These cells can eventually form a tumor. Some tumor suppressor genes are BRCA1 , BRCA2 , and p53 or TP If a mutation in the BRCA1 or BRCA2 genes is passed down from your parents, you have a greater risk of hereditary breast, ovarian , pancreatic, and prostate cancer.

Mutations in these genes also raise the risk of melanoma. The most common tumor suppressor gene that mutates in people with cancer is p53 or TP This gene is missing or damaged in more than half of all cancers. Inherited p53 mutations are rare. If you have one, you have a greater risk of many different types of cancer.

Mutations in certain genes can turn a healthy cell into a cancer cell. These mutations are not known to be inherited. This gene makes a protein that controls cancer growth. It is found in some cancer cells, such as some breast and ovarian cancer cells. If a breast cancer is "HER2 negative," it means the HER2 gene is not making this needed protein.

The RAS family of genes. These genes make proteins that help cells communicate with each other, grow normally, and die when they are supposed to. Mutations in these genes can turn healthy cells into cancer cells. DNA repair genes. DNA repair genes fix mistakes that happen when your DNA is copied.

DNA copying happens when cells divide normally to make new cells. Many DNA repair genes work like tumor suppressor genes do. They limit how much the cell can grow. BRCA1 , BRCA2 , and p53 are all DNA repair genes. If one of your DNA repair genes has a mutation, mistakes in DNA do not get corrected.

This means mutations can develop, and eventually lead to cancer. This is especially true if the mutation is in a tumor suppressor gene or an oncogene. You can inherit a mutation in a DNA repair gene, or it can happen on its own over your lifetime. Lynch syndrome is caused by an inherited DNA repair mutation.

Mutations in BRCA1 , BRCA2 , and p53 are inherited too. Learn more about hereditary cancer-related syndromes. Researchers have learned a lot about how genes are involved in cancer.

But many cancers are not linked with any specific gene. When cancer develops, several gene mutations are probably involved, not just one.

Also, gene mutations are not the only cause of cancer. Your everyday environment can also affect how genes and gene mutations work. Together, your genes and your environment can determine if you develop cancer. Researchers continue to study genes and cancer.

What they have learned so far has helped improve cancer care. It has given doctors new ways to find cancer early, lower your risk, treat cancer with new treatments such as targeted therapy , and live longer.

More studies about genes and cancer may help researchers learn how to do the following in the future:. Did one of my parents pass on the genetic mutation that caused my cancer? Is my cancer part of an inherited syndrome? Should other people in my family have genetic testing? Who can we talk to about this?

Genetic Testing for Cancer Risk. Understanding Cancer Risk. What Is Personalized Cancer Medicine? Introduction to Cancer Research.

Expert Perspectives about Genetics and Cancer. National Cancer Institute: The Genetics of Cancer. NCI: How Genetic Changes Lead to Cancer Infographic. Comprehensive information for people with cancer, families, and caregivers, from the American Society of Clinical Oncology ASCO , the voice of the world's oncology professionals.

org Conquer Cancer ASCO Journals Donate. Home Types of Cancer Navigating Cancer Care Cancer Basics What is Cancer? The Cancer Care Team What is Metastasis? Veterans Prevention and Healthy Living Cancer. Net Videos Coping With Cancer Research and Advocacy Survivorship Blog About Us.

Genes and Cancer Approved by the Cancer. Some things your genes control are: How fast each cell grows How often it divides to make new cells How long it lives The genes in each cell are arranged in structures called chromosomes.

What are chromosomes? How do our genes work? Types of gene mutations or pathogenic variants There are 2 basic types of gene mutations or pathogenic variants. Common factors causing sporadic mutations include the following: Tobacco use Ultraviolet UV radiation Viruses Aging Germline mutations.

Do mutations always cause cancer? Are there specific "cancer genes? They do this by: Controlling how fast cells divide to make new cells Fixing mistakes in DNA Controlling when a cell dies Variants in a tumor suppressor gene can allow cells to grow out of control.

Two common oncogenes are: HER2. What is known about genes and cancer? More studies about genes and cancer may help researchers learn how to do the following in the future: Predict a person's individual risk of cancer Find cancer more easily when it is smaller Treat different types of cancer better Questions to ask the health care team Is my cancer caused by a genetic mutation?

A Immunofluorescence of B16 cells treated with PTU and tyrosine with phosphorylated H2AX antibody. Nuclear DNA stained with DAPI blue and γH2AX in red.

Experiment was performed with two biological replicates and a representative image is depicted. Scale bar 10 μm.

B Quantitation of mean fluorescence intensity per cell of γH2AX from two biological replicates of pigmented day 7 PTU or tyrosine treated cells shown in A.

Data represented as a box plot, horizontal line represents mean and whiskers represent SEM. Ordinary one-way ANOVA was performed for multiple comparisons.

Cells were left untreated for control treated with tyrosinase inhibitor μM phenylthiourea PTU or 1mM tyrosinase substrate L-tyrosine Tyr for 7 days. Number of cells, mean ± SEM across three biological replicates is depicted below the image of the cell pellet. Numbers represent mean ± SEM cell counts across biological triplicates.

