Detection of cis- and trans-acting Factors in DNA Structure-Induced Genetic Instability Using In silico and Cellular Approaches

Abstract

Sequences that can adopt alternative DNA structures (i.e., non-B DNA) are very abundant in mammalian genomes, and recent studies have revealed many important biological functions of non-B DNA structures in chromatin remodeling, DNA replication, transcription, and genetic instability. Here, we provide results from an in silico web-based search engine coupled with cell-based experiments to characterize the roles of non-B DNA conformations in genetic instability in eukaryotes. The purpose of this article is to illustrate strategies that can be used to identify and interrogate the biological roles of non-B DNA structures, particularly on genetic instability. We have included unpublished data using a short H-DNA-forming sequence from the human c-MYC promoter region as an example, and identified two different mechanisms of H-DNA-induced genetic instability in yeast and mammalian cells: a DNA replication-related model of mutagenesis; and a replication-independent cleavage model. Further, we identified candidate proteins involved in H-DNA-induced genetic instability by using a yeast genetic screen. A combination of in silico and cellular methods, as described here, should provide further insight into the contributions of non-B DNA structures in biological functions, genetic evolution, and disease development.

Keywords: DNA repair; genetic instability; non-B DNA; replication; triplex.

FIGURE 1

Enrichment of non-B DNA structure at human mutation hotspots. (A) Co-localization of H-DNA- and Z-DNA-forming sequences with mutation hotspots in the human c-MYC promoter. Search results for H-DNA- or Z-DNA-forming sequences in the promoter region of the human c-MYC gene are shown. P0, P1 and P2, Exon 1, and Intron 1 of the human c-MYC gene are shown in the center. Potential H-DNA-forming sequences are shown on the left and potential Z-DNA-forming sequences are shown on the right. Chromosome breakpoints involved in c-MYC translocations in previous reports, or single-strand-specific S1 nuclease sensitive sites in cultured human cells are marked as red stars. (B) Co-localization of H-DNA-forming sequences within the human BCL-2 major breakpoint region. Search results for H-DNA-forming sequences in the upstream 70-bp from major breakpoint region of the human BCL-2 gene are shown on the left. Chromosome breakpoints involved in BCL-2 translocations in previous reports are marked as red stars on the right.

FIGURE 2

H-DNA stalls DNA replication forks in mammalian cells. Replication intermediates of pCON and pMYC plasmids recovered from mammalian COS-7 cells (24 h post-transfection) were separated via 2-D gel electrophoresis and the region containing the H-DNA-forming or control sequence was probed by Southern blotting. The SV40 Ori is not included in the probed regions, thus the products resulted in a typical Y-arc. The arrow designates the bulge on the Y-shaped replication arc, indicative of the accumulation of stalled replication intermediates. The lighter signal on the left arm of the arc (indicated with a bracket) on the pMYC sample suggests fewer replication forks passing through the H-DNA-containing region reducing the full-length products. A representative image of three independent repeats is shown.

FIGURE 3

Replication-independent H-DNA-induced genetic instability in HeLa cell extracts. (A) H-DNA-induced mutation frequencies in the presence (SV40T+) or absence (SV40T-) of replication in HeLa cell-free extracts. The number of mutants observed and the total number of colonies screened are listed below the corresponding bars. Error bars show the standard errors of the mean value of three independent experiments. (B) H-DNA-induced cleavage in replication-incompetent HeLa cell extracts. A radiolabeled 400-bp fragment containing SSBs generated in HeLa cell extracts (+) is indicated by an arrow, and the radiolabeled shorter fragments indicative of H-DNA-induced DSBs in HeLa cell extracts are marked with “^∗”. Plasmids incubated in the absence of HeLa cell extracts (-) served as controls.