| Germline mutations in breast cancer susceptibility gene, BRCAl predispose women to breast cancer and ovarian cancer. Lower than normal expression of BRCAl is thought be an important contributing factor in sporadic breast cancer. BRCAl is a large protein comprising 1863 amino acids with a molecular weight of 220 kDa in human. BRCAl contains two important protein interaction domains. In the amino terminus of BRCA1, there exists a zinc - binding RING domain. BARD1, the BRCAl - associated RING domain has been identified as protein that associated with the amino terminus of BRCA1. The amino terminus of BRCAl has ubiquitin polymerase activity by itself, and the BARD1 association enhances the ubiquitin polymerase activity. In the carboxy terminus of BRCAl, there are two tandem copies of BRCT domain, which are highly conserved , are found in DNA repair proteins, and behave as phosphopeptide binding modules.A number of evidences have shown that BRCAl is involved in the cellular processes of DNA repair, transcription, cell cycle regulation, and chromatin remodeling, apoptosis, and protein ubiquitination. Mouse and human cells deficient for BRCAl are sensitive to various type of DNA damage, indicating that BRCAl have functions as a caretaker of genome integrity through its role in repairing DNA damage.DNA double - strand breaks ( DSBs) are a common form of DNA damage. DSBs are produced directly by ionizing radiation or some chemicals, and indirectly by a product of blocked replication forks. Repairing DSBs correctly is a fundamental mechanism for genome protection. As DSB repair pathways, HR,NHEJ, and single - stand annealing ( SSA) have been already known. It is thought that BRCAl is mainly involved in the repair pathways of HR. In spite of many reports describing BRCAl response to DSBs, it has been still unknown when BRCAl accumulate at the damage sites and how they play a role for DNA repair system.SSBs are discontinuities in the sugar - phosphate backbone of one strand of a DNA duplex. Hundreds of thousands of cellular SSBs arise from DNA damage each day, and if these are not repaired, they can be converted into potentially clastogenic and/or lethal DNA double - strand breaks ( DSBs ). The two major sources of endogenous SSBs are sugar damage and DNA base damage arising primarily from attack by reactive oxygen species (ROS) and other _electrophilic molecules, and from the intrinsic instability of DNA. It is generally accepted that SSBs are far less toxic to cells than their double - stranded counterparts;however, this largely reflects the ability of cells to rapidly repair large numbers of SSBs, rather than an intrinsic lack of toxicity of these lesions. Indeed, any SSB not repaired prior to DNA replication or actively tolerated'in the appropriate way during DNA replication will become a DSB. Given the frequency of spontaneous ' SSBs, it is thus not surprising that cells have evolved highly efficient mechanisms to minimize their impact. Perhaps the most compelling experimental evidence for this emerges from mammalian cells in which there is a defect in SSB repair (SSBR). The loss of SSBR leads to cell genetic deletion, chromosome aberrations, and final premature aging, genetic instability which ultimately lead to tumorigenesis.We have already established a laser light micro - irradiation system to create various DNA damage, including DSB, SSB, and base damage in living cells. Here we analyzed accumulations of endogenous BRCAl and green - fluorescent -protein (GFP) -tagged several BRCAl deletion mutants and missense mutants at the site of DSB in a real time course. We found that both the amino terminus and the carboxy terminus of BRCAl are important for the accumulation to the site of DSB. BARD1, the important patterner protein of BRCAl, also accumulates to the DSBs, and its kinetics of accumulation mimics the one of WT BRCAl. Furthermore, the short fragment, amino acid 101 -200 of BRCAl inthe amino terminus could accumulate to DSBs. Several cancers associated mis-sense mutations in the amino terminus and BRCT domain, lost or weakened the accumulation. In the absence of ATM,H2AX and DNA - Pkcs cell, BRCA1 could accumulate at DSBs sites normally. On the other hand, in the absence of NBS1 cell, full length of BRAC1 could accumulate normally but the carboxy terminus lost the responsibility. And in the absence of Ku80 cell, the N - terminus lost the responsibility. Our results suggest that BRCA1 accumulate at the laser -induced DSBs