Bottom Number of abasic sites in the genomic DNA was estimated by an aldehyde specific conjugation of biotin and subsequent detection using streptavidin based detection. Using standards, abasic sites per 10 5 bp is estimated. Bars represent mean ± SEM across duplicate biological experiments, each conducted in triplicates.

D Single cell electrophoresis followed by comet analysis of B16 cells undergoing varying levels of pigmentation in the presence of PTU and tyrosine alkaline comet assay. Experiment was carried out at mid phase day 5 and late phase day 7 of pigmentation.

Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Two-way ANOVA was performed. E Neutral comet assay on B16 unpigmented day 0 and pigmented day 7 cells.

Student's unpaired t-test was performed. P values ns non-significant. F Single cell electrophoresis followed by comet analysis of B16 cells untreated, treated with DMSO for 24 h, melanin synthesis ex-cellulo L-tyrosine and tyrosinase added to cell media for 24 h or cells treated with 1 mM dihydroxyindole DHI for 24 h alkaline comet assay.

Ordinary one-way ANOVA was performed. Surprisingly not only the γH2AX phosphorylation was elevated with tyrosine treatment and decreased with PTU, the total H2AX levels also followed a similar trend Supplementary Figure S1F. In alkaline comet assay, the level of associated DNA nicks was high in day 7 pigmented cells, tyrosine treated hyperpigmented cells demonstrated maximal DNA nicks.

The mean tail moment progressively increased in control and tyrosine treated cells on day 7, compared to day 5 as the melanin content increased. We then proceeded to assess DNA breaks by neutral comet assay.

In the neutral comet assay we observed very little breaks and the tail moment was close to 1—2 units in both pigmented and unpigmented cells, and did not observe significant differences between these two states Figure 1E.

In all, these experiments suggest that ongoing melanogenesis is associated with DNA damage. On a closer look the following points emerge. Melanogenesis causes free radical generation but does not increase 8-OHdG levels in the cells. Instead, there is a marked elevation in abasic sites, which is clearly dependent on pigmentation.

The increased intensity of γH2AX puncta classically associated with double stranded breaks, could also mark single stranded DNA regions as well as other DNA lesions 27 , Since the neutral comet assay does not show comet formation upon pigmentation, double stranded breaks would not account for the observed DNA breaks.

However, the alkaline comet assay increases the tail moment, progressively and predictably in a pigmentation dependent manner Figure 1D. It is interesting to note that the abasic sites are prone to strand cleavage and result in enhanced comets as seen in alkaline comet assay Therefore, the elevated tail moment observed in alkaline comet assay could be contributed by the abasic sites alkali labile as well as single stranded DNA lesions, if any, generated by melanin-based damage and the relative contribution is difficult to assess at this stage.

Finally, to confirm the role of melanin intermediates in causing the DNA damage, we subjected depigmented B16 cells to 1mM Dihydroxy Indole DHI , one of the intermediates of eumelanin synthesis reaction, as well as exogenously increased melanin synthesis through ex-cello synthesis of melanin with the substrate L-DOPA and enzyme Tyrosinase added in the culture medium and performed alkaline comet assay.

We observed an increase in the comet tail length with both the treatments, confirming that melanin intermediates can cause DNA damage Figure 1F. The DNA damage due to melanin intermediates was known 5—9.

By the systematic investigation of melanogenesis induction and its pharmacological perturbation, we unequivocally demonstrate the DNA damage using this cell-based model system.

Since melanin is a complex polymer, we could not demonstrate direct evidence for covalent conjugation of melanin intermediates to DNA. However, plasmid DNA incubated with melanin synthesis reaction which was further purified to remove unbound melanin intermediates demonstrated quinone-DNA adduct formation using coupled Nitro-blue tetrazolium NBT based detection Supplementary Figure S1H.

In support, DNA incubated with melanin intermediates during melanin synthesis showed altered mobility in agarose gel, while the same was not observed when the DNA is incubated with pre-synthesized melanin Supplementary Figure S1I. Having observed that melanogenesis causes DNA damage, we then proceeded to decipher the nature of response used to combat this unique challenge faced by melanocytes.

Towards this we treated either unpigmented day 0 or pigmented day 7 B16 cells with known DNA damaging agents. UVA and UVB to assess photo-oxidative damage, hydroxyurea for replication stress and H 2 O 2 for oxidative damage were employed.

Pigmented and unpigmented cells that were not treated with any DNA damaging agents were included, and the expression of a panel of DNA repair genes were analyzed by qRT-PCR analysis after 24 h treatment.

Signature pattern of expression for each of the damage was clearly distinct, and notably the pattern of gene expression induced by pigmentation closely paralleled that of hydroxyurea treatment that results in replication stress Figure 2A.

Melanogenesis induces replication stress response. A Fold change in mRNA levels of a panel of DNA repair genes by qRT-PCR analysis. The heat map represents fold change and compares DNA repair gene signature of depigmented left , pigmented middle B16 cells to various DNA damaging agents.

