIV reverse transcriptase (RT). Incorporation of the NRTIs into the viral genome by HIV RT terminates the replication process due to the lack of a 3Ј-hydroxyl in these drugs. Unfortunately, host DNA polymerases (Pols), organized into the A, B, X, and Y families, are susceptible to drug inhibition, because these enzymes catalyze a nucleotidyl transfer reaction similar to that of HIV RT. Incorporation of the drug analog into human DNA will inhibit DNA replication and possibly lead to cell death and drug toxicity. A correlation has been established between the kinetics of nucleotide analog incorporation catalyzed by human DNA Pol ␥, an A-family member, and the observed clinical toxicity that presents as mitochondrial dysfunction (12,13,23,26). However, some drug toxicity occurs via Pol ␥-independent mechanisms (36, 52), such as the bone marrow toxicity as...

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“…These results can be partially explained by base-pairing modes and the structures in the active site of each polymerase. For pols and , which require Watson-Crick base pairing for efficient catalysis (50,51), dCTP is more efficiently incorporated opposite N 2 -G adducts than O 6 -G adducts, because optimal Watson-Crick hydrogen bonding is possible only with N 2 -G adducts but not with O 6 -G adducts. This view would be consistent with recent reports that the S. solfataricus Y family DNA polymerase Dpo4 forms a wobble base pair between C and O 6 -MeG (or O 6 -BzG), and thus the polymerization is inhibited (52,53 may uniquely utilize an Arg for the optimal hydrogen bonding with an incoming dCTP, as shown with yeast Rev1 (34).…”

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confidence: 99%

Kinetic Analysis of Translesion Synthesis Opposite Bulky N2- and O6-Alkylguanine DNA Adducts by Human DNA Polymerase REV1

Choi

Guengerich

2008

Journal of Biological Chemistry

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REV1, a Y family DNA polymerase (pol), is involved in replicative bypass past DNA lesions, so-called translesion DNA synthesis. In addition to a structural role as a scaffold protein, REV1 has been proposed to play a catalytic role as a dCTP transferase in translesion DNA synthesis past abasic and guanine lesions in eukaryotes. To better understand the catalytic function of REV1 in guanine lesion bypass, purified recombinant human REV1 was studied with two series of guanine lesions, N 2 -alkylG adducts (in oligonucleotides) ranging in size from methyl (Me) to CH 2 (6-benzo[a]pyrenyl) (BP) and O 6 -alkylG adducts ranging from Me to 4-oxo-4-(3-pyridyl)butyl (Pob). REV1 readily produced 1-base incorporation opposite G and all G adducts except for O 6 -PobG, which caused almost complete blockage. Steadystate kinetic parameters (k cat /K m ) were similar for insertion of dCTP opposite G and N 2 -G adducts but were severely reduced opposite the O 6 -G adducts. REV1 showed apparent pre-steadystate burst kinetics for dCTP incorporation only opposite N 2 -BPG and little, if any, opposite G, N 2 -benzyl (Bz)G, or O 6 -BzG. The maximal polymerization rate (k pol 0.9 s ؊1 ) opposite N 2 -BPG was almost the same as opposite G, with only slightly decreased binding affinity to dCTP (2.5-fold). REV1 bound N 2 -BPG-adducted DNA 3-fold more tightly than unmodified G-containing DNA. These results and the lack of an elemental effect ((S p )-2-deoxycytidine 5-O-(1-thiotriphosphate)) suggest that the late steps after product formation (possibly product release) become rate-limiting in catalysis opposite N 2 -BPG. We conclude that human REV1, apparently the slowest Y family polymerase, is kinetically highly tolerant to N 2 -adduct at G but not to O 6 -adducts.Cellular DNA is continuously attacked by various endogenous and exogenous agents. Although the resulting lesions can be removed by versatile cellular repair systems, many DNA lesions escape repair and are usually present in replicating DNA. Facing DNA lesions during DNA replication, DNA polymerases often show unusual behavior, such as misinsertion, slippage, and blockage, which can give rise to mutations or cell death (1). Therefore, the characterization of interaction of DNA polymerases with DNA lesions is crucial for understanding the mechanism of mutagenesis in cells in detail (2). Human cells possess at least 15 different DNA polymerases, the physiological functions of most of which are still unclear. Replicative DNA polymerases, such as pol 2 ␣, ␦, and ⑀, are intolerant of DNA distortions caused by many DNA lesions and thus are blocked (3). As a tolerance mechanism to this replication blockade, cells utilize the specialized translesion synthesis (TLS) DNA polymerases, which have a spacious active site to replicate past replication fork-blocking lesions (4). Many of human TLS DNA polymerases belong to the recently discovered Y family, including pol , pol , pol , and REV1 (5). Y family members often have different properties of bypass ability and fidelity opposite various DNA ...

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

Cited by 54 publications

References 49 publications

Structure and Activity of Y-class DNA Polymerase DPO4 from Sulfolobus solfataricus with Templates Containing the Hydrophobic Thymine Analog 2,4-Difluorotoluene

Structure and Activity of Y-class DNA Polymerase DPO4 from Sulfolobus solfataricus with Templates Containing the Hydrophobic Thymine Analog 2,4-Difluorotoluene

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

Kinetic Analysis of Translesion Synthesis Opposite Bulky N2- and O6-Alkylguanine DNA Adducts by Human DNA Polymerase REV1

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