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Nucleic Acids Res
2007 Jan 01;3516:5422-9. doi: 10.1093/nar/gkm552.
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Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts.
Liao S, Matsumoto Y, Yan H.
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Cellular DNA is under constant attack from numerous exogenous and endogenous agents. The resulting DNA lesions, if not repaired timely, could stall DNA replication, leading to genome instability. To better understand the mechanism of DNA lesion replication at the biochemical level, we have attempted to reconstitute this process in Xenopus egg extracts, the only eukaryotic in vitro system that relies solely on cellular proteins for DNA replication. By using a plasmid DNA that carries a site-specific apurinic/apyrimidinic (AP) lesion as template, we have found that DNA replication is stalled one nucleotide before the lesion. The stalling is temporary and the lesion is eventually replicated by both an error-prone mechanism and an error-free mechanism. This is the first biochemical system that recapitulates efficiently and faithfully all major aspects of DNA lesion replication. It has provided the first direct evidence for the existence of an error-free lesion replication mechanism and also demonstrated that the error-prone mechanism is a major contributor to lesion replication.
Figure 1. Establishment of the AP lesion replication system. (A) Experimental design for AP lesion replication. A plasmid DNA that carries a synthetic AP site (Δ) is first incubated with the dominant negative human APE (dnAPE) to protect the AP lesion and then with cytosol to assemble the pre-replication complex. Replication was initiated by the addition of NPE and monitored by 32P dATP. (B) Protection of AP containing DNA by dnAPE. The presence of AP site was detected by digestion with restriction enzymes PstI and EarI plus or minus AP endonuclease (wtApe). The EarI ends were then filled in with 32P TTP by Klenow and the DNA fragments were separated on a 5% urea polyacrylamide gel. (C) The replication of the AP lesion-containing DNA. Δ:C plasmid was replicated in the presence of dnAPE (AP lesion protected) or buffer (AP lesion repaired). Samples taken at the indicated times were de-proteinized with SDS and proteinase K and separated on a 1% TAE agarose gel and the gel was dried for exposure to phosphoimager. R: relaxed; S: supercoiled; L: linear. (D) Effect of dnAPE on the replication of the normal plasmid DNA pBS-Trx. The DNA was replicated in the presence of dnAPE or buffer and analyzed in the same way as in (C).
Figure 2. Restriction enzyme mapping of the replication intermediate. (A) Probable products of the lesion-carrying strand and lesion-free strand after replication. (B) The relaxed and supercoiled products of the 20 min time point were gel-purified, digested with the indicated restriction enzymes and separated on 1% agarose gels. The gels were stained with SYBR Gold to detect the control DNA pET28a and then dried for exposure to Phosphoimager to detect the 32P signal of the replication products. pET28a lacks a PstI site, but the enzyme caused some nicking to the DNA. PvuII cuts Δ:C at 2 sites. HincII and NgoM1V cut pET28a at 2 and 4 sites, respectively. (C) Plot of the restriction digestion pattern of the relaxed product. (+): cut; (−) uncut.
Figure 3. Effect of ClaI hemi-methylation. (A) The predicted methylation pattern of the ClaI site in Δ:C after one round of replication. (B) ClaI digestion of the two hemi-methylated, ClaI-containing NdeI fragments copied from Δ:C.
Figure 4. Determination of the stalling sites of DNA replication. (A) Experimental strategy to clone the stalled intermediates. The arrows illustrate the primers used for PCR. The templates for tailing and PCR were the gel-purified relaxed DNA from the 20 min time point. (B) Sequencing data of the PCR products showing the position of stalling.
Figure 5. Determination of the nucleotides opposite the AP site in the final replication products (after 75 min of incubation in NPE). (A) Restriction digestion of the gel-purified supercoiled final replication products (detected by 32P) and the control pET28a DNA (detected by SYBR Gold). R: relaxed; L: linear; S: supercoiled. (B) Transformation efficiency of pET28a (kanR; expressed in percentages of the number of transformants of the uncut DNA) and AP DNA (ampR; expressed in absolute colony numbers). (C) The nucleotides found at the position opposite the AP lesion in the plasmids isolated from the transformants of the DpnI and ClaI-digested Δ:C and Δ:G replication products. (D) The average ratios and absolute deviations of each nucleotide inserted at the position opposite the AP lesion for the Δ:C and Δ:G replication products. The data were from two independent experiments for each substrate. The right-most column listed the expected ratios if the AP lesion was replicated by random insertion of the 4 nt.
Figure 6. Analysis of the replication products that still carried the AP lesion (after 75 min of incubation in NPE). (A) Six potential types of DNA and their sensitivity to various enzymes. The lesion-carrying DNA would be nicked on the AP strand but intact on the complementary strand. BER: base excision repair; H: non-G; D: non-C. (B) Sequence analysis of the cloned PCR products amplified from Δ:G replication products that had been digested with DpnI, ClaI, APE and KpnI. (C) Sequence analysis of the cloned PCR products amplified from Δ:T replication products that had been digested with DpnI, ClaI, APE and KpnI.
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