Untreated pigmented cells compared to depigmented control is depicted as a heat map right. C Heat map of expression changes fold change in mRNA levels of a panel of known DNA replication stress response genes by qRT-PCR analysis in B16 melanoma pigmented day 7 cells treated with PTU or tyrosine across three biological replicates.

D Western blot images and quantitation of p-RPA2 and total RPA2 in B16 melanoma unpigmented day 0 cells compared to pigmented day 7 cells.

Numbers below represent beta-actin normalized fold changes wrt shNT at day 0. Two biological replicates were performed. E DNA fiber assay of unpigmented day 0 and pigmented day 7 B16 mouse melanoma cells. Top Representative images of the DNA fiber tracts are shown.

Bottom Quantification of fork length μm and fork speed in kb per minute is shown as bar graphs. Bars represent mean ± SEM across triplicate biological experiments. Student's unpaired t -test was performed.

Scale bar 5 μm. F Cell cycle analysis carried out using propidium iodide staining detected by FACS is depicted as stacked bars.

Sub G0 population is marked as apoptotic. Experiment was carried out in biological triplicates and mean ± SEM is depicted. Molecularly, replication stress is slowing or stalling of replication fork progression associated with DNA replication Although the importance of replication stress response is well-recognized, molecular events associated with this remain enigmatic.

Validation of this set of genes in depigmented day 0 and pigmented day 7 B16 cells confirmed that these genes were induced during pigmentation Figure 2B. Expression of these genes was further augmented with tyrosine and reduced with PTU treatment confirming elicitation of replication stress response during pigmentation Figure 2C.

Protein levels as well as the phosphorylation status of RPA2 at Ser 33 residue that are hallmarks of replication stress response also showed a pigmentation dependent induction Figure 2D. Based on DNA fiber assay, we further demonstrate decreased fork length and fork speed Figure 2E.

When compared to control pigmented cells, a consistent reduction in the number of cells on day 7 upon hyper-pigmentation with tyrosine, and an increase upon depigmentation with PTU was observed Figure 1C , top. Annexin V- PI based apoptosis assay confirmed that higher uncontrolled hyperpigmentation results in an increase in the apoptotic population.

While this cell death was seen to be higher in tyrosine treated cells which are hyperpigmented, control pigmented cells also showed apoptosis compared to depigmented cells Supplementary Figure S2C, D.

Taken together we conclude that melanin causes DNA damage, and a key response is elicitation of replication stress resulting in the arrest of cell cycle progression.

Response and repair mechanisms would be operational in the pigmented cell to sense and correct the DNA damage. Cells use specialized polymerases to resume DNA synthesis by bypassing it.

For achieving this a battery of translesion polymerases are known to play an important role We therefore studied the expression changes of all the DNA translesion polymerases during pigmentation in B16 melanoma cells.

Profiling of expression changes using the earlier microarray data, in DNA translesion polymerases on different days of progressive pigmentation revealed that three of the translesion polymerases Polk , Polh and Rev3l were increased in expression at early and late-phase of pigmentation Supplementary Figure S3A.

To investigate whether their regulation is critically dependent on pigmentation, B16 cells were treated with PTU, tyrosine or both and on day 7 were subject to qRT-PCR analysis for these three candidate genes. Regulation of Polk paralleled the observed pigmentation Figure 3A. Whereas, the other two translesion polymerases Polh and Rev3l were minimally altered and did not show an expected trend in their regulation with pigmentation and its suppression with PTU Supplementary Figure S3B, C.

Thereby, Polκ emerged as a promising candidate that could have an important role in DNA damage response during pigmentation. Melanogenesis induces Polκ via ATR-CHK1 signaling axis.

A mRNA levels of Polk in B16 cells that are allowed to pigment differentially in the presence of μM PTU, 1 mM tyrosine or both. Bars represent percent mRNA levels compared to control as mean ± SEM across five biological replicates. B Immunofluorescence of B16 cells treated with μM PTU, 1 mM tyrosine or both.

Top panel is the bright field BF and dark granules are the pigment accumulation. Nuclear DNA stained with DAPI blue , Polκ in red and γH2AX in green. Experiments were performed in triplicates.

C Quantitation of mean nuclear fluorescence intensity of Polκ of differentially pigmented day 7 B16 cells treated with PTU, tyrosine or both shown in B. Bars represent mean ± SEM across two biological replicates. D Western blot analysis of nuclear and the post-nuclear cytoplasmic lysates of B16 cells on day 5, day 6 and day 7 of pigment accumulation.

Numbers below the blot correspond to day 5 normalized expression of the indicated protein. Experiment was performed in duplicates. E Western blot analysis of B16 cells treated with DMSO or 50 nM AZ20, a selective inhibitor of ATR kinase.

Numbers below the blot correspond to control normalized expression of the indicated protein wrt GAPDH. Experiments were performed in duplicates. Immunocytochemical localization of Polκ and labelling by γH2AX puncta was simultaneously carried out in differentially pigmenting B16 cells.

We observed high Polκ as well as γH2AX labelling in tyrosine treated hyperpigmented cells Figure 3B , C. Prevention of pigmentation by PTU reversed the levels of both Polκ and γH2AX staining, substantiating the induction to be pigmentation dependent.