sites by its amino terminus and the carboxy terminus using different mechanism to function for the DNA repair in living cells, and lost or weakened function at DSB sites of mutants used here explicated why these point mutations cause carcinomas.Although BRCA1 is associated with various types of DNA damage response, it is unknown whether BRCA1 play a role for SSB repair. It might be due to that there is little experimental procedure that induces SSB alone in cells. We have already established a laser light micro - irradiation system to create various DNA damage, including DSB, SSB, and base damage in living cells. Here we analyzed accumulations of endogenous BRCA1 and green - fluorescent -protein (GFP) —tagged several BRCA1 deletion mutants and at the site of SSB in a real time course. We found that the carboxy terminus of BRCA1 plays a critical role to move to the site of SSB. Loss of the carboxy terminus of BRCA1 could let cell sensitive for the drug MMS, this perhaps is one of the tumorige-neitc mechanism of BRCA1;Materials and MethodsPlasmid Construction of GFP - Fused Genes:All BRCA1 mutant and deletant tested in this study are constructed and confermed. Full -length GFP-tagged BRCA1 (WT) and BRCA1 containing a RING domain mutation C61G or a BRCT domain mutation M1775R, were sub-cloned from constructs in the pEGFP - Cl vector. The a. a. 1 -302, a. a. 305 -770, a. a. 775 - 1292, a. a. 1527 - 1863 deletants were constructed as previously scribed. The a. a. 303 - 1526 fragment was generated by digestion of the a. a.1 -302 deletant with Sac I and Not I , and then the rest fragment BRCA1 -303 -1526 - pEGFP was blunted and ligated. The 1527 -1863 fragment was constructed by digestion of full - length GFP - tagged BRCA1 with Xho I and Sac I , then the rest fragment pEGFP - BRCA1 - 1527 - 1863 was blunted and ligated. The a. a. 1 - 100, a. a. 1 - 200, a. a. 1 - 304, a. a. 101 - 200, a. a. 101 -304, a. a. 201 - 304 fragments amplified from full - length GFP - tagged BRCA1 with PCR were cloned into pEGFP - C3 vectors.Cell lines and Transfection ?.Saos2 cells, ATM,H2AX, DNA -Pkcs NBS1 and Ku80 wild type and deficient cells, were planted in DMEM and 293T cells were planted in RPI - 1640 supplemented with 10% FBS at 37T!, in a 5% CO2/95% air atmosphere. SaoS2 cells were planted on 35 - mm glass - bottom dishes at 50% confluence 24 h before the transfection and irradiated with laser light under the microscope 48h after transfection.Immunocytochemistry and Chemicals;Saos2 cells were laser - light irradiated and stained with antibodies raised against human -yH2AX and BRCA1. Saos2 cells were fixed with methanol - acetone for 10 minutes after irradiation, and then dried. Anti - phosphorylated H2AX, anti - BRCA1 - M were used. Fixed cells were washed with 0. 05% PBS -T for three times. After blocking by 1.5% skin milk at room temperature for 30 minutes, cells were incubated with antibodies dilution in 1% BSA -PBS at 37 X. for 1 hour. After washing treatment, cells were incubated with Alexa Fluor 594 goat anti - rabbit immunoglobulin G conjugate, FITY goat anti -mouse immunoglobulin G conjugate at 1;400 dilution in 1% BSA -PBS at 37X1 for 30 minutes. Cell samples were then mounted in drops of mounting buffer. Confocal imaging was performed with an Olympus FV - 500 Confocal laser system connected to an Olympus microscope with a 40 x oil immersion objective lens.Microscopy and laser - light irradiation:Florence images were obtained and processed using an Fv - 500 con - focal scanning laser microscopy system (Olympus). A 405nm scan laser system (O-lympus) for irradiation of cells in the epi - fluorescence path of the microscopesystem was used here. One scan of the laser light at full power delivers energy of around 1600nW. We used only a full power from the 405nm laser in this study, and used 500 scanning times which mainly induced DSB as described previously. A 365nm pulse laser microscope was used as previously described. We used a lower dose (0. 75jxJ) , which was obtained by passing laser light through a F20 filter, in front of the lens. By using this system, SSBs were produced at restricted nuclear regions of mammalian cells. 