A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

Abstract

Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis-syn thymidine-thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases.

Scheme 1.

Lesion bypass products were initially generated by extension of a control or damaged primer/template pair by an individual polymerase. The newly synthesized strands were then isolated by denaturing PAGE and subsequently amplified by two rounds of PCR amplification with primers containing a four nucleotide barcode sequence, and the adapter sequences necessary for next-generation sequencing. The adapter sequences are shown as white bars. The four nucleotide barcode is shown as ‘XXXX’ in green. The sampled sequence and the flanking sequences are shown in red and blue, respectively. The sequencing primer used for next-generation sequencing anneals to the PCR products within Adapter 1 and the 42 nucleotides of sequencing information obtained are indicated by a bracket. The position of the cis-syn TT dimer within the damaged template is underlined and the nucleotide incorporations opposite from the lesion are shown as ‘??’.

Figure 1.

Comparison of the preferred actions of human Y-family DNA polymerases opposite a cis–syn TT-dimer. The relative frequency of nucleotide incorporations opposite the (a) 3′-dT or (b) 5′-dT of the cis–syn TT-dimer is indicated. For comparison, relative frequency of nucleotide incorporations opposite the corresponding (c) 3′-dT or (d) 5′-dT of the control template is indicated.

Figure 2.

Histogram of the relative percent error as a function of template position. The relative number of base insertions (striped bar), substitutions (black bar) and deletions (white bar) as a percentage of the total dNTP incorporations is shown at each template position. The indicated template position is relative to the cis–syn TT dimer site within the 77-mer-TT template. The template bases are indicated and the cis–syn TT dimer is represented as T-T. Lesion bypass analysis for (a) hPolη, (c) hPolκ and (e) hPolι with the cis–syn TT dimer-containing DNA substrate is shown. The relative error as a function of template position with the control DNA substrate containing a pair of undamaged template dTs was also analyzed for (b) hPolη, (d) hPolκ and (f) hPolι.