Whole cell lysate confirmed the induction of Polκ with progressive pigmentation Supplementary Figure S3D. Subsequent cell fractionation followed by western blot analysis on different days of pigmentation confirmed induction of Polκ in the progressive pigmentation model Figure 3D.

From these lysates, a clear induction of nuclear localized phosphorylated ATR and phosphorylated CHK1 during pigmentation further indicated activation of DNA repair response pathway during progressive pigmentation. Having observed an induction of Polκ during pigmentation and a concomitant increase in the ATR-CHK1 signaling axis, we hypothesized that the ATR-CHK1 pathway could be responsible for Polκ induction.

To test this, during pigmentation, we treated the cells with 50 nM of ATR inhibitor AZ Western blot analysis confirmed decrease in p-CHK1 levels and reduction in Polκ Figure 3E. Hence we identify Polκ induction to be under the control of ATR-CHK1 signaling axis.

Thereby, this translesional polymerase is likely to function as a key sensor or a responder in combating the pigmentation induced DNA damage.

It is likely that treatment with chemicals such as tyrosine and PTU could trigger the changes in Polκ levels in cells independent of pigmentation.

Hence, we genetically ablated Tyrosinase, the key enzyme involved in melanin synthesis using CRISPR based methodology Supplementary Figure S4A. Individual depigmented and control pigmented colonies derived from a single cell, were then trypsinized and screened for their ability to pigment, TYR protein levels and in vitro enzyme activity involving L-DOPA zymography Supplementary Figure S4B, C.

Sequence confirmation identified a single base deletion downstream to the sgRNA sequence, resulting in a frameshift mutation Tyr fs encoding a truncated protein Supplementary Figure S4A. Thereby these mutant cells were compromised in their ability to make melanin and could be compared with B16 WT cells for studying DNA damage under conditions of low-density culturing wherein progressive pigmentation is induced.

We first assessed the staining of day 7 B16 WT and Tyr fs cells with γH2AX foci and detection of newly synthesized DNA using EdU coupled fluorophore labelling Figure 4A. B16 WT pigmented cells have a significant overlap of EdU with γH2AX signals indicating that these are regions of DNA damage where new DNA synthesis is ongoing.

Interestingly, these wild type cells showed heterogeneity in double stained population. On closer analysis, the heavily pigmented cells had several double positive foci, whereas cells with moderate pigmentation had lower levels of colocalized puncta Figure 4B.

Analysis of the EdU and γH2AX colocalized spots clearly suggested that the pigmented cells had a higher proportion of DNA breaks, that are in the process of repair wherein fresh DNA synthesis is ongoing.

Whereas B16 Tyr fs remain depigmented and had lower level of γH2AX staining concomitant to a high EdU puncta per cell. This suggested higher proliferation, which was confirmed by the growth curve analysis of B16 WT and B16 Tyr fs cells Figure 4C.

DNA breaks determined by comet analysis indicated that the mutant depigmented cells had minimal DNA damage, as inferred from alkaline comet assay Figure 4D. Furthermore, western blot analysis showed sustained induction of Polκ only in B16 WT pigmented cells but not in the depigmented B16 Tyr fs cells, in which the levels decreased from day 5 to day 7 Figure 4E.

Thereby the overall DNA damaging effects of melanin and the cellular response of melanocytes by invoking Polκ is confirmed using this genetic model. While it is tempting to speculate that the difference in proliferation is due to the lack of sustained Polκ induction in Tyr fs cells, it cannot be unequivocally concluded.

This establishes the causal link between melanogenesis, DNA damage and Polκ during pigmentation. CRISPR-based genetic ablation of tyrosinase prevents melanogenesis, reduces DNA breaks and curtails Polκ induction. A Immunofluorescence images of B16 WT and B16 Tyrosinase mutant Tyr fs cells that were allowed to pigment for 7 days.

Bright field images indicate the presence of melanin granules. Merged images show co-localization of γH2AX with EdU yellow.

A zoomed in view of a single cell is shown as an inset. Scale bars represent 10 μm. B Top Cell pellets of B16 WT and Tyrosinase mutant Tyr fs cells on days 5, 6 and 7 of pigmentation.

Bottom Quantitation of average of the number of γH2AX puncta that are single positive or double positive colocalizing with EdU puncta per cell across 50 cells in B16 WT and B16 Tyr fs cells is depicted as a violin plot across three biological replicates.

The percent population of cells with indicated puncta is mentioned alongside. C Growth curve analysis of B16 WT and Tyr fs cells on days 0, 5, 6 and 7 of pigmentation.

Each point represents mean ± SEM across biological triplicates. D B16 WT and Tyr fs cells on days 5 and 7 of pigmentation were subjected to single cell electrophoresis and alkaline comet assay was performed.

Mean tail moment distribution across each population of duplicate biological experiments with at least 50 comets analyzed is depicted by a violin plot. E Western blot analysis of whole-cell lysates of B16 WT and B16 Tyr fs cells on day 5 and day 7 stages of pigmentation.

