Fig 1.
Exposure to different heavy metals alters the selection of DNA repair pathway used to repair exogenously induced double strand breaks.
A. A schematic of the four green fluorescent protein (GFP)-based reporter cassettes are shown. These reporter cassettes are designed to provide quantitative information on the outcome of the double strand break (DSB) repair [23]. The exogenously induced DSBs are repaired by Homologous Recombination (HR) or Non-homologous End Joining (NHEJ). Depending on which pathway is utilized to repair the break the outcome will be faithful restoration of the DNA sequence or a mutated outcome (error-prone repair). The four reporter cassettes are: 1) DR-GFP for homology-directed repair (HR), 2) SA-GFP for single strand annealing (SSA) repair or non-allelic homologous repair (NAHR), 3) EJ2-GFP for alternative non-homologous end joining (alt-EJ) and 4) EJ5-GFP for total end joining (total EJ). The DR-GFP cassette contains the SceGFP cassette that is interrupted by an I-SceI site (black bar) and followed downstream by a 5’and 3’ truncated fragment of GFP (iGFP). The I-SceI induced DSB is repaired by HDR using the iGFP sequence as the template, which can lead to a GFP+ product. The SA-GFP cassette contains a 5’ fragment of GFP (5’GFP), and a 3’ fragment of GFP (Sce3’GFP) disrupted by an I-SceI site. Repair of the DSB in Sce3’GFP by single strand annealing will generate a GFP+ product and cause a deletion (unfaithful repair). The EJ2-GFP cassette contains an expression cassette for a tagged version of an I-SceI disrupted GFP and a series of stop codons, which is flanked by eight bases of homology. Repair by alternative end joining (alt-EJ) that deletes the stop codons, restores the GFP coding frame, and bridges the eight bases of flanking homology, leads to a GFP+ product. The EJ5-GFP cassette contains the pCAGGS promoter separated from the GFP coding sequence by a puromycin selection cassette (puro) flanked by I-SceI sites. Shown is the outcome of a distal-EJ (total EJ) event where the puromycin sequence is removed by the joining of the two flanking sequences in a manner that leads to the expression of the GFP+ product. Only the HR pathway through homology-directed repair can restore the genomic damage faithfully. In contrast, the other repair outcomes reflect mutagenic changes that occurred during repair of the DSB. Cell lines containing the GFP-reporter systems DR-GFP for homologous recombination (HR, -♦ -), SA-GFP for single strand annealing (SSA -□-), EJ2-GFP for alternative non-homologous end joining (NHEJ, --▲--) and EJ5-GFP for non-homologous end joining (total EJ, …x…) were treated with different amounts of: B. cadmium chloride, C. nickel chloride and D. arsenic trioxide and. The Y-axis shows the percent fluorescence (% GFP) normalized using the results from the I-SceI transfected and untreated cells as baseline, which was set to 100%, and highlighted as a solid horizontal line across the graph. Heavy metal treatments increasing a particular repair outcome will show signals above 100% while inhibitory effects will show signals below the 100%. Asterisks indicate results significantly different from untreated cells t-test, P < 0.05; n ≥ 3. Contrasting results between different metal exposures are highlighted in red and blue.
Fig 2.
Exposure to heavy metals induces deletion events.
A. A schematic of the 0% AARP cassette is shown. The cassette is stably integrated into an FRT site of the HEK293 cell line. The basic construct consists of a promoter upstream of two identical Alu sequences (i.e. 0% divergence between Alu1 and Alu2) separated by approximately 1.1 kb which contains an I-SceI site. The vector is designed so that only Neo (blue) is expressed driven by the pEF5 promoter (gray arrow). DSBs can be exogenously induced by transfection of an I-SceI expression vector or to evaluate induction of DSBs by xenobiotics like heavy metals. In this approach, only those DSBs generated by metal exposure that occur near the cassette sequences are detectable. Events that are repaired by deleting the sequence between the Alus will allow expression of PuroR (light gray). All events detected by this assay are repair events that result in a mutagenic outcome. Neo = neomycin resistance gene, pA = polyadenylation signal, Puro = puromycin resistance gene, pEF5 = elongation factor 5 promoter B. A schematic of the protocol of the time line of the assay is shown. Cells are seeded and 16–24h later they are exposed to medium with or without the heavy metal for 48 h. Then the medium is removed and replaced puromycin medium for selection. Fourteen days post-selection, cells are stained in crystal violet solution and the puroR colonies are counted. Three heavy metals were evaluated: C. 100 μM NiCl2 D. 1 μM CdCl2 and E. 1 μM AsO3. Data represent the mean with standard error of the mean from at least three independently experiments for each condition. Statistical differences are indicated as *P<0.05; **P<0.001 (two-tailed two sample Student’s T-test).
