BACKGROUND
a major product of dna damage, 8-oxoguanine , is generated in dna following oxidative damage by reactive oxygen species .
if not repaired, this 8-oxog results in a transversion from g:c to t:a  <cit> .
the human muty glycosylase homolog  is involved in base excision repair ; which is initiated by recognition and removal of adenine residues from dna.
hmyh cleaves the n-glycosidic bond between a target base and its deoxyribose sugar.
this results in an apurinic/apyrimidinic  site  <cit> .
hmyh has multiple forms that arise from multiple transcription initiation sites and alternative splicing of hmyh mrna transcripts  <cit> .
rad9-rad1-hus <dig> forms a heterotrimeric protein complex called 9-1- <dig>  the structure of this complex is similar to that of proliferating cell nuclear antigen , a replication clamp  <cit> .
previous studies have shown that rad <dig> recruits the 9-1- <dig> complex to dna damage sites and the rad17-rfc2- <dig> complex loads the 9-1- <dig> complex onto primed dna  <cit> .
the 9-1- <dig> complex has several functions, such as dna repair, cell cycle checkpoint control, ber, homologous recombination, mismatch repair, apoptosis, and 3′–5′ exonuclease activity  <cit> .
the complex also interacts with and/or stimulates components of the ber pathway, including polymerase β , flap endonuclease  <dig> , replication protein a , and dna ligase  <dig>   <cit> .
hrad <dig> is regulated by phosphorylation and by the differential interactions with different protein partners, which likely determines the multiple functions of hrad <dig>  <cit> .
protein-protein interactions can be studied by several methods.
bimolecular fluorescence complementation  is based on the complementation of two non-fluorescent fragments of a fluorophore.
interaction between proteins fused to the fragments facilitates the association of the non-fluorescent fragments  <cit> .
dronpa is a green fluorescent protein -like protein that photoswitches between a fluorescent “on” state and a non-fluorescent “off” state in response to light irradiation  <cit> .
previously, we had successfully generated a dronpa-bifc system to visualize the interaction between hhus1-hmyh and an intercomplex interaction between hrad1-hhus <dig>  <cit> .
in saccharomyces pombe, each component of the 9-1- <dig> complex interacts directly with spmyh, and these interactions were increased after treatment with h2o <dig> <cit> .
furthermore, human 8-oxoguanine dna glycosylase , another dna glycosylase, can interact with each subunit of the 9-1- <dig> complex.
the interaction between the 9-1- <dig> complex and hogg <dig> increase hogg <dig> enzyme activity  <cit> .
however, according to previous report by shi et al., hmyh interacts with the 9-1- <dig> complex through hhus <dig> and interacts to a lesser degree with hrad <dig>  but not with hrad <dig>  <cit> .
the glycosylase activity of hmyh is stimulated by hhus <dig> and the 9-1- <dig> complex, and this interaction is enhanced by dna damaging treatments  <cit> .
therefore, it would be interesting to study the different interactions between 9-1- <dig> and hmyh under various conditions.
in this study, we have used a dronpa-bifc system and shown for the first time that hrad <dig> interacts with the n-terminal region of hmyh.
we also examined the physiological effects of the hrad <dig> and hmyh interaction by exposing cells to damaging agents such as hu.
the mrna levels of hmyh and hrad <dig> were increased after treatment with hu, but not after treatment with h2o <dig>  moreover, the interaction between hmyh and hrad <dig> was also indicated by fret , which increased after hu treatment.
cell cycle arrest, induced by p-chk <dig> , is evident from the increase in the inactive form of cyclin-dependent kinase  <dig> .
phosphorylation of chk <dig> and cdk <dig> decreased in hmyh and hrad <dig> knockdown cells; this decrease was more pronounced after hu treatment.
hrad <dig> interacted with hmyh as a component of the 9-1- <dig> complex because depletion of hus <dig> reduced the interaction.
we hypothesize that this interaction occurs during the early stages of dna damage repair.