Numbers below the blot correspond to day 5 GAPDH normalized expression of the indicated protein. Experiments were performed in biological duplicates with similar results. Since B16 cells are derived from mouse melanoma, it is likely that the observed response could be restricted to melanoma cells and not a physiological response of melanocytes.

Hence, we subjected already pigmented normal human epidermal melanocytes NHEM that are derived from healthy skin and represent primary melanocytes, to differential pigmentation with PTU and tyrosine for seven days Figure 5A.

Assessment of H2AX phosphorylation by western blot analysis revealed a similar pattern of increased phosphorylation and a concomitant increase in total protein level with hyperpigmentation Figure 5A.

This was recapitulated in the foci formation detected by immunofluorescence analysis Figure 5B , C. Alkaline comet assay revealed that the DNA breaks were highest in hyperpigmented cells and lowest in depigmented cells Figure 5D.

Hence, these observations indicate that pigmentation is indeed a strong physiological response that causes DNA breaks in non-transformed primary cells.

We therefore propose that melanogenesis is potentially genotoxic and causes DNA damage in melanocytes. Normal human epidermal melanocytes NHEM respond to pigmentation induced DNA breaks by elevating Polκ.

A NHEM cells were treated with μM PTU or 1mM tyrosine for 7 days for differential pigmentation. Top Cell pellet, bottom western blot analysis of cell lysates with POLK, HSC70, phosphorylated H2AX, total H2AX and beta actin antibodies.

Numbers below the blot correspond to control normalized expression of the indicated protein. Experiments were performed in biological duplicates. B Immunofluorescence of NHEM treated with PTU or tyrosine with phosphorylated H2AX antibody. Experiments were performed with two biological replicates.

C Quantitation of mean fluorescence intensity per cell of γH2AX from two biological replicates of NHEM treated with PTU or tyrosine shown in B. D PTU and tyrosine treated NHEM cells were subjected to single cell electrophoresis and comet analysis alkaline comet assay.

E Heat map of expression fold change in mRNA levels of top two translesion polymerases that were enriched in B16 microarray top , and a panel of known DNA replication stress response genes by qRT-PCR analysis in NHEM Control, PTU or tyrosine treated. Data represented as mean of triplicate biological experiments.

F Western blot analysis of NHEM treated with DMSO or 50 nM AZ20, a selective inhibitor of ATR kinase, for 24 h. Numbers below the blot correspond to control normalized expression of the indicated protein wrt beta-actin.

Bars represent percent mRNA levels compared to control across biological triplicates. Experiments were performed in biological triplicates.

While POLK showed a correlation with pigmentation response, POLH showed an opposite trend. Validating the B16 based observations, several of the replication stress response genes were elevated in primary melanocytes treated with tyrosine, strengthening the possibility of DNA damage associated with a Polκ response elicitation at the RNA level.

Polκ at the protein level was induced upon hyperpigmentation and reduced upon depigmentation in cultured primary human melanocytes Figure 5A. Involvement of ATR-CHK1 axis in this induction was confirmed using ATR inhibitor AZ20 Figure 5F. As the control primary cells are constitutively pigmented, the reduction observed with AZ20 was lower as compared to B16 cells wherein the progressive pigmentation could be induced in presence of the inhibitor.

We then assessed the ability of melanin modified DNA to elicit a Polκ response in melanocytes that do not make melanin. Transfection of plasmid DNA incubated with melanin synthesis reaction column purified after 16 h of incubation in depigmented B16 cells induced Polκ protein expression Figure 5G.

We could observe the induction of Polκ at the RNA level in these cells only when transfected with plasmid DNA incubated with melanin synthesis reaction or extracellular melanogenesis achieved with 1mM tyrosine and tyrosinase enzyme in the culture medium. Whereas, mere incubation of pre-synthesized melanin with plasmid DNA just before transfection, pre-synthesized melanin alone, or melanin synthesis reaction mixture purified through the column did not elicit Polκ response in these depigmented cells.

Similar to the RNA level changes we could confirm melanin-modified plasmid DNA to elevate protein levels of Polκ Figure 5G , inset. The melanin intermediate DHI, that caused DNA breaks in B16 cells Figure 1F , was also able to increase POLK mRNA expression. Hence, the response of melanocytes to pigmentation is the induction of Polκ through melanin-mediated DNA damage.

We then proceeded to investigate the role played by Polκ in maintaining genome integrity and cellular homeostasis during sustained pigmentation. We resorted to silencing Polκ in B16 cells stably expressing shRNA targeting Polκ shPolκ and this was compared to non-targeting shRNA against luciferase shNT.

B16 cells were freshly transfected and used as an enriched pool to prevent multiple consequences of Polκ depletion Under pigmenting conditions both shNT and shPolκ showed comparable pigmentation by day 7 Figure 6A. We observed that the depigmented day 0 cells had minimal γH2AX puncta, and these were comparable across shNT and shPolκ cells.

Pigmented day 7 shPolκ cells have more mean fluorescence intensity of the foci indicating higher DNA damage Figure 6B. With progressive pigmentation the level of DNA damage increased more in the shPolκ cells compared to shNT cells Figure 6C.