405nm laser light was focused through a 40 x objective lens. Cells were incubated with Opti - medium in glass- bottom dishes, which were places in chambers to prevent evaporation, on a 37*^0 hot plate. The energy of fluorescent light at the irradiated site was measured with a Laser power/energy monitor. The mean intensity of each focus was obtained after subtraction of the background intensity in the irradiated cell. Each experiment was performed at least three times and the data presented here are mean values obtained in a given experiment.Immunoprecipitation and Western - blotHEK - 293 T cells were grown in RPI - 1640 medium supplemented with 10% fetal bovine serum, 100 jxg/ml penicillin and streptomycin and transfected with expression vector for checking the expression by western - blot. And GFP- BRCA1 - 1 - 100, GFP - BRCA1 - 1 -200, GFP - BRCA1 - 101 -200 were transfected with FLAG - BARD1 to HEK -293T cells for immunoprecipitation. Two days post - transfection, cell lysates were prepared in 1 ml of wash buffer. For immunoprecipitation, 3 jxl of anti - GFP monoclonal antibody was used. For Western blot analysis, samples were subjected to electrophoresis in 5 or 5 ~ 20% SDS - polyacrylamide gels and immunoblotted using the anti - BRCA1 - M the anti - GFP antibody MMS - 118P, or the anti - FLAG M2 monoclonal antibody.Local UV irradiationLocal UV irradiation was performed by UV cross machine. XPA - Vector and XPA - UVDE Cells mono - layered in 35 - mm glass - bottom dishes were treated with ethanol or actinomycin D at 10|xg/ml final concentration 1 hour before being covered with a polycarbonate isopore membrane filter with pores of 3u,m in diameter (Millipore) and 254nm UV irradiated with a dose of 60J/m2.The polycarbonate blocked the 254nm UV - light, and cells were exposed only though the pore of the filter.ImmunocytochemistryXPA - Vector and XPA - UVDE cells were local UV irradiated and double stained with antibodies raised against human CPD and BRCA1, or transfected with GFP-XRCC1 48h before local UV irradiation and then stained with antibodies raised against BRCA1. XPA - Vector and XPA - UVDE cells were fixed and dried as described above 1 hour after irradiation. And then cells were co -dyed with antibodies.RNA interferenceShort interference - RNA ( siRNA) was synthesized by Silencer siRNA contraction Kit. The sense sequence: 5'-TGACTGGCGCTTTGAAACC -3'was used for designing siRNA for hBRCAl. Negative control was used silenserTM negative control siRNA template set. Final concentration 10 nM of siRNA was used for transfection by using oligofectamineTM regent (Invitrogene ). Immuno- blot assay was performed 48 hrs after transfection.Colony formation assayBRCA1 - deficient HCC1937 cell lines or HeLa which BRCA1 expression was blocked using small interfering RNA and WT BRCA1 - expressing HCC1937 cell lines were used in this assay. Eight hours after plating, cells were treated with methylmethanesulfonate for 1 h. Cells were then washed twice with PBS and medium was added. Colonies were fixed and stained with 0. 3% crystal violet for counting. Survival was expressed as the numbers of colonies formed from MMS- treated cells as a percentage of those from control cells. Three independent experiments were carried out for each point and the standard errors were indicated by an error bar.ResultEndogenous BRCA1 and 7H2AX accumulated at the sites of DSBs induced by laser micro -irradiation.In order to detect the production of DSBs, -yH2AX was immunostained withBRCAl after irradiation with the 405nm laser 500 scanning times. We used antibody raised against human BRCAl and 7H2AX. Both ten minutes and 1 hour after irradiation, 7H2AX were detected as clear lines in irradiated cells. 5 hours after irradiation, the lines of 7H2AX had faded away, and 10 hours after irradiation, these lines were almost indiscernible. Using the same laser scanning, ten minutes after irradiation, lines of BRCAl were detected. 1 hour after irradiation, intensity of lines are stronger than 10 min. 5 hours after irradiation, BRCAl accumulation became weak. In contrast with -yH2AX, 10 hours after irradiation, we could find that BRCAl still accumulated at the irradiated site, while the lines of *yH2AX had disappeared already. In 10 min after irradiation, 7H2AX become foci faster tan BRCAl.BRCAl accumulated at laser - induced DSBs site via its amino terminus and/or its carboxy terminus.To determine which domain is responsible for the accumulation of BRCAl at laser - induced DSBs site, we constructed several GFP - tagged deletion mutants of BRCAl, and confirmed their expressions by Western blots using antibody against BRCAl. The full -length, amino acid ( a. a. ) 1 -302, 305 -770, 775 - 1292 and 1527 - 1863 deleted mutants all could accumulate to the sites immediately after 