RESULTS
hmyh physically interacts with hrad9
to determine whether hrad <dig> interacts with hmyh, co-immunoprecipitation  was performed using hek <dig> cells transfected with c-myc and flag-tagged hrad <dig> or c-myc-tagged hmyh and flag-tagged hrad <dig>  after incubation for 24 h, cell lysates were extracted and immunoprecipitated with an anti-c-myc antibody.
precipitated samples were analyzed by immunoblotting with anti-flag and anti-c-myc antibodies.
flag-hrad <dig> was precipitated through its interaction with c-myc-hmyh .
in western blot analysis, hrad <dig> was visualized as several bands due to differences in the phosphorylation states.
these results may indicate a physical interaction between hmyh and hrad <dig>  the endogenous interaction between hrad <dig> and hmyh was confirmed by co-ip with anti-hmyh antibody.
hrad <dig> immunoprecipitated with hmyh , indicating that endogenous hmyh interacts with hrad <dig> to determine the involvement of the 9-1- <dig> complex in the interaction between hmyh and hrad <dig>  hus <dig> expression was knocked down using specific sirna.
in hus1-depleted cells, the interaction between hmyh and hrad <dig> was decreased .
this result indicates that hrad <dig> interacts with hmyh as part of the 9-1- <dig> complex.
bacterially expressed and purified his-hrad <dig> and gst-hmyh were used in gst pull-down assays to demonstrate the physical interaction between hmyh and hrad <dig>  gst or gst-hmyh was immobilized on glutathione-sepharose beads and used to pull-down purified his-hrad <dig>  we observed a band for his-hrad <dig> in the pull-down assay with gst-hmyh, but not with gst alone, indicative of a direct interaction between hmyh and hrad <dig> .
our results differ from those obtained by shi et al.
<cit> , which did not observe an interaction between hmyh and hrad <dig> in gst pull-down assays.
hrad <dig> interacts with the n-terminal region of hmyh
the n-terminal region of hmyh contains sites for rpa binding  and function in substrate specificity, and the c-terminal region is important for glycosylase activity  <cit> .
we created hmyh mutants with n-, c-, or n- and c-terminal deletions  to determine the functional impact of the hmyh and hrad <dig> interaction on substrate specificity and glycosylase activity.
we successfully used the dronpa-bifc system developed in our lab to identify the region of hmyh that interacts with hrad <dig> and to visualize the interaction between hmyh-hhus <dig>  the optimum fragments for dronpa cleavage were selected according to a structural analysis of gfp and mrfp <dig>  bifc vectors for visualization of the interaction between hrad <dig> and hmyh were constructed as described in the methods section  <cit> .
the dronpa c-terminus was fused to hrad <dig>  and the dronpa n-terminus was fused to either full-length hmyh  or hmyh deletion mutants .
the vector used for the dronpa-bifc system is shown in figure 2a.
the transient expression of transfected proteins in cells was confirmed by immunoblotting .hek <dig> cells were transfected with different vector sets of plasmids .
cells were incubated for 24 h, and dronpa fluorescence was visualized by fluorescence microscopy.
dronpa fluorescence was observed in cells transfected with native dronpa and in cells co-transfected with hmyh-ldn/dcl-hrad <dig> and hmyh-∆c-ldn/dcl-hrad <dig>  but no fluorescence was observed in cells transfected with hmyh-∆n-ldn/dcl-hrad <dig> and hmyh-∆nc-ldn/dcl-hrad <dig> .
these results showed that hrad <dig> interacts with the n-terminal region of hmyh.
the dronpa fluorescence observed in cells transfected with dcl-hrad <dig> and hmyh-ldn or hmyh-∆c-ldn was due to reconstitution of functional dronpa facilitated by the interaction between hrad <dig> and hmyh.
photoswitching activity used to identify dronpa protein  <cit> .
to evaluate the photoswitching activity of native or complemented dronpa fragments, hek <dig> cells were co-transfected with vectors expressing dronpa or hmyh-ldn and dcl-hrad <dig>  the cells were incubated for 24 h and analyzed by confocal fluorescence microscopy.
after visualization, cells exhibiting dronpa fluorescence were irradiated at 488 nm for 2 min to induce photobleaching and then photoactivated at 430 nm for 30 s. the similar results were obtained with native and complemented dronpa fragments .to confirm this interaction, we transfected different sets of vectors into hek <dig> cells .
after incubation for 24 h, cell lysates were extracted and immunoprecipitated with anti-c-myc antibody .
immunoblot analysis was performed with anti-c-myc or anti-flag antibody.
in agreement with figure 2c, flag-hrad <dig> precipitated with c-myc-hmyh-full and c-myc-hmyh-Δc .
to further demonstrate the direct interaction between hrad <dig> and hmyh, we performed a gst pull-down assay, as previously described.
