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. 2008 Jun 20;283(25):17075-82.
doi: 10.1074/jbc.M801238200. Epub 2008 Apr 22.

Role for DNA polymerase kappa in the processing of N2-N2-guanine interstrand cross-links (VSports最新版本)

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Role for DNA polymerase kappa in the processing of N2-N2-guanine interstrand cross-links

Irina G Minko et al. J Biol Chem. .

VSports手机版 - Abstract

Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N(2)-guanines, similar to cross-links formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was approximately 97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) kappa not only catalyzed accurate incorporation opposite the cross-linked guanine but also replicated beyond the lesion, thus providing the first biochemical evidence for TLS past an ICL. The efficiency of TLS was greatly enhanced by truncation of both the 5 ' and 3 ' ends of the nontemplating strand. Further analyses showed that although yeast Rev1 could incorporate a dCTP opposite the cross-linked guanine, no evidence was found for TLS by pol zeta or a pol zeta/Rev1 combination. Because pol kappa was able to bypass these ICLs, biological evidence for a role for pol kappa in tolerating the N(2)-N(2)-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol kappa-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol kappa in the processing of N(2)-N(2)-guanine ICLs VSports手机版. .

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VSports app下载 - Figures

FIGURE 1.
FIGURE 1.
Oligodeoxynucleotide structures. A, structure of model acrolein-derived N2-N2-guanine cross-link. B, schematic of the cross-linked oligodeoxynucleotides used for mutagenesis assays. C, schematic of the cross-linked and nondamaged template oligodeoxynucleotides. Template deoxyguanosines associated with cross-link and the corresponding unmodified deoxyguanosines are underlined. Arrows indicate the direction of DNA synthesis. To prevent DNA synthesis off of the shorter strand of the duplex oligodeoxynucleotide, either a 3′-glycerol unit (gl) or a dideoxycytidine (dd) were incorporated. To inhibit DNA synthesis off of the shorter strand in ND2, a double mismatch was placed at the end of the duplex region. D, primer oligodeoxynucleotides.
FIGURE 2.
FIGURE 2.
Replication bypass of ICLs by human pol κ. A, primer extensions by pol κ (2 nm) were conducted for a period of time as indicated. B, single nucleotide incorporations by pol κ (1 nm) were carried out for 30 min.
FIGURE 3.
FIGURE 3.
Cellular responses to MMC exposure in pol κ-depleted GM639 cells. A, pol κ transcript levels following treatment by specific siRNA. B, relative colony-forming ability. C, radial formation. Error bars represent standard errors for at least four independent experiments, and data points are marked by asterisk with p < 0.05 (relative to corresponding control).
FIGURE 4.
FIGURE 4.
Replication bypass of ICLs by yeast Rev1. Primer extensions (marked all) and single nucleotide incorporations by Rev1 (10 nm) were carried out for 30 min.
FIGURE 5.
FIGURE 5.
Replication bypass of ICLs by yeast polζ. A, primer extensions by pol ζ (5 nm) were conducted for a period of time as indicated. B, single nucleotide incorporations by pol ζ (5 nm) were carried out for 30 min. C, primer extensions were carried out for 30 min at increasing concentrations of pol ζ (5, 10, or 20 nm) and Rev1 (5, 10, or 20 nm). Polymerases were present in reactions either individually or in combination. D, primer extensions in the presence of pol ζ (10 nm) or a combination of pol ζ (10 nm) and Rev1 (10 nm) were carried out for 45 min.

References

    1. Noll, D. M., Mason, T. M., and Miller, P. S. (2006) Chem. Rev. 106 277–301 - PMC - PubMed
    1. Lehoczky, P., McHugh, P. J., and Chovanec, M. (2007) FEMS Microbiol. Rev. 31 109–133 - PubMed
    1. Kozekov, I. D., Nechev, L. V., Moseley, M. S., Harris, C. M., Rizzo, C. J., Stone, M. P., and Harris, T. M. (2003) J. Am. Chem. Soc. 125 50–61 - PubMed
    1. Wang, X., Peterson, C. A., Zheng, H., Nairn, R. S., Legerski, R. J., and Li, L. (2001) Mol. Cell. Biol. 21 713–720 - PMC - PubMed
    1. Zheng, H., Wang, X., Warren, A. J., Legerski, R. J., Nairn, R. S., Hamilton, J. W., and Li, L. (2003) Mol. Cell. Biol. 23 754–761 - PMC - PubMed

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