Polymerase incorporation and miscoding properties of 5-chlorouracil

Abstract

Inflammation-mediated hypochlorous acid (HOCl) can damage DNA, DNA precursors, and other biological molecules, thereby producing an array of damage products such as 5-chlorouracil (ClU). In this study, we prepared and studied 5-chloro-2'-deoxyuridine (CldU) and ClU-containing oligonucleotide templates. We demonstrate that human K-562 cells grown in culture with 10 muM CldU incorporate substantial amounts of CldU without significant toxicity. When in the template, ClU residues pair with dATP but also with dGTP, in a pH-dependent manner with incorporation by human polymerase beta, avian myeloblastosis virus reverse transcriptase (AMV-RT), and Escherichia coli Klenow fragment (exo(-)) polymerase. The enhanced miscoding of ClU is attributed to the electron-withdrawing 5-chlorine substituent that promotes the formation of an ionized ClU-G mispair. When mispaired with G, ClU is targeted for removal by human glycosylases. The formation, incorporation, and repair of ClU could promote transition mutations and other forms of heritable DNA damage.

Figure 1. Schematic diagram of CldU and CldU-phosphoramidite

Figure 2. Template and primer sequences

Sequences for studies of (A) Incorporation of dATP or dGTP opposite template ClU or T residue. (B) Repair by hSMUG1 glycosylase.

Figure 3. GC/MS analysis of DNA bases from DNA extracted from K-562 cells following growth in T or CldU containing media

Total ion chromatogram of DNA bases in K-562 cells exposed to (A) 10 µM T or (B) 10 µM CldU. (C) Mass spectrum of the TBDMS derivative of ClU.

Figure 4. Gel electrophoretic analysis of the incorporation by pol β of dGTP against template ClU and T residues as a function of solution pH

(A) Template ClU. (B) Template T. A labeled primer is extended by the incorporation of two T deoxynucleotides (TTP is present at a saturating concentration of 15 µM) to reach the template target site ClU or T. Lane 1 includes the primer only while dGTP is present in the following concentrations: Lane 2: 0 µM; Lane 3: 15 µM; Lane 4: 30 µM; Lane 5: 60 µM; Lane 6: 125 µM; Lane 7: 250 µM; Lane 8: 1000 µM; Lane 9: 3000 µM. Template sequences corresponding to Figure 2A and the incorporated bases are shown on the right side of the figure.

Figure 5. Kinetic parameters of misincorporation as a function of solution pH corresponding to Table 2

(A) Pol β misincorporation. (B) AMV-RT misincorporation. (C) Klenow misincorporation. T:dGTP (▼), ClU:dGTP (■).

Figure 6. Proposed base pairing schemes for ClU

(A) Neutral wobble configuration. (B) Ionized pseudo-Watson-Crick configuration.

Figure 7. Relationship between polymerase kinetic parameters and base ionization

Pol β (■): The apparent pK_a is 8.9; the slope of the line is 1.89 × 10⁻⁴ with an intercept of 4.89 × 10⁻⁴ and r² is 0.99. AMV-RT (●): The apparent pK_a is 8.5; the slope of the line is 1.90 × 10⁻⁵ with an intercept of 8.50 × 10⁻⁶ and r² is 0.99. Klenow (▼): The apparent pK_a is 8.7; the slope of the line is 6.32 × 10⁻⁵ with an intercept of 6.32 × 10⁻⁴ and r² is 0.99.

Figure 8. Activity of hSMUG1 on synthetic oligonucleotides containing T-G, ClU-A or ClU-G

MALDI spectra of oligonucleotides. (A) Mixture of T-G, ClU-A and ClU-G oligonucleotide without enzyme reaction. (B) T–G oligonucleotide reacted with hSMUG1. (C) ClU-A oligonucleotide reacted with hSMUG1. (D) ClU-G oligonucleotide reacted with hSMUG1, generating an oligonucleotide with an abasic site. The cleaved ClU residue is too small to be seen with this method.

Figure 9. Repair schemes for T and ClU mispaired with guanine

The T–G and ClU-G mispairs can be repaired by either the mismatch repair (MMR) or the base-excision repair (BER) pathways. ClU is rapidly repaired by BER, potentially promoting a transition mutation. The asterisk (*) denotes the gap in the opposing strand generated as a repair intermediate.