as same with ip results, purified his-hrad <dig> was pulled down by gst-hmyh-full and gst-hmyh-Δc, but not by gst alone, gst-hmyh-Δn, and gst-hmyh-Δnc .
these results indicate that the n-terminal region of hmyh facilitates the interaction between hmyh and hrad <dig>
hu treatment increases the expression of hrad <dig> and hmyh, leading to cell cycle arrest
we examined the physiological effects of the hrad <dig> and hmyh interaction by exposing cells to damaging agents .
hmyh and hrad <dig> mrna levels were examined by rt-pcr.
after normalization to actin, hmyh intensity was higher in hu-treated samples , compared to the intensity in untreated  and h2o2-treated  samples.
the same result was observed for hrad <dig>  which increased after hu treatment .
thus, we noted a marked increase in hrad <dig> and hmyh mrna levels after treatment with hu, but not after h2o <dig> treatment .
moreover, hmyh and hrad <dig> protein expression also increased in hu-treated cells, but not in h2o2-treated cells.
hu arrests cell cycle progression by inhibiting ribonucleotide diphosphate reductase.
p-chk <dig>  an indicator of dna damage, was detected in both hu- and h2o2-treated cells .
p-chk <dig> induces cell cycle arrest.
consistently, the phosphorylated, inactive form of cdk <dig> , which does not promote cell cycle progression, was higher in hu-treated cells.to further study the functional impact of the interaction between hrad <dig> and hmyh in hu-treated cells, we knockdown the expression of hmyh and/or hrad <dig> using sirna.
sigfp-transfected cells were used as a control.
after transfection, cells were treated with hu, and protein expression was analyzed by immunoblotting.
in sigfp-transfected cells, p-chk <dig>  and p-cdk <dig>  were detected, indicating that hu treatment induced cell cycle arrest .
p-chk <dig> and p-cdk <dig> levels were lower in hu-treated cells transfected with sirad <dig> and/or simyh .
the reduction in p-cdk <dig> was more pronounced in cells in which both hmyh and hrad <dig> were knocked down.
the interaction and co-localization of hmyh and hrad <dig> in nuclear foci after exposure to genotoxic stress
the 9-1- <dig> complex and hmyh participate in dna damage repair  <cit> .
the interaction between hrad <dig> and hmyh after hu treatment was examined by fret analysis.
hek <dig> cells were transfected with ecfp/hmyh and eyfp/hrad <dig>  individually or together.
using confocal fluorescence microscopy, we detected ecfp and eyfp fluorescence in all transfected cells.
fret fluorescence was only observed in cells co-transfected with ecfp/hmyh and eyfp/hrad <dig>  moreover, higher intensity fret fluorescence was seen in cells treated with hu, suggesting that hu treatment enhances the interaction between hrad <dig> and hmyh .the interaction between hrad <dig> and hmyh exhibited a relatively high fret intensity .
given this result, we determined whether endogenous hmyh and hrad <dig> localized to the same nuclear foci after hu treatment.
in immunofluorescence analysis, hmyh and hrad <dig> staining in the nucleus of untreated cells was faint .
however, in hu-treated cells, hmyh and hrad <dig> formed discrete nuclear foci, and a significant fraction of the hmyh nuclear foci co-localized with hrad <dig>  indicating that hmyh and the 9-1- <dig> complex translocated to the same lesions after dna damage.
CONCLUSIONS
in this study, we showed that hrad <dig> interacts with hmyh by co-ip of over-expressed c-myc-hmyh and flag-hrad <dig>  and co-ip of endogenous hmyh and hrad <dig> .
we confirmed the interaction in gst pull-down assays using gst-hmyh and his-hrad <dig> .
moreover, we observed that interaction between hmyh and hrad <dig> was independent with dna .
the results showed that the proteins interact directly.
we also observed that hmyh interacts with hhus <dig>  <cit> , and hrad <dig> .
in addition, the dronpa-bifc system, used previously to visualize the intercomplex interactions between hhus <dig> and hrad <dig> and between hmyh and hhus <dig>  <cit> , was used to show that hrad <dig> interacts with hmyh .
our results differ from those of a previous study by shi et al., in which hmyh was not detected in gst pull-down assays using gst-hrad <dig>  <cit> .
shi et al.
investigated whether gst-hhus <dig>  gst-hrad <dig>  and gst-hrad <dig> pulled down his-hmyh.
