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, 8 (7), 852-6

Construction of a Circular Single-Stranded DNA Template Containing a Defined Lesion

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Construction of a Circular Single-Stranded DNA Template Containing a Defined Lesion

Kiyonobu Karata et al. DNA Repair (Amst).

Abstract

We report a concise and efficient method to make a circular single-stranded DNA containing a defined DNA lesion. In this protocol, phagemid DNA containing Uracil is used as a template to synthesize a complementary DNA strand using T7 DNA polymerase and an oligonucleotide primer including a site-specific DNA lesion. The ligated lesion-containing strand can be recovered after the phage-derived template DNA is degraded by treatment with E. coli Uracil DNA glycosylase and Exonucleases I and III. The resulting product is a circular single-stranded DNA containing a defined DNA lesion suitable for in vitro translesion replication assays.

Conflict of interest statement

Conflict of interest statement: The authors declare that there are no conflicts of interest.

Figures

Fig. 1
Fig. 1
Flow chart for the construction of pSOcpd. All reactions were performed in one tube, except for the preparation of phage DNA and the purification of the final product. The approximate time to complete each step is shown in parentheses.
Fig. 2
Fig. 2
Verification of each step during second strand DNA synthesis and glycosylase/nuclease treatments. Samples were separated on 1% agarose gel containing 2 μg/ml ethidium bromide. Lane M, DNA size markers. Lane 1, 200ng of sspAVR88. Lane 2, product after annealing of the 2nd strand primer. Lane 3, product after second strand synthesis and ligation. Lane 4, product after glycosylase and nuclease treatment. Lane 5 and 6 are different amounts of purified pSOcpd. Equal portions corresponding to 200ng of sspAVR88 were loaded in lanes 1-5.
Fig. 3
Fig. 3
(A) Sequence of pSOcpd surrounding the cis-syn CPD (indicated as T-T in bold font). The binding site of the labeled primer M13-TT, is shown as an arrow. (B) In vitro DNA replication assay with pSOcpd. The TLS reactions were performed in the presence of pol I (Kf) (lane 1), T7 DNA polymerase (lane 2), pol V(R391) + RecA protein in the absence (lane 3), or presence of the β-clamp and γ-clamp loader complex (lane 4), or human polη (lane 5). The position of the labeled primer (lane 6), is shown on the right of the gel (P), while the local template sequence context and position of the T-T CPD (in bold font) is shown on the left side of the gel.

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