405nm laser 500 times scans. So, we constructed the GFP - tagged - BRCAl 303 - 1526 fragment, this fragment of BRCAl did not accumulate to the laser - irradiated DSB sites. On the other hand, the amino terminus and the carboxy terminus of BRCAl could accumulate the laser - induced DSB sites. These results indicate that the amino terminus or carboxy terminus is enough to accumulate at the laser - irradiated DSB sites.By using a computer - aided analysis system, the amount of the accumulated GFP - tagged proteins was quantified. Interestingly, the a. a. 1 -304, amino terminus fragment immediately accumulated to the DSB sites after irradiation, while a. a. 1527 - 1863, carboxy terminus fragment gradually accumulated. These results suggest that both fragments of the amino terminus and the carboxy terminus of BRCAl have ability to move to the laser - induced DSB sites, but that their mechanisms for accumulation have different.C61G and M1775R are well - characterized cancer associated missense mu-tations. We observed that neither C61G mutant nor M1775R mutant accumulated to irradiated sites.BARD1 accumulated to the laser - induced DSB sites as well as BRCA1To analyze BARD1 DSB responsibility, we constructed the GFP - BARD1 protein, and also transfected to SaoS2 cell line. After 20 seconds of irradiation, the accumulation of the GFP - BARD1 protein was observed clearly at the irradiated sites. So BARD1 also have the activity to accumulate to the laser - induced DSBs lesions. The kinetics for accumulation of BARD1 mimics that of BRCA1, which indicated that the two proteins perhaps work together at DSBs sites.BRCA1 fragments, a. a. 1 -100 and a. a. 101 -200 accumulate to the laser - induced DSB sites, respectively. In the amino terminus of BRCA1, the RING domain is located in a. a. 24 - 64, to analyze whether this RING domain is responsible for the accumulation to the laser - induced DSB sites, we constructed GFP - tagged BRCA1 fragments of a. a. 1 - 100, 1 - 200, 1 - 300, 101 -200, 101 -300 and 201 -300. As expected, BRCA1 fragments containing RING domain, a. a. 1 - 100, 1 -200, and 1 -300 could accumulate to the laser - induced DSB sites. These suggest that the RING domain is supposed to be responsible for the accumulation to the laser - induced DSB sites.However, to our surprise, GFP - tagged BRCA1 fragments, a. a. 101 -200 and 101 - 300 also accumulated to the irradiated sites. Only GFP - tagged BRCA1 fragment of a. a. 201 - 300 did not accumulate to the irradiated sites. These results indicate that BRCA1 fragment of a. a. 101 - 200 might contain some domains contributing the accumulation.We selected four missense mutations in a. a. 101 -200, whose reported numbers are relatively high, Y105E, P142H, P143K, and E179C. We constructed eight 'GFP - tagged BRCA1 fragments, a. a. 101 -200 and 101 -300, containing these four missense mutations. All mutants could accumulate to the laser - induced DSB sites, intensity of fluorescent lines of BRCA1 mutants seems to be weaker than wild - type BRCA1 fragments, especially the P142H mutation, although all mutants have the same expression with wild - type fragments in SaoS2 cell lines checked by western - blot.ATM was not the unique kinase for BRCA1 and NBS1 influence the C - ter-minus of BRCAl accumulation, Ku80 influence the N - terminus of BRCAl accumulation at double - strand breaksWe also found that BRCAl can accumulate to the laser - induced DSB sites normally in AT1KY/T - n ( ataxia - telangiectasia patient cells, ATM - mutant) and H2AX deficient cell lines. Furthermore both the amino terminus and the carboxy terminus can accumulate to the DSB sites respectively. This data coincided with the reported ones that BRCAl is multi - phosphorylated by multi - protein in response to DNA damage.NBS1 is responsible for Nijimegen Breakage Syndrome. In our experiment, the accumulation at double - strand breaks sites of BRCAl full length are normal in NBS1 deficient cell line, as well as BRCAl 775 -1292 deletant and the N -terminus of BRCAl. In contrast, the C - terminus of BRCAl lost the responsibility. Ku80 is essencial for DSBs repair pathway NHEJ,the N - terminus of BRCAl lost the responsibility.BRCAl also accumulated at the irradiated sites of SSB induced by laser micro - irradiation, and only BRCT domains are essential for the accumulation.About 2 minutes after irradiated, the accumulated GFP - BRCAl - wt is barely visible, which was slower than other SSB response proteins as we reported