Similarly, single stranded DNA breaks and alkali sensitive abasic sites assessed by comet assay showed a progressive increase in the mean tail moment with pigmentation. This increase was more in the shPolκ cells on day 7 when the pigmentation was the highest Figure 6D. Sequence independent silencing RNA siRNA also confirmed this increase in mean tail moment in pigmented cells Supplementary Figure S5B.

shPolκ cells resulted in an increase in the number of abasic sites, confirming a role for Polκ in resolving these lesions Supplementary Figure S5C. Abasic sites arise from melanin-modifications Figure 1C , and this result confirms a critical role for Polκ in its repair perhaps by its TLS activity.

These results indicate that Polκ is required to resolve the DNA damage caused by melanin. Silencing of Polκ during pigmentation prevents replication stress response despite elevated DNA damage.

A Cell pellets of control non-targeting shNT and Polκ silenced shPolκ B16 cells on day 0 left and day 7 right of pigmentation. B Immunofluorescence analysis of day 7 pigmented shNT and shPolκ cells with phosphorylated H2AX antibody puncta labelled in green and the nucleus is counterstained with DAPI blue.

C Quantitation of mean fluorescence intensity of γH2AX shown in B from two biological replicates of shNT and shPolκ cells across day 0, 5 and 7 of pigmentation induction. Two-way ANOVA was performed for multiple comparisons. D shNT and shPolκ expressing pigmented B16 cells were subjected to single cell electrophoresis and comet analysis alkaline comet on days 0, 5 and 7 of pigmentation induction.

E Growth curve analysis of shNT and shPolκ expressing B16 cells on days 0, 5, 6 and 7 of pigmentation. F Western blot analysis of DNA repair and cell cycle related proteins in shNT and shPolκ cells. Numbers below represent tubulin normalized fold changes wrt shNT.

The volume of the tumor was non-invasively monitored and plotted over time of biological triplicates mean ± SEM.

H Heat map of expression fold change in mRNA levels of a panel of known DNA replication stress response genes by qRT-PCR analysis in shNT day 0-unpigmented and day 7-pigmented and shPolκ day 0-unpigmented and day 7-pigmented B16 cells.

I Western blot images and analysis of p-RPA2 and total RPA2 in shNT and shPolκ B16 cells day 0 unpigmented and day 7 pigmented. J Analysis of melanoma samples from TCGA data for mRNA expression of POLK high, low or not detected segregated into bar plots and proportion of mutations were plotted on y-axis.

K Survival plot of melanoma patients with low or high expression of POLK from TCGA data. Analysis from Human Protein Atlas database. Paired t -test P value 0. Pigmenting melanocytes elicit induction of Polκ as a response through ATR-CHK1 axis Figure 3E.

Further, silencing studies indicated that Polκ is necessary to keep a check on the DNA damage caused during pigmentation. Based on these results we anticipated that silencing of Polκ would result in more cell cycle arrest and cell death as a consequence. Interestingly, the number of cells were significantly higher upon Polκ knockdown during progressive pigmentation Figure 6E.

While the growth differences were non-significant at earlier days, the differences become prominent on day 7, when the pigmentation was highest. Additionally, we verified that pigmented shNT and shPolκ cells exhibited comparable PI-stained populations, indicating similar cell viability, and the annexin-V labeled population showed similar levels of apoptosis between them Supplementary Figure S5E, F.

This unexpected observation prompted us to look at the battery of proteins associated with cell cycle. A panel of orchestrators and effectors were analyzed by western blot on day 7 of pigmentation upon knockdown of Polκ. Confirming the silencing, levels of Polκ protein was downregulated Figure 6F.

PCNA is an effector of Polκ and a marker of proliferating cells. However, upon Polκ silencing PCNA levels remained unaltered, perhaps due to opposing effects of proliferation and the absence of key repair polymerase.

However, both p53 and p21 that function to couple DNA damage to cell cycle arrest were low and CDK2 was elevated, further substantiating that more cells are in the proliferative phase of cell cycle.

We consistently observed that shPolκ cells formed tumors with higher volume, confirming that under this pigmenting condition as well, insufficient induction of Polκ results in lack of cell cycle arrest Figure 6G , Supplementary Figure S5G, H. Hence, Polκ knockdown does not result in cell cycle arrest or cell death as anticipated, instead the DNA-damaged cells continue to proliferate more.

We then assessed the replication stress response genes and observed a pronounced induction in pigmented day 7 shNT cells as observed earlier for control B16 cells Figures 6H , 2B. Pigmented day 7 shPolκ cells failed to mount such a response and several of the genes were mildly downregulated compared to shNT day 0 cells.

Indicating that Polκ is a crucial elicitor of replication stress response when cells are challenged with melanin modified DNA. This was further strengthened by the loss of induction of phosphorylated and total RPA2 levels in day 7 shPolκ cells Figure 6I.