Fig 3.
Heavy metal exposure favors NAR repair over NHEJ repair of induced DSBs.
Cells containing two different AARP constructs with sequence diverged Alus (5 and 10% sequence variation between Alu1 and Alu2) were used. The cells were transfected with an I-SceI expressing construct and allowed to repair in the presence or absence of heavy metals. Individual puroR colonies were selected for DNA extraction. PCR products of the repaired event from DNA extracted from the individual colonies were sequenced and analyzed. The DSB repair can occur by: non allelic Alu/Alu recombination (NAR) or by Non Homologous End joining (NHEJ) driven repair through alt-NHEJ or classic NHEJ (C-NHEJ). The type of repair (NAR or NHEJ represented in A) is verified by sequencing PCR products of the genomic region from individual puroR clones (primers F5 and R5 are shown as black arrows). An example of a PCR analysis is shown in B, with colonies 1–3 showing a 1.5 kb product consistent with NAR repair and colony 4 with a smaller product consistent with EJ type of repair. The Alu sequence variation allows for mapping of recombination sites in the recovered events and the events were classified as NAR (black) or EJ (white). Three heavy metals were evaluated: C. 1 μM CdCl2 D. 1 μM AsO3 and E. 100 μM NiCl2 Statistical differences are indicated as *P<0.05; (Fisher’s exact test). Numbers above each column represent the total colonies analyzed, details in Table 1.
Table 1.
Number of recovered events.
Fig 4.
Distribution of single and complex chimeras.
Alu-Alu recombination can generate two types of outcomes: simple chimera or complex chimera. A. A simple chimera shows only one recombination site (left portion of the Alu sequence chimera derived from Alu1 and right portion derived from Alu2). B. A complex chimera shows multiple recombination sites with intermixed regions of the chimera derived from the two Alu sequences. Schematic of actual recovered outcomes are shown below the diagrams. C. The 5 and 15%AARP HEK 293 cell line was incubated with media containing 100 μM NiCl2. Untreated cells were used as the reference control. Sequence analyses of the recovered events showed that two types of Alu-Alu recombination products can be observed: simple and complex. NAR events recovered from the nickel treated cells were further classified as single (gray) or complex chimeras (white). The results are expressed as mean ± SD of three independent experiments. Statistical differences are indicated as *P<0.05; (Fisher’s exact test); ns = not significant. Numbers above each column represent the total colonies analyzed; sequence alignments are shown in S7 Fig.
Fig 5.
Heavy metal exposure alters the sequence characteristics of EJ DSB repair outcomes.
A. A schematic of the protocol of the time line of the assay is shown. B. Table showing the number of recovered events classified as end-joining products of I-SceI induced breaks of cells exposed to: no treatment control, 1 μM CdCl2, 100 μM NiCl2, and 1 μM AsO3. Events rescued from all experimental conditions were pooled and classified into events showing microhomology (MH), no microhomology (noMH) and insertions (Ins) at the repair site. Note that no insertions were ever observed in the EJ events from unexposed cells. C. Stacked column chart shows the relative proportion of the types of end-joining products rescued from repaired I-SceI induced breaks of cells exposed to 100 μM NiCl2, 1 μM CdCl2 and 1 μM AsO3. “Other” represents one event showing the presence of one inverted chimeric Alu sequence at the DSB site. Sequence data obtained from untreated cells were used as the no treatment control.
Fig 6.
Role of heavy metals as promotors of genetic instability through the accumulation of genetic damage by favoring error-prone DNA repair.
A. DSBs are consistently occurring in cells either as a byproduct of natural processes or due to external insults such as heavy metal exposure. DSBs near repetitive sequences, such as Alu elements (shown as orange and blue boxes) will result in different repair outcomes usually with one type being favored. B. When compared to an untreated control (no Tx), our data show that exposure to different heavy metals can differentially alter the choice between pathways to promote error-prone repair by favoring spontaneous recombination between repeat sequences (e.g.Alu); homeology-mediated deletions which occur through the NAR or Alt-NHEJ repair; and untemplated insertions during repair by alt-NHEJ. The proportion of the different types of repair outcomes varies between the different heavy metal treatments. The overall effect of heavy metal exposure will favor the accumulation of specific genomic changes and loss of genomic sequence contributing to signature mutagenic changes as a manifestation of the exposure. ND = not detected.