their results showed that his-hmyh interacted directly with hhus <dig> and hrad <dig>  but not with hrad <dig>  they also found that gst-spmyh pulled down the 9-1- <dig> complex.
we suggest that the differences between their results and ours may be due to the different protein constructs used or the weak binding of hrad <dig> to hmyh  <cit> .
in our lab, we found that disruption of hmyh and the 9-1- <dig> complex sensitizes cells to hydroxyurea  and ultraviolet  irradiation  <cit> .
it is already known that 9-1- <dig> complex function in the early detection of dna damage and assists other protein to bind to the site of damage  <cit> .
on the other hand, hmyh function in creating ap-site in the early ber pathway  <cit> .
moreover, knockdown of hmyh and hrad <dig> induced the decrease of phosphorylation in the chk <dig> and cdk <dig>  an upstream in the dna damage repair pathway .
therefore, we suggest that the interaction between hmyh and 9-1- <dig> occurs early in the dna damage response and functions as an adaptor for other proteins at lesion sites to activate checkpoint control.
the hrad <dig> and hmyh interaction was studied by fluorescence resonance energy transfer .
fret occurs upon energy transfer from a donor molecule  to an acceptor molecule   <cit> .
cells that over-expressed ecfp/hmyh and eyfp/hrad <dig> were treated with hu.
fret increased significantly in cells treated with hu .
we also analyzed the interacting region of hmyh using the dronpa-bifc system.
hmyh is significantly larger than the bacterial protein and comprises the entire muty sequence plus extended n- and c-terminal flanking domains  <cit> .
these 50– <dig> amino acids terminal domains are involved in subcellular targeting and interactions with other proteins.
rpa and pcna binding motifs, for example, map to the n- and c-terminus, respectively.
these interactions suggest coupling of hmyh to the dna replication machinery  <cit> .
the dronpa-bifc system showed that hrad <dig> interacts with the n-terminal region of hmyh .
subsequently, co-ip and gst pull-down assays showed that hrad <dig> interacts directly with the n-terminal region of hmyh .
hmyh and hrad <dig> mrna and protein levels increased after hu treatment, but not after h2o <dig> treatment .
hu inhibits ribonucleotide diphosphate reductase, thereby blocking dna synthesis and repair  <cit> .
when dna is damaged, cell cycle progression arrests or slows to allow time for dna repair  <cit> .
the cell cycle checkpoint proteins rad9-rad1-hus <dig> complex play important roles in both cell cycle checkpoint control and dna repair  <cit> .
deletion of each gene encoding the three proteins in the fission yeast s. pombe inactivates s/m, intra-s, and g2/m checkpoint controls.
endogenous hmyh in hek <dig> cells increased during s phase and decreased in m phase  <cit> .
however, a basal level of hmyh was maintained during m phase.
disruption of hmyh levels reduced the amount of chk <dig> activated by hu.
these results indicate that, although mainly induced from late g <dig> to s phase, chk <dig> activation can also be induced by dna damage during g2/m phase and attenuated by hmyh disruption  <cit> .
chk <dig> on chromatin undergoes atr-dependent phosphorylation in response to dna damage.
phosphorylation appears to disrupt intramolecular interactions, leading to an open conformation of chk <dig> and to checkpoint activation  <cit> .
cdks  are heterodimeric serine/threonine protein kinases that control cell cycle progression.
among them, the cdk1-cyclin b complex controls cell cycle progression in g2/m phase, and cdk2-cyclin e/a complexes function in the g1/s and s/g <dig> transitions  <cit> .
cell cycle entry into mitosis is regulated by cdk <dig> activation, which is controlled by cyclin binding and phosphorylation at t <dig>  on the other hand, cdk <dig> is activated during the progression of mitosis by dephosphorylation at t <dig> and y <dig>  thus, phosphorylation of cdk <dig>  is indicative of cell cycle arrest  <cit> .in cells depleted of hmyh and hrad <dig> by sirna knockdown, p-chk <dig> and p-cdk <dig> expression levels decreased .
the decrease in p-chk <dig> in single and double knockdowns indicates that hmyh and hrad <dig> are defective in promoting atr activity.
therefore, we can conclude that hmyh and hrad <dig> interaction are important for the dna damage response.
we suggest that hmyh and hrad <dig> interact early in the process of cell cycle arrest.
further studies to elucidate the mechanism that regulates interactions between hmyh and the 9-1- <dig> complex in response to different types of dna damage are required.