In the absence of Polκ, despite the presence of elevated DNA damage, cells aberrantly progress through the cell cycle and proliferate more. This could potentially render these pigmenting cells vulnerable to genome instability. Substantiating this possibility, a recent systematic investigation of combination of DNA damaging agents and repair gene knockouts carried out in C.

elegans highlighted an increased mutation frequency for alkylating agents MMS and DMS upon deletion of the Polκ ortholog In the context of melanocytes, we would expect that Polκ would be the primary mitigating response to melanin-induced DNA damage, instead we observe that Polκ is the primary mediator of cellular response caused by this damaged DNA.

At a molecular level the mechanism by which a translesion polymerase like Polκ would be able to elicit a replication stress response remains enigmatic. It is likely that Polκ based flagging of melanin lesions may elicit this response by invoking other repair proteins like CHK1.

Having established the role of sustained melanogenesis in causing DNA damage and mapping the Polκ response by melanocytes, we decided to explore whether this is relevant in the human melanoma context.

We extracted the cutaneous melanoma samples from The Cancer Genome Atlas TCGA and segregated the samples based on the mRNA expression of POLK While 83 melanoma samples did not have detectable expression of POLK mRNA by sequencing, FPKM based binning resulted in patients with high levels of POLK and patients had lower expression of POLK Overall number of somatic mutations were highest in high- POLK group and least in samples that did not have detectable POLK Figure 6J.

Thereby, suggesting that expression of POLK could explain a fraction of mutations in human melanoma, and provide physiological relevance to the observations in cultured cells.

The status of pigmentation of melanoma compared to cognate skin-derived melanocytes was not available in the TCGA meta-data, to empirically assess this we looked at the expression of several pigmentation genes for which the data was available. We observed that most of the pigmentation genes were not significantly different across the three groups, suggesting similar distribution of pigmentation Supplementary Figure S5G.

Subsequently, our focus shifted towards identifying distinct signatures within the array of somatic mutational changes associated with varying levels of Polκ expression. In a prior study, we had already compared the somatic mutational spectrum of different cutaneous malignancies with both lesional and non-lesional vitiligo skin samples In this investigation, we reanalyzed the data, specifically focusing on melanoma samples from TCGA, grouped according to their respective Polκ expression levels.

Notably, SBS7a and SBS7b were prevalent in both groups of melanoma samples, as well as in various other cutaneous tissues, including healthy skin Supplementary Figure S5J.

These signatures were likely linked to sun exposure, a well-known factor in cutaneous mutational patterns 20 , 38 , Moreover, we identified additional signatures enriched in melanoma samples.

Among them, SBS6 and SBS21 were enriched exclusively in low Polκ tumors, and interestingly, they are associated with genome instability. Conversely, SBS31 was found to be enriched solely in high Polκ tumors, and it is linked to prior chemotherapy with Platinum drugs, which induce DNA adducts, resembling the proposed mechanism involving melanin in this study.

Although it would have been intriguing to explore the correlation of SBS signatures with Polκ levels in the context of patients' prior chemotherapy history, unfortunately, due to a lack of available information, we could not establish conclusive evidence in this regard.

Thereby, in this study we reinforce the correlation of DNA damage and possible mutations with pigmentation. This provides a compelling reason to further investigate this aspect of eumelanogenesis and its implication in melanoma in future.

While the UV protection role is likely to be overarching, the current work unravels a rather unwanted side-effect during synthesis of the esoteric biopolymer melanin. This was earlier alluded to, but the details of the DNA damage and the response elicited by melanocytes remained elusive so far 5 , 6.

In this work, using cell-bases models, we demonstrate that uncontrolled melanin synthesis, via its intermediates causes DNA damage. The key response of pigmenting cells is the replication stress response which critically involves induction of translesion polymerase Polκ via the ATR-Chk1 signaling axis.

Among translesion repair enzymes, Polκ is necessary to bypass large bulky adducts on the nucleotide bases that are not amenable for repair by other DNA repair systems 40 , Additionally other non-canonical, non-TLS functions of Polκ such as stabilizing replication forks via Chk-1 and repair of oxidized dNTPs are well-known Herein we report yet another unexpected, crucial cellular function of Polκ in eliciting replication stress response in melanocytes undergoing pigmentation induced DNA damage.

Supported by the co-localization of EdU and γH2AX positive foci in pigmented cells, our initial notion was that the role of Polκ would be restricted to translesional bypass of melanin modified DNA adducts.

Some TLS enzymes such as Polζ help cancer cells come out of replication stress However, Polκ elicits replication stress response upon melanin-induced DNA damage.

This is strikingly different to the reported role of TLS enzymes as a mitigator of replication stress This could be attributed to the melanin-based DNA lesions, as opposed to hydroxyurea induced replication fork stalling, as the cause of replication stress It is also likely that additional factors associated with melanogenesis such as protein oxidation could also contribute to the replication stress response and this would need to be independently assessed In an earlier study, authors report subcellular localization of Polκ inside nucleus of human melanoma cells and this involves mTOR pathway The work further demonstrated Polκ to provide survival advantage upon pharmacological inhibition of the oncogenic BRAF, independent of creating somatic mutations in the genome.

In their study, overexpression of Polκ did not significantly increase the mutation burden. However, this remains to be seen in the context of ongoing melanogenesis.