before. Then we determined which domain is responsible for the accumulation of BRCAl at SSB sites. Different from DSB, without BRCT domains, no accumulation could be observed at the SSB irradiated sites. The GFP - BRCAl - 1527 ~ 1863 fragment accumulated to the irradiated sites clearly. From the kinetics of 775 - 1292aa J527 ~ 1863 deletants and 1527 ~ 1863 fragment, we could know that BRCT domains were essential for the BRCAl accumulation of SSB, but the 775 - 1292aa region perhaps had some domain to influence this accumulation.Endogenious BRCAl also accumulated to the SSB sites induced by UV irradiation in the XPA - UVDE cell.When there was no treatment to the cell lines, BRCAl accumulated to the UV irradiated spots where GFP - XRCC1 accumulated to in XPA - UVDE cell line, while BRCAl also formed foci at UV irradiated sites where GFP -XRCC1 didnt in XPA - Vector cell line. So we added a polymerase II inhibitor actino-mycin D which could reduce the foci of BRCAl about 80% in SaoS2 cell line af-ter UV irradiation to these couple cell lines. Then contracted with the methanol control, there was few BRCAl foci detected in XPA - Vector cell line with acti-nomycin D treatment. But in XPA - UVDE cell line, despite the inhibition of actinomycin D for UV irradiation, co - localization of BRCAl and CPD could be seen almost 70% of the irradiated area. From these data, we could ensure that BRCAl was responsible for SSB and perhaps had some important function in SSB repair.Knockdown the expression of BRCAl could let cells sensitivity to MMS which induced SSBs in cellsWe investigated the sensitivity of the BRCAl C - terminus mutated HCC1937 cell lines,BRCAl -deficient HCC1937 cell lines which BRCAl expression was blocked using small interfering RNA and WT BRCAl - expressing HCC1937 cell lines to methyl methanesulfonate (MMS) which induced single strand breaks (SSBs). HCC1937 and BRCAl -deficient HCC1937 cell lines showed similar high sensitivity to MMS, and BRCAl - expression prevented HCC1937 cell lines from this sensitivity. The similar data were also from HeLa cell lines. These data was consistent with the idea that C - terminus of BRCAl is essential for SSB repair. This at least partially participate cancer formation and deteriorates.DiscussionAlthough BRCAl has been implicated in multiple cellular responses, the precise mechanism that determines its tumor suppressor activity is not defined. BRCAl plays an important role in maintaining genomic integrity by protecting cells from DSBs that arise after DNA damage and during DNA replication.Here, using laser micro - irradiation system, we showed that BRCAl accumulated at the laser - induced DSB sites in living cells. By analysis using several deletion mutants, we found that the amino terminus and the carboxy terminus of BRCAl could accumulate independently to the laser - induced DSB sites. BRCAl comprises a RING domain at the amino terminus and two BRCT domains at the carboxy terminus. Recently, Au et al have reported that both RING do-main and BRCT domain of BRCAl cooporate to form nuclear foci after treatment with ionizing radiation. They mapped the BRCAl foci - targeting domains of yellow fluorescence protein (YFP) tagged BRCAl in breast cancer cells exposed to ionizing radiation. They have shown that YFP - BRCT domain itself localized poorly at irradiation induced foci and that RING domain and BRCT domain combined BRCAl fragment formed foci as efficiently as full -length BRCAl. Their findings are similar with our results. However they have reported that only RING domain could not form nuclear foci after irradiation, while in our laser irradiation analysis, almost same fragment of the amino terminus could accumulate to the laser irradiated DSB site. Because actually, irradiation causes various DNA damage, DSB, SSB, and base damage, they have observed mixed response for the various DNA damage. Furthermore, they examined the nuclear foci four hour after irradiation. What we observed is the BRCAl accumulation to the DNA damage site, while nuclear foci that they observed might be where many proteins are actually repairing the DNA damage.Someone propose the model for the role of Brcal during DSB repair in a two phase modle. In the primary phase of response to DSB, Atm/Atr, in cooperation with sensor proteins ( MRN or Rpa) , becomes activated to phosphorylate H2AX. Mdcl/Nfbdl and53BPl translocate to 7H2AX, and sequesters Brcal to form the "repairsome" complex. At this time, Brcal surveys the