Error-prone Translesion Synthesis Past DNA-Peptide Cross-links Conjugated to the Major Groove of DNA via C5 of Thymidine

Wickramaratne, Susith; Boldry, Emily J.; Buehler, Charles J.; Wang, Yen Chih; Distefano, Mark D.; Tretyakova, Natalia

doi:10.1074/jbc.m114.613638

Cited by 34 publications

(49 citation statements)

References 73 publications

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“…Higher catalytic efficiency of hPol than hPol is consistent with previous reports for other bulky nucleobase lesions such as exocyclic adducts (35,(71)(72)(73) and DNA-glutathione conjugates (74). Unlike C5-thymine-peptide lesions, which induce high numbers of frameshift and base substitution mutations upon primer extension in the presence of hPol and hPol (36), C7-G conjugated DPCs were not miscoding. Our gel electrophoresis and mass spectrometry experiments have shown that replication bypass of the 10-mer peptide cross-linked to C7-G by hPol was essentially error-free, whereas hPol incorporated the correct base (dC) with 500-fold preference over the mispair (dT) ( Table 2 and Figs.…”

Section: 64supporting

confidence: 78%

“…DPCs had been hypothesized to completely block DNA polymerases, leading to stalling of replication forks (43,56). In our earlier study, we found that model DPCs generated by copper-catalyzed [3 ϩ 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides completely blocked DNA replication, whereas the corresponding 10-mer peptide conjugate was bypassed in an errorprone manner (29,36). In these model DPCs, peptides were conjugated to the C5 position of thymidine via a six-carbon linker (29).…”

Section: Discussionmentioning

confidence: 99%

“…Capillary HPLC-ESI Ϫ -MS/MS analyses were conducted as described previously (36). An Agilent 1100 HPLC system (Agilent Technologies, Wilmington, DE) coupled to a Thermo LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA) was operated in the negative ion ESI-FTMS/MS mode.…”

Section: Methodsmentioning

confidence: 99%

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Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

Wickramaratne

Mukherjee

et al. 2016

Journal of Biological Chemistry

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DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases and . DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50 -100-fold lower than for native G. Both human polymerase (hPol) and hPol inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol . Molecular dynamics simulation of an hPol ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.

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Section: 64supporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

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Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

Wickramaratne

Mukherjee

et al. 2016

Journal of Biological Chemistry

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“…Residual DNA-peptide crosslinks stall DNA polymerase in the leading strand and continuing DNA unwinding generates ss-DNA gaps, triggering ATR-mediated CHK1 phosphorylation. Bypass of residual crosslinks by error-prone polymerases can produce mutations (Minko et al, 2008; Wickramaratne et al, 2015). …”

Section: Figurementioning

confidence: 99%

Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

Ortega-Atienza

Green

Zhitkovich

2015

Toxicology and Applied Pharmacology

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Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA-protein crosslinks (DPC) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions.

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“…To gain insight into the ability of hpol to extend beyond the lesion, an LC-MS/MS method previously developed in this laboratory (34 -36) and applied extensively (37)(38)(39)(40)(41)(42)(43) was used for sequence analysis of the extension products. dU-containing primers were extended by hpol in the presence of all four dNTPs, followed by treatment with UDG and piperidine to cleave the fully extended products into shorter fragments for LC-MS/MS analysis (35,36).…”

Section: Lc-ms/ms Analysis Of Primer Extensionmentioning

confidence: 99%

Structural and Kinetic Analysis of Miscoding Opposite the DNA Adduct 1,N6-Ethenodeoxyadenosine by Human Translesion DNA Polymerase η

Patra¹,

Su²,

Zhang³

et al. 2016

Journal of Biological Chemistry

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1,N 6-Ethenodeoxyadenosine (1,N 6 -⑀dA) is the major etheno lesion formed in the reaction of DNA with epoxides substituted with good leaving groups (e.g. vinyl chloride epoxide). This lesion is also formed endogenously in DNA from lipid oxidation. Recombinant human DNA polymerase (hpol ) can replicate oligonucleotide templates containing 1,N 6 -⑀dA. In steady-state kinetic analysis, hpol preferred to incorporate dATP and dGTP, compared with dTTP. Mass spectral analysis of incorporation products also showed preferred purine (A, G) incorporation and extensive ؊1 frameshifts, suggesting pairing of the inserted purine and slippage before further replication. Five x-ray crystal structures of hpol ternary complexes were determined, three at the insertion and two at the extension stage. Two insertion complexes revealed incoming non-hydrolyzable dATP or dGTP analogs not pairing with but instead in a staggered configuration relative to 1,N 6 -⑀dA in the anti conformation, thus opposite the 5-T in the template, explaining the proclivity for frameshift misincorporation. In another insertion complex, dTTP was positioned opposite 1,N 6 -⑀dA, and the adduct base was in the syn conformation, with formation of two hydrogen bonds. At the extension stage, with either an incorporated dA or dT opposite 1,N 6 -⑀dA and 2-deoxythymidine-5-[(␣,␤)-imido-]triphosphate opposite the 5-A, the 3-terminal nucleoside of the primer was disordered, consistent with the tendency not to incorporate dTTP opposite 1,N 6 -⑀dA. Collectively, the results show a preference for purine pairing opposite 1,N 6 -⑀dA and for ؊1 frameshifts.

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Error-prone Translesion Synthesis Past DNA-Peptide Cross-links Conjugated to the Major Groove of DNA via C5 of Thymidine

Cited by 34 publications

References 73 publications

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

Structural and Kinetic Analysis of Miscoding Opposite the DNA Adduct 1,N6-Ethenodeoxyadenosine by Human Translesion DNA Polymerase η

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