Division of labor of Y-family polymerases in translesion-DNA synthesis for distinct types of DNA damage

Abstract

Living organisms are continuously under threat from a vast array of DNA-damaging agents, which impact genome DNA. DNA replication machinery stalls at damaged template DNA. The stalled replication fork is restarted via bypass replication by translesion DNA-synthesis polymerases, including the Y-family polymerases Polη, Polι, and Polκ, which possess the ability to incorporate nucleotides opposite the damaged template. To investigate the division of labor among these polymerases in vivo, we generated POLη-/-, POLι-/-, POLκ-/-, double knockout (KO), and triple knockout (TKO) mutants in all combinations from human TK6 cells. TKO cells exhibited a hypersensitivity to ultraviolet (UV), cisplatin (CDDP), and methyl methanesulfonate (MMS), confirming the pivotal role played by these polymerases in bypass replication of damaged template DNA. POLη-/- cells, but not POLι-/- or POLκ-/- cells, showed a strong sensitivity to UV and CDDP, while TKO cells showed a slightly higher sensitivity to UV and CDDP than did POLη-/- cells. On the other hand, TKO cells, but not all single KO cells, exhibited a significantly higher sensitivity to MMS than did wild-type cells. Consistently, DNA-fiber assay revealed that Polη plays a crucial role in bypassing lesions caused by UV-mimetic agent 4-nitroquinoline-1-oxide and CDDP, while all three polymerases play complementary roles in bypassing MMS-induced damage. Our findings indicate that the three Y-family polymerases play distinctly different roles in bypass replication, according to the type of DNA damage generated on the template strand.

Fig 1. Role of Y-family polymerases in cellular tolerance to DNA-damaging agents.

TK6 cells were assessed for sensitivity to six DNA-damaging agents. Cell viability was assessed by ATP assay, as described in the Materials and Methods. The dose of the indicated DNA-damaging agent is displayed on the x-axis on a linear scale, while the percentage of cell survival is displayed on the y-axis on a logarithmic scale. Error bars represent the standard deviation from three independent measurements. The p-value was calculated by Student’s t-test (*p < 0.05 and n.s [not significant]).

Fig 2. The relationship between Polη, Polι, and Polκ in cellular tolerance to MMS, CDDP, and UV.

(A-C) Indicated TK6 cells were assessed for sensitivity to MMS (A), CDDP (B), and UV (C) as in Fig 1. Data for the CDDP sensitivity for POLI^−/− were completely overlayed by the data for POLI^−/−/POLK^−/−. The dose of DNA-damaging agents is displayed on the x-axis on a linear scale, while the percentage of cell survival is displayed on the y-axis on a logarithmic scale. Error bars represent the standard deviation from three independent experiments. The p-value was calculated by Student’s t-test (*p < 0.05).

Fig 3. Contribution of Polη, Polι, and Polκ in preventing chromosomal breakage after exposure to MMS, CDDP, or UV.

(A) Representative images showing chromosomal aberrations. The arrow and arrowhead indicate a chromatid break and an isochromatid break, respectively. (B) TK6 cells were cultured in a medium containing CDDP (0.8 μM) or MMS (5 μg/mL) for 12 h or exposed to UV (4 J/m²) and cultured for 12 h. Cells were treated with colcemid for the last 3 h. The number of chromosomal aberrations (CAs) per 100 mitotic cells before and after the 12-h treatment was scored three times; the average and standard deviation from three experiments are presented in S6 Fig. The number of spontaneous CAs was subtracted from the number of DNA-damaging agent-induced CAs and is shown in the histogram. Error bars represent the standard deviation from three independent measurements. The p-value was calculated by Student’s t-test (*p < 0.05, ** p < 0.01).

Fig 4. Relationship between Polη, Polι, and Polκ in replication-fork progression after exposure to MMS-, CDDP-, or 4NQO-damaged DNA.

(A) Representative image showing stained DNA fibers. TK6 cells were labeled sequentially with CldU and IdU and treated with MMS (50 μg/mL), CDDP (1.5 μM), or 4NQO (5 μg/mL) after CldU labeling. (B) The lengths of the CldU and IdU tracts were measured, and the CldU/IdU ratio for each replication fork was calculated for at least 100 replication forks. The assay was carried out independently 2 times. The averages and standard deviations of medians are shown in the histograms. The p-value was calculated by Student’s t-test (**p < 0.01). Each data set is shown in S7 Fig.

Fig 5. Increased number of SCEs due to TLS failure across DNA damage induced by MMS, CDDP, or UV.

(A) Representative image showing SCEs in TK6 cells. (B) TK6 cells were continuously cultured in a medium containing BrdU (10 μM) and either CDDP (0.2 μM) or MMS (1 μg/mL) for 24 h. For UV irradiation, cells were exposed to UV (1 J/m²), then cultured in a medium containing BrdU for 24 h. Cells were treated with colcemid (0.1 μg/ml) to enrich for mitotic cells for the last 3 h of incubation. The number of spontaneous and induced SCEs in the macrochromosomes of 50 metaphase cells was scored at least two times. Error bars represent standard deviations from at least two independent analyses. Each data set is shown in S8 Fig. (C) The number of MMS-, CDDP-, or UV-induced SCEs was calculated by subtracting the number of spontaneous SCEs. Error bars represent standard deviations from at least two independent analyses. The p-value was calculated by Student’s t-test using all scored SCE data (*p < 0.05, **p < 0.01).