DNA Repair Enzyme Shields Mouse Tissues from Chemotherapy-Induced Breaks

A DNA repair enzyme called polymerase kappa acts as a body-wide shield against the DNA damage caused by the chemotherapy drug mitomycin C, according to a study in mice published in the journal Genes and Environment. When researchers inactivated the enzyme, the drug’s ability to trigger DNA double-strand breaks rose across many organs, with the strongest effects seen in the liver, bladder, adrenal cortex, thyroid, and sperm-forming cells of the testis.

Mitomycin C is a crosslinking agent widely used as a first-line chemotherapeutic drug; it generates intrastrand cross-links at GpG sites in the same strand of DNA and interstrand cross-links at CpG sites in opposite strands. Even when double-strand breaks are patched up by homologous recombination or non-homologous end joining, the repairs are sometimes imperfect, leaving short deletions or large allele losses that impair cell function.

Polymerase kappa belongs to the Y-family of specialized DNA polymerases that carry out translesion DNA synthesis, a process in which the enzyme inserts DNA building blocks opposite lesions in the template strand and continues synthesis, allowing replication forks to bypass damage that would otherwise stall them. In vitro, polymerase kappa can bypass a striking variety of structurally unrelated lesions, including benzo[a]pyrene-induced guanine adducts, abasic sites, thymidine glycol, and interstrand cross-links. The enzyme is also believed to contribute to replication-independent repair of cross-links, a pathway in which endonucleases first unhook the cross-link from one strand and a translesion polymerase then traverses the gap. Notably, no reports suggest a relation between polymerase kappa deficiency and human disease, and differences between knockout or knock-in mice and wild-type mice in general conditions such as appearance, body weight, and behavior have not been reported, although knockout mice show elevated spontaneous mutation frequencies of base substitutions mainly at G:C base pairs in kidney, liver, and lung in old age.

To test the enzyme’s protective role across the whole body, the team used mice in which polymerase kappa had been catalytically inactivated by knock-in, alongside wild-type controls. Nine- to twelve-week-old animals of both genotypes received daily intraperitoneal injections of mitomycin C at 1 milligram per kilogram for five days, or saline as a control, with six mice per group split evenly between males and females. Organs were collected three hours after the final dose, fixed, and stained immunohistochemically for gamma-H2AX, a phosphorylated histone variant that marks DNA double-strand breaks. More than thirty organs and tissues were scored semi-quantitatively for staining intensity, and five tissues showing the clearest effects—liver, bladder, adrenal cortex, thyroid, and spermatids—were then counted cell by cell for precise quantification.

The semi-quantitative survey showed that mitomycin C raised gamma-H2AX scores in almost every tissue examined in both genotypes, confirming that the drug damages DNA systemically. Crucially, when the drug-treated groups were compared directly, scores were higher in the enzyme-inactivated mice than in wild-type mice across many tissues, including liver, bladder, and adrenal cortex. The pattern was not uniform. Enhancement was obvious in liver, bladder, adrenal cortex, thyroid, and spermatids, but weak or absent in brain regions such as the hippocampus, cerebral cortex, and cerebral white matter, and in the retina. In those neural tissues, scores were actually lower in the drug-treated enzyme-inactivated mice than in drug-treated wild-type mice; the authors note that spontaneous gamma-H2AX phosphorylation has been reported in the mouse brain, possibly driven by reactive oxygen species from catecholamine oxidation, and that enhanced double-strand break repair pathways might mitigate break induction, although the exact reasons for the lower scores remain unknown. The limited ability of mitomycin C to penetrate the blood-brain barrier has been reported, further complicating interpretation in the brain.

The quantitative counts sharpened the picture. In liver hepatocytes, the fraction of gamma-H2AX-positive cells after drug treatment rose from 1.3 percent to 20.1 percent in wild-type males and from 0.6 percent to 25.1 percent in wild-type females, but reached 41.1 percent in enzyme-inactivated males and 40.9 percent in enzyme-inactivated females, all differences statistically significant at p less than 0.01 by Fisher’s exact test. Liver sinusoidal cells showed an even starker contrast: drug-treated wild-type mice reached 43.7 percent positive cells in males and 46.0 percent in females, while enzyme-inactivated mice climbed to 67.0 percent and 55.9 percent respectively. In bladder epithelium, a rapidly proliferating tissue, drug-treated enzyme-inactivated females reached 56.1 percent positive cells compared with 27.2 percent in drug-treated wild-type females. Thyroid cells followed the same pattern, with enzyme-inactivated females jumping from a 7.6 percent drug response in wild-type animals to 36.8 percent. In round spermatids at specific stages of the spermatogenic cycle, the drug pushed wild-type mice to 45.4 percent positive cells and enzyme-inactivated mice to 66.1 percent.

