Short-patch correction of C/C mismatches in human cells - PubMed (original) (raw)

. 2004 Dec 21;32(22):6696-705.

doi: 10.1093/nar/gkh990. Print 2004.

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Short-patch correction of C/C mismatches in human cells

Regula Muheim-Lenz et al. Nucleic Acids Res. 2004.

Abstract

We examined whether the human nucleotide excision repair complex, which is specialized on the removal of bulky DNA adducts, also displays a correcting activity on base mismatches. The cytosine/cytosine (C/C) lesion was used as a model substrate to monitor the correction of base mismatches in human cells. Fibroblasts with different repair capabilities were transfected with shuttle vectors that contain a site-directed C/C mismatch in the replication origin, accompanied by an additional C/C mismatch in one of the flanking sequences that are not essential for replication. Analysis of the vector progeny obtained from these doubly modified substrates revealed that C/C mismatches were eliminated before DNA synthesis not only in the repair-proficient background, but also when the target cells carried a genetic defect in long-patch mismatch repair, in nucleotide excision repair, or when both pathways were deleted. Furthermore, cells deficient for long-patch mismatch repair as well as a cell line that combines mismatch and nucleotide excision repair defects were able to correct multiple C/C mispairs, placed at distances of 21-44 nt, in an independent manner, such that the removal of each lesion led to individual repair patches. These results support the existence of a concurrent short-patch mechanism that rectifies C/C mismatches.

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Figures

Figure 1

Figure 1

Assay for in vitro human NER activity. Internally 32P-labeled linear substrates of 147 bp were incubated with HeLa whole cell extract. The substrate contained various mismatches as indicated (lanes 2–6), a site-specific AAF-dG adduct (lane 8) or a site-specific pivaloyl backbone adduct (lanes 9) in the central BstBI sequence. UM, unmodified homoduplex substrate. The gel has been overexposed to demonstrate the absence of detectable activity in response to the mismatches.

Figure 2

Figure 2

Excision of mismatches in an in vitro assay for human MMR activity. Heteroduplex bacteriophage substrates, containing a G/T or C/C mismatch, were incubated with cytoplamic extracts (CE), whole cell extracts (WCE) or nuclear extracts (NE) prepared from different cell lines. MMR efficiency was quantified after transformation of an E.coli mutS strain with the in vitro repair products. The mock-treated sample resulted from an incubation without cell extract. Complementation was performed by the addition of hMutLα to 293T cytoplasmic extracts. Lovo and HCT15 are human cell lines deficient for hMSH2 and hMSH6, respectively.

Figure 3

Figure 3

Vector pS189 derivatives containing two C/C mismatches. (A) Scheme illustrating the location of C/CSfiI (position 3578) in the SV40 origin of pS189. The pentanucleotide recognition sequences for T antigen binding are indicated by the arrows. (B) Composition of substrates 1 and 2. (C) Restriction analysis of substrate 1 (lanes 4–6) and substrate 2 (lanes 9–11). The homoduplex vector was prepared by extension and ligation of unmodified primers. Control, no restriction endonuclease added.

Figure 4

Figure 4

Independent processing of two neighboring C/C mismatches in human fibroblasts. (A) SfiI and Acc65I restriction analysis of clones 1–4 isolated after transfection of substrate 1 in wild-type fibroblasts. Control, no enzyme added. (B) Schematic view of the two different repair products isolated after transfection of substrate 1. (C) AvrII restriction analysis of clones number 1–14 isolated after transfection of substrate 2 in wild-type fibroblasts. (D) Scheme of the two different repair products of substrate 2.

Figure 5

Figure 5

Processing of different lesions in long-patch MMR-deficient 293T cells. (A) Scheme of substrate 3 (pS189 containing two neighboring A/C mismatches) and substrate 4 (pS189 containing two neighboring single nucleotide deletions). (B) Relative yield of progeny after transfection of pS189 derivatives, carrying the indicated lesions, in 293T cells. Colony numbers (mean values of two independent experiments) are expressed as the percentages of bacterial colonies obtained after transfection of the same cells with unmodified control vector.

Figure 6

Figure 6

Repair of C/C mismatches in long-patch MMR-deficient 293T cells. (A) SfiI and Acc65I restriction analysis of clones 1–4 isolated after transfection of substrate 1 in 293T cells. (B) Restriction analysis by AvrII of clones 1–20 isolated after transfection of substrate 2 in 293T cells.

Figure 7

Figure 7

Short-patch repair of C/C mismatches. (A) Schematic view of substrate 6 containing three closely spaced C/C mismatches. (B) Sequence of a representative clone isolated after transfection of substrate 6 in MMR- and NER-defective XP12ROB4 fibroblasts. Red arrows indicate the original position of C/C mismatches in the substrate.

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