 a major product of dna damage, 8-oxoguanine, is generated in dna following oxidative damage by reactive oxygen species. if not repaired, this 8-oxog results in a transversion from g:c to t:a. the human muty glycosylase homolog is involved in base excision repair; which is initiated by recognition and removal of adenine residues from dna. hmyh cleaves the n-glycosidic bond between a target base and its deoxyribose sugar. this results in an apurinic/apyrimidinic site. hmyh has multiple forms that arise from multiple transcription initiation sites and alternative splicing of hmyh mrna transcripts. rad9-rad1-hus forms a heterotrimeric protein complex called 9-1- the structure of this complex is similar to that of proliferating cell nuclear antigen, a replication clamp. previous studies have shown that rad recruits the 9-1- complex to dna damage sites and the rad17-rfc2- complex loads the 9-1- complex onto primed dna. the 9-1- complex has several functions, such as dna repair, cell cycle checkpoint control, ber, homologous recombination, mismatch repair, apoptosis, and 3′–5′ exonuclease activity. the complex also interacts with and/or stimulates components of the ber pathway, including polymerase β, flap endonuclease, replication protein a, and dna ligase. hrad is regulated by phosphorylation and by the differential interactions with different protein partners, which likely determines the multiple functions of hrad. protein-protein interactions can be studied by several methods. bimolecular fluorescence complementation is based on the complementation of two non-fluorescent fragments of a fluorophore. interaction between proteins fused to the fragments facilitates the association of the non-fluorescent fragments. dronpa is a green fluorescent protein -like protein that photoswitches between a fluorescent “on” state and a non-fluorescent “off” state in response to light irradiation. previously, we had successfully generated a dronpa-bifc system to visualize the interaction between hhus1-hmyh and an intercomplex interaction between hrad1-hhus. in saccharomyces pombe, each component of the 9-1- complex interacts directly with spmyh, and these interactions were increased after treatment with h2o. furthermore, human 8-oxoguanine dna glycosylase, another dna glycosylase, can interact with each subunit of the 9-1- complex. the interaction between the 9-1- complex and hogg increase hogg enzyme activity. however, according to previous report by shi et al., hmyh interacts with the 9-1- complex through hhus and interacts to a lesser degree with hrad but not with hrad. the glycosylase activity of hmyh is stimulated by hhus and the 9-1- complex, and this interaction is enhanced by dna damaging treatments. therefore, it would be interesting to study the different interactions between 9-1- and hmyh under various conditions. in this study, we have used a dronpa-bifc system and shown for the first time that hrad interacts with the n-terminal region of hmyh. we also examined the physiological effects of the hrad and hmyh interaction by exposing cells to damaging agents such as hu. the mrna levels of hmyh and hrad were increased after treatment with hu, but not after treatment with h2o moreover, the interaction between hmyh and hrad was also indicated by fret, which increased after hu treatment. cell cycle arrest, induced by p-chk, is evident from the increase in the inactive form of cyclin-dependent kinase. phosphorylation of chk and cdk decreased in hmyh and hrad knockdown cells; this decrease was more pronounced after hu treatment. hrad interacted with hmyh as a component of the 9-1- complex because depletion of hus reduced the interaction. we hypothesize that this interaction occurs during the early stages of dna damage repair.  hmyh physically interacts with hrad9 to determine whether hrad interacts with hmyh, co-immunoprecipitation was performed using hek cells transfected with c-myc and flag-tagged hrad or c-myc-tagged hmyh and flag-tagged hrad after incubation for 24 h, cell lysates were extracted and immunoprecipitated with an anti-c-myc antibody. precipitated samples were analyzed by immunoblotting with anti-flag and anti-c-myc antibodies. flag-hrad was precipitated through its interaction with c-myc-hmyh. in western blot analysis, hrad was visualized as several bands due to differences in the phosphorylation states. these results may indicate a physical interaction between hmyh and hrad the endogenous interaction between hrad and hmyh was confirmed by co-ip with anti-hmyh antibody. hrad immunoprecipitated with hmyh, indicating that endogenous hmyh interacts with hrad to determine the involvement of the 9-1- complex in the interaction between hmyh and hrad hus expression was knocked down using specific sirna. in hus1-depleted cells, the interaction between hmyh and hrad was decreased. this result indicates that