We do observe that in our study the induction of Polκ in response to melanogenesis is independent of mTOR pathway, as the pharmacological inhibition by PP does not alter Polκ levels Supplementary Figure S3E.

In another study, role of Polκ in promoting DNA synthesis and recovery of replication stress at stalled forks upon nucleotide deprivation was elucidated The link between CHK-1 and Polκ seems to be 2-fold, while in some cells loss of Polκ abrogates CHK-1 phosphorylation 42 , 45 , whereas in multiple cell types knock down of Polκ did not alter CHK-1 phosphorylation In these studies, effect of CHK-1 phosphorylation on Polκ induction is not systematically investigated.

Our observations highlight a third interplay wherein Polκ induction is under the control of ATR-CHK-1 axis. Functions of Polκ during DNA damage response may be different to its homeostatic role in DNA fork stalling.

Most of the work on UV-induced melanoma formation has concentrated on direct DNA damage. This is substantiated by the prevalence of UV-signature mutations in several melanoma samples.

This link is further strengthened by the recent data suggesting an increase in the incidence of melanoma among users of artificial tanning beds Surprisingly, despite deep-seated within the epidermis and loaded with melanin, melanocytes are not that well-protected from UV and are vulnerable to DNA damage.

It is apparent that there are both UV-signature mutations and unexplained large-scale genomic alterations associated with malignant cells that could be as important as point mutations.

Suggesting that melanin damage followed by translesion bypass by Polκ could account for at least a fraction of the known mutations in melanoma. It is likely that UV-induced melanogenesis could result in these driver mutations adding a layer of complexity to the link between UV and melanoma.

While the activation of melanocytes in conditions such as tanning enable adaptive pigmentation response, repeated or uncontrolled activations could alter these cells and possibly promote mutagenesis and facilitate their transformation into melanoma. The role of Polκ in restoring melanocyte homeostasis seems to be 2-fold, with TLS and more importantly as a check point controller.

While the former may involve some errors, the latter effect of Polκ is primarily protective against deleterious mutations, providing reasons for its evolutionary conservation.

Thereby, our study establishes a hitherto unknown, undesirable side of melanin and its possible involvement in melanoma. The data underlying this article are available in the article and in its online supplementary material.

Supplementary Data are available at NAR Online. and M. acknowledge CSIR for Research Fellowship. We thank the FACS facility at CSIR-IGIB for extending support for the conduct of experiments pertaining to apoptosis and cell-cycle seamlessly.

Council for Scientific and Industrial Research CSIR , India, through grant RegenX [MLP]; Department of Biotechnology [GAP] provided support to the execution. Funding for open access charge: Council for Scientific and Industrial Research.

Conflict of interest statement. is the co-founder of Vyome Biosciences Pvt Ltd, a biopharmaceutical company working in the dermatology area. Brenner M. The protective role of Melanin against UV damage in Human skin.

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Here, we determine the genome-wide effects of MMR on mutation. We first identify almost mutations accumulated over five generations in eight MMR-deficient mutation accumulation MA lines of the model plant species, Arabidopsis thaliana We then show that MMR deficiency greatly increases the frequency of both smaller-scale insertions and deletions indels and of single-nucleotide variant SNV mutations.

Most indels involve A or T nucleotides and occur preferentially in homopolymeric poly A or poly T genomic stretches. In addition, we find that the likelihood of occurrence of indels in homopolymeric stretches is strongly related to stretch length, and that this relationship causes ultrahigh localized mutation rates in specific homopolymeric stretch regions.

ABOVE: Scanning electron microscopy image of Arabidopsis thaliana © ISTOCK. Specifically, genes playing a crucial role in survival and reproduction mutate far less often than those that are less important. Arabidopsis is a small flowering plant that has a comparatively small genome, making it a popular system for genetic research.

The study, which began at the Max Planck Institute for Biology in Germany and was carried over to University of California, Davis, when lead author and plant scientist J.

Grey Monroe got a job there, finds a 58 percent lower mutation rate within genes than in the areas of the genome around them. On top of that, genes considered essential had a 37 percent lower mutation rate than those in which modifications would be less likely to prove disastrous.

Mutations are distributed in a way that seems to be beneficial to the plant, Monroe tells The Scientist. He adds that his paper is the first comprehensive analysis in a eukaryotic species that connects the mechanisms driving the variability in mutation rate at the cellular level with the finding that more important genes seem protected from mutation.

It also clarifies and reinforces earlier studies that indicated nonrandom mutation rates, but whose results were less clear-cut or obtained with older techniques. Monroe and his colleagues found evidence of specific epigenetic characteristics such as cytosine methylation that prevent mutations from occurring in those regions, not unlike protective barriers.

He says that it provides an important next step, a reinforcement of earlier findings using newer, better technology, in a long history of mutation research. Monroe and his team ran mutation accumulation MA lines, in which organisms are separated from one another and carefully inbred across multiple generations, and the genomes of all individuals are sequenced and scrutinized for changes.

The technique became possible with the advent and increasing accessibility of high-throughput gene sequencing and allows researchers to pinpoint small and uncommon mutations with greater precision and ease than prior techniques.

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