situation, and recruits the NHEJ or HR protein machinery for repair. If the damage reaches a certain level, Brcal acts as an adaptor between Atm/Atr and Chkl/Chk2 to establish the cascade towards a checkpoint in the cell cycle. Brcal will also participate in the transcriptional activation of certain DNA repair and cell cycle regulator genes, all in an effort to promote the secondary response phase. Finally, if the damage is too great, cells will undergo. Although the molecular mechanism remains to be clarified, Brcal appears to act a modulator at the boundary between the primary and secondary waves of the response pathway. This important role provides an explanation for the pleiotropic DNA repair phenotypes seen in cells deficient for functional Brcal.There are still many unknown about BRCAl, for example, what function it lost could cause breast cancer, and how it woks. Our data just give a new in-sight about DSBs repair function of BRCAl, further works need to be done to find the mechanisms about BRCAl.Since both the C - terminus and the N - terminus of BRCAl are important for DSBs repair response, it seems that we can not explain enough why more than 80% of known breast associated mutations result in a truncated form of the BRCAl protein. Here we show one of the possible mechanisms about it. Using micro - irradiation in the nucleus to produce " pure" single strand breaks at where in our previous experiments only SSB repair proteins accumulated, we found that there was a slowly, but high - level BRCAl recruitment at SSBs irradiated sites. And did not like accumulated rapidly to DSBs sites, BRCAl accumulated to the SSBs sites about 3 minutes after irradiation. By comparing the accumulation kinetics of BRCAl after irradiation through F20 filter with other SSBs response proteins, we found that BRCAl showed a similar kinetics as PCNA and CAFl - pl50 which accumulated more slowly than POL(3 and LIGHa. Because PCNA is involve in the late step of the long - patch BER pathway and CAFl has a role in chromatin assembly in the late steps of SSB repair in vitro, BRCAl is perhaps also involve in the SSB late repair processes with PCNA and/or CAFl -pi50 in living cells.Domain analysis indicated that only C - terminus of BRCAl was essential for SSBs responsibility. Deletion of BRCT domains of BRCAl prevented its accumulation at single - strand breaks and M1775R mutant protein lost the most accumulation even co - transfected with BARD1 protein. Caffeine could inhibit the accumulation of BRCAl at SSBs sites indicated that BRCAl participate the repair pathway by its phosphorylated form. We investigated the sensitivity of the BRCAl C -terminus mutated HCC1937 (derived from a BRCAl related hereditary tumor) cell lines3RCAl -deficient HCC1937 cell lines which BRCAl expression was blocked using small interfering RNA and BRCAl - expressing HCC1937 cell lines to methyl methanesulfonate (MMS) which induced single strand breaks (SSBs). HCC1937 and BRCAl -deficient HCC1937 cell lines showed similar high sensitivity to MMS, and BRCAl - expression prevented HCC1937 cell lines from this sensitivity. These data was consistent with the idea that C - terminus of BRCAl is essential for SSB repair. This at least partiallyparticipate cancer formation and deteriorates. The experiments shown here demonstrate the responsibility of Brcal for DNA single - strand breaks, but how this activity is integrated into the pathways of DNA repair in vivo has yet to be determined.Conclution1. BRCAl accumulated at laser - induced DSBs site via its amino terminus and/or its carboxy terminus, and they use different mechanism.2. BARD1 accumulated to the laser - induced DSB sites as well as BRCAl.3. BRCAl fragments, a. a. 1 -100 and a. a. 101 -200 accumulate to the laser - induced DSB sites, respectively.4. Y105E, P142H, P143K, and E179C could accumulate to the laser - induced DSB sites;intensity of fluorescent lines of BRCAl mutants seems to be weaker than wild —type BRCAl fragments, especially the P142H mutation.5. NBS1 influence the C -terminus of BRCAl accumulation, Ku80 influence the N - terminus of BRCAl accumulation at double - strand breaks.6. Both endogenious and transfected BRCAl have responsibility to SSBs.7. BRCAl accumulated at laser -induced SSBs site via its amino terminus.8. Knockdown the expression of BRCAl could let cells sensitivity to MMS which induced SSBs in cells. This at least partially participate cancer formation and deteriorates.
|
PDF Full Text Request