hrad interacts with hmyh as part of the 9-1- complex. bacterially expressed and purified his-hrad and gst-hmyh were used in gst pull-down assays to demonstrate the physical interaction between hmyh and hrad gst or gst-hmyh was immobilized on glutathione-sepharose beads and used to pull-down purified his-hrad we observed a band for his-hrad in the pull-down assay with gst-hmyh, but not with gst alone, indicative of a direct interaction between hmyh and hrad. our results differ from those obtained by shi et al., which did not observe an interaction between hmyh and hrad in gst pull-down assays. hrad interacts with the n-terminal region of hmyh the n-terminal region of hmyh contains sites for rpa binding and function in substrate specificity, and the c-terminal region is important for glycosylase activity. we created hmyh mutants with n-, c-, or n- and c-terminal deletions to determine the functional impact of the hmyh and hrad interaction on substrate specificity and glycosylase activity. we successfully used the dronpa-bifc system developed in our lab to identify the region of hmyh that interacts with hrad and to visualize the interaction between hmyh-hhus the optimum fragments for dronpa cleavage were selected according to a structural analysis of gfp and mrfp bifc vectors for visualization of the interaction between hrad and hmyh were constructed as described in the methods section. the dronpa c-terminus was fused to hrad and the dronpa n-terminus was fused to either full-length hmyh or hmyh deletion mutants. the vector used for the dronpa-bifc system is shown in figure 2a. the transient expression of transfected proteins in cells was confirmed by immunoblotting.hek cells were transfected with different vector sets of plasmids. cells were incubated for 24 h, and dronpa fluorescence was visualized by fluorescence microscopy. dronpa fluorescence was observed in cells transfected with native dronpa and in cells co-transfected with hmyh-ldn/dcl-hrad and hmyh-∆c-ldn/dcl-hrad but no fluorescence was observed in cells transfected with hmyh-∆n-ldn/dcl-hrad and hmyh-∆nc-ldn/dcl-hrad. these results showed that hrad interacts with the n-terminal region of hmyh. the dronpa fluorescence observed in cells transfected with dcl-hrad and hmyh-ldn or hmyh-∆c-ldn was due to reconstitution of functional dronpa facilitated by the interaction between hrad and hmyh. photoswitching activity used to identify dronpa protein. to evaluate the photoswitching activity of native or complemented dronpa fragments, hek cells were co-transfected with vectors expressing dronpa or hmyh-ldn and dcl-hrad the cells were incubated for 24 h and analyzed by confocal fluorescence microscopy. after visualization, cells exhibiting dronpa fluorescence were irradiated at 488 nm for 2 min to induce photobleaching and then photoactivated at 430 nm for 30 s. the similar results were obtained with native and complemented dronpa fragments.to confirm this interaction, we transfected different sets of vectors into hek cells. after incubation for 24 h, cell lysates were extracted and immunoprecipitated with anti-c-myc antibody. immunoblot analysis was performed with anti-c-myc or anti-flag antibody. in agreement with figure 2c, flag-hrad precipitated with c-myc-hmyh-full and c-myc-hmyh-Δc. to further demonstrate the direct interaction between hrad and hmyh, we performed a gst pull-down assay, as previously described. as same with ip results, purified his-hrad was pulled down by gst-hmyh-full and gst-hmyh-Δc, but not by gst alone, gst-hmyh-Δn, and gst-hmyh-Δnc. these results indicate that the n-terminal region of hmyh facilitates the interaction between hmyh and hrad hu treatment increases the expression of hrad and hmyh, leading to cell cycle arrest we examined the physiological effects of the hrad and hmyh interaction by exposing cells to damaging agents. hmyh and hrad mrna levels were examined by rt-pcr. after normalization to actin, hmyh intensity was higher in hu-treated samples, compared to the intensity in untreated and h2o2-treated samples. the same result was observed for hrad which increased after hu treatment. thus, we noted a marked increase in hrad and hmyh mrna levels after treatment with hu, but not after h2o treatment. moreover, hmyh and hrad protein expression also increased in hu-treated cells, but not in h2o2-treated cells. hu arrests cell cycle progression by inhibiting ribonucleotide diphosphate reductase. p-chk an indicator of dna damage, was detected in both hu- and h2o2-treated cells. p-chk induces cell cycle arrest. consistently, the phosphorylated, inactive form of cdk, which does not promote cell cycle progression, was higher in hu-treated cells.to further study the functional impact of the interaction between hrad and hmyh in hu-treated cells, we knockdown the expression of hmyh and/or hrad using sirna. sigfp-transfected