Strikingly, the enzyme’s protection extended beyond drug-induced damage. In saline-treated animals with no mitomycin C exposure, enzyme-inactivated mice showed higher gamma-H2AX levels than wild-type mice in the adrenal cortex, prostate, and retina, and in hepatocytes and sinusoidal cells of females. The adrenal cortex result was dramatic: saline-treated enzyme-inactivated females had 19.2 percent positive adrenal cortex cells versus 1.9 percent in wild-type females, a roughly ten-fold difference that was absent in males. The authors note that a three-fold higher cell turnover in the adrenal cortex of female mice compared to male mice has been reported, and suggest that polymerase kappa may prevent the accumulation of spontaneous breaks, which may be induced in highly proliferating adrenal cortex cells in female mice. The authors note that intrinsic reactive oxygen species generation has been reported in these tissues, and that intrinsic reactive oxygen species can generate lipid peroxidation products such as malondialdehyde, which is known to induce DNA cross-links. The findings support the authors' suggestion that polymerase kappa may protect DNA from endogenous and inflammation-induced mutagens, and may also protect against endogenously generated double-strand breaks in some organs.

Because the enhancement appeared in both proliferative cells, such as bladder epithelium, and in slowly dividing or non-proliferative cells, such as hepatocytes, the authors conclude that polymerase kappa acts through both replication-dependent and replication-independent mechanisms. In dividing cells, replication forks stall at mitomycin C cross-links, and polymerase kappa-mediated translesion synthesis resumes synthesis past the block; without the enzyme, the stall persists and breaks accumulate. In less or slowly proliferative cells, the authors suggest the enzyme is involved in replication-independent repair of interstrand cross-links, in which endonucleases first unhook the cross-link from one of the two DNA strands and polymerase kappa then facilitates bypass of the unhooked cross-link; this pathway has been reported in Xenopus egg extracts. The authors also point out that another specialized polymerase, polymerase zeta, is suggested to be involved in translesion synthesis in both types of interstrand cross-link repair, and that its inactivation enhances killing sensitivity against mitomycin C in human cells; this involvement may offset the contribution of polymerase kappa, potentially explaining the relatively moderate increases in gamma-H2AX-positive cells observed with polymerase kappa inactivation across organs and tissues.

The study also revealed sex differences that the authors could not fully explain. In the adrenal cortex, the protective effect of the enzyme against spontaneous breaks was evident in females but not in males. In the thyroid, saline-treated wild-type males showed an eight-fold higher incidence of positive cells than females, despite reports of higher oxidative stress in female rat thyroid. In the bladder, saline-treated enzyme-inactivated females showed roughly twice the positive-cell frequency of males. No reports describe sex differences in reactive oxygen generation or proliferation in the bladder, so the mechanisms remain unclear. In the liver, no consistent sex-specific pattern emerged between hepatocytes and sinusoidal cells. The authors also observed that polymerase kappa expression levels, which are very high in bladder and testis and low in liver and stomach, do not necessarily correlate with the enzyme’s contribution to break prevention, since the liver showed strong enhancement despite low expression.

Linking the molecular findings to known drug toxicities, the authors note that the heart, liver, kidney, lung, bone marrow, and testis are established targets of mitomycin C toxicity, and that the organs showing the clearest break induction in this study—liver, bone marrow, and testis—overlap with those targets. The authors suggest that mitomycin C-induced organ or tissue toxicity may be linked to double-strand break induction and that polymerase kappa plays a role, at least partially, in preventing such toxicities in these organs or tissues. In kidney, heart, and lung, the induction frequency of gamma-H2AX positive cells was moderate; the authors note that mitomycin C-induced renal toxicity is suggested to be unrelated to DNA damage, instead being mediated by thrombotic microangiopathy, and that although reactive oxygen generation is believed to contribute to cardiotoxicity and pulmonary toxicity, cumulative dosing is required to induce this toxicity, so the five-day treatment duration might be insufficient for detecting DNA damage in heart and lungs, or the toxicities may not be directly linked to DNA damage. The work was conducted under protocols approved by AAALAC International, and the authors caution that the exact reasons for the reduced gamma-H2AX responses in the brains of drug-treated enzyme-inactivated mice remain unknown. Overall, the study establishes polymerase kappa as a systemic guardian against chemotherapy-induced and endogenous DNA breaks, with tissue-specific contributions that do not simply mirror its expression levels.

Subject of Research: The role of DNA polymerase kappa in preventing mitomycin C-induced DNA double-strand breaks in mouse tissues

Article Title: Mitomycin C-induced DNA double-strand breaks are enhanced by catalytical inactivation of DNA polymerase κ in mice

Article References: Wada, N. A., Takeiri, A., Motoyama, S., Matsuzaki, K., Tanaka, K., Matsuo, S., Fujii-Takeiri, E., Tateishi, H., Matsumoto, K., Niimi, N., Sassa, A., Grúz, P., Masumura, K., Mishima, M., Jishage, K.-I., Sugiyama, K.-I., & Nohmi, T. (2025). Mitomycin C-induced DNA double-strand breaks are enhanced by catalytical inactivation of DNA polymerase κ in mice. Genes and Environment, 47(1), Article 22. https://doi.org/10.1186/s41021-025-00343-x

Image Credits: AI Generated

DOI: 10.1186/s41021-025-00343-x

Keywords: DNA polymerase kappa, mitomycin C, DNA double-strand breaks, translesion DNA synthesis, gamma-H2AX, DNA cross-links, genome integrity, chemotherapy toxicity, knock-in mice, interstrand cross-link repair, reactive oxygen species, Genes and Environment