cells were used as a control. after transfection, cells were treated with hu, and protein expression was analyzed by immunoblotting. in sigfp-transfected cells, p-chk and p-cdk were detected, indicating that hu treatment induced cell cycle arrest. p-chk and p-cdk levels were lower in hu-treated cells transfected with sirad and/or simyh. the reduction in p-cdk was more pronounced in cells in which both hmyh and hrad were knocked down. the interaction and co-localization of hmyh and hrad in nuclear foci after exposure to genotoxic stress the 9-1- complex and hmyh participate in dna damage repair. the interaction between hrad and hmyh after hu treatment was examined by fret analysis. hek cells were transfected with ecfp/hmyh and eyfp/hrad individually or together. using confocal fluorescence microscopy, we detected ecfp and eyfp fluorescence in all transfected cells. fret fluorescence was only observed in cells co-transfected with ecfp/hmyh and eyfp/hrad moreover, higher intensity fret fluorescence was seen in cells treated with hu, suggesting that hu treatment enhances the interaction between hrad and hmyh.the interaction between hrad and hmyh exhibited a relatively high fret intensity. given this result, we determined whether endogenous hmyh and hrad localized to the same nuclear foci after hu treatment. in immunofluorescence analysis, hmyh and hrad staining in the nucleus of untreated cells was faint. however, in hu-treated cells, hmyh and hrad formed discrete nuclear foci, and a significant fraction of the hmyh nuclear foci co-localized with hrad indicating that hmyh and the 9-1- complex translocated to the same lesions after dna damage.  in this study, we showed that hrad interacts with hmyh by co-ip of over-expressed c-myc-hmyh and flag-hrad and co-ip of endogenous hmyh and hrad. we confirmed the interaction in gst pull-down assays using gst-hmyh and his-hrad. moreover, we observed that interaction between hmyh and hrad was independent with dna. the results showed that the proteins interact directly. we also observed that hmyh interacts with hhus, and hrad. in addition, the dronpa-bifc system, used previously to visualize the intercomplex interactions between hhus and hrad and between hmyh and hhus, was used to show that hrad interacts with hmyh. our results differ from those of a previous study by shi et al., in which hmyh was not detected in gst pull-down assays using gst-hrad. shi et al. investigated whether gst-hhus gst-hrad and gst-hrad pulled down his-hmyh. their results showed that his-hmyh interacted directly with hhus and hrad but not with hrad they also found that gst-spmyh pulled down the 9-1- complex. we suggest that the differences between their results and ours may be due to the different protein constructs used or the weak binding of hrad to hmyh. in our lab, we found that disruption of hmyh and the 9-1- complex sensitizes cells to hydroxyurea and ultraviolet irradiation. it is already known that 9-1- complex function in the early detection of dna damage and assists other protein to bind to the site of damage. on the other hand, hmyh function in creating ap-site in the early ber pathway. moreover, knockdown of hmyh and hrad induced the decrease of phosphorylation in the chk and cdk an upstream in the dna damage repair pathway. therefore, we suggest that the interaction between hmyh and 9-1- occurs early in the dna damage response and functions as an adaptor for other proteins at lesion sites to activate checkpoint control. the hrad and hmyh interaction was studied by fluorescence resonance energy transfer. fret occurs upon energy transfer from a donor molecule to an acceptor molecule. cells that over-expressed ecfp/hmyh and eyfp/hrad were treated with hu. fret increased significantly in cells treated with hu. we also analyzed the interacting region of hmyh using the dronpa-bifc system. hmyh is significantly larger than the bacterial protein and comprises the entire muty sequence plus extended n- and c-terminal flanking domains. these 50– amino acids terminal domains are involved in subcellular targeting and interactions with other proteins. rpa and pcna binding motifs, for example, map to the n- and c-terminus, respectively. these interactions suggest coupling of hmyh to the dna replication machinery. the dronpa-bifc system showed that hrad interacts with the n-terminal region of hmyh. subsequently, co-ip and gst pull-down assays showed that hrad interacts directly with the n-terminal region of hmyh. hmyh and hrad mrna and protein levels increased after hu treatment, but not after h2o treatment. hu inhibits ribonucleotide diphosphate reductase, thereby blocking dna synthesis and repair. when dna is damaged, cell cycle progression arrests or slows to allow time for dna repair. the cell cycle checkpoint proteins rad9-rad1-hus complex play important roles in both cell cycle checkpoint control and dna repair. deletion of each gene encoding the three proteins in the fission yeast s. pombe inactivates s/m, intra-s, and g2/m checkpoint controls. endogenous hmyh in hek cells increased during s phase and decreased in m phase. however, a basal level of hmyh was maintained during m phase. disruption of hmyh levels reduced the amount of chk activated by hu. these results indicate that, although mainly induced from late g to s phase, chk activation can also be induced by dna damage during g2/m phase and attenuated by hmyh disruption. chk on chromatin undergoes atr-dependent phosphorylation in response to dna damage. phosphorylation appears to disrupt intramolecular interactions, leading to an open conformation of chk and to checkpoint activation. cdks are heterodimeric serine/threonine protein kinases that control cell cycle progression. among them, the cdk1-cyclin b complex controls cell cycle progression in g2/m phase, and cdk2-cyclin e/a complexes function in the g1/s and s/g transitions. cell cycle entry into mitosis is regulated by cdk activation, which is controlled by cyclin binding and phosphorylation at t on the other hand, cdk is activated during the progression of mitosis by dephosphorylation at t and y thus, phosphorylation of cdk is indicative of cell cycle arrest.in cells depleted of hmyh and hrad by sirna knockdown, p-chk and p-cdk expression levels decreased. the decrease in p-chk in single and double knockdowns indicates that hmyh and hrad are defective in promoting atr activity. therefore, we can conclude that hmyh and hrad interaction are important for the dna damage response. we suggest that hmyh and hrad interact early in the process of cell cycle arrest. further studies to elucidate the mechanism that regulates interactions between hmyh and the 9-1- complex in response to different types of dna damage are required. 
rad9-rad1-hus is a heterotrimeric protein complex that plays a role in cell cycle checkpoint control and dna repair. in humans, hmyh and 9-1- interact through hus and to a lesser degree with rad in the presence of dna damage. mrna and protein levels of hmyh and hrad were increased following hu treatment. this interaction is enhanced by hu treatment. in saccharomyces pombe, each component of the 9-1- complex interacts directly with spmyh. co-ip and bifc indicates that hmyh interacts with hrad as shown by gst-pull down assay, this interaction is direct. the glycosylase activity of hmyh is stimulated by hus and the 9-1- complex and enhanced by dna damage treatment. since both protein functions in the early detection of dna damage, we suggest that this interaction occurs early in dna damage pathway. furthermore, bifc with deletion mutants of hmyh showed that hrad interacts with n-terminal region of hmyh. our data reveal that hrad interacts directly with n-terminal region of hmyh. human muty glycosylase homolog, a component of the base excision repair pathway, is responsible for the generation of apurinic/apyrimidinic sites.
 human muty glycosylase homolog, a component of the base excision repair pathway, is responsible for the generation of apurinic/apyrimidinic sites. rad9-rad1-hus is a heterotrimeric protein complex that plays a role in cell cycle checkpoint control and dna repair. in humans, hmyh and 9-1- interact through hus and to a lesser degree with rad in the presence of dna damage. in saccharomyces pombe, each component of the 9-1- complex interacts directly with spmyh. the glycosylase activity of hmyh is stimulated by hus and the 9-1- complex and enhanced by dna damage treatment. cells respond to different stress conditions in different manners. therefore, we investigated whether rad interacted with hmyh under different stresses. here, we identified and visualized the interaction between hrad and hmyh and investigated the functional consequences of this interaction.  co-ip and bifc indicates that hmyh interacts with hrad as shown by gst-pull down assay, this interaction is direct. furthermore, bifc with deletion mutants of hmyh showed that hrad interacts with n-terminal region of hmyh. the interaction was enhanced by hydroxyurea treatment. mrna and protein levels of hmyh and hrad were increased following hu treatment. a marked increase in p-chk and p-cdk was observed. but this phosphorylation decreased in simyh- or sirad9-transfected cells, and more pronounced decrease observed in co-transfected cells.  our data reveal that hrad interacts directly with n-terminal region of hmyh. this interaction is enhanced by hu treatment. knockdown of one or both protein result in decreasing chk and cdk phosphorylation. since both protein functions in the early detection of dna damage, we suggest that this interaction occurs early in dna damage pathway. human myhhuman rad9bimolecular fluorescence complementationfretimmunofluorescenceprotein-protein interaction 