BACKGROUND
reactive oxygen species and radiation cause dna strand breaks and base lesions, thus affecting genomic integrity.
particularly, the c-g-rich telomeres are highly susceptible to oxidative damage  <cit> .
a frequent and highly mutagenic oxidative lesion is 8-oxo- <dig> -dihydroguanine , which mispairs with adenine during dna replication, resulting in g:c to t:a mutations  <cit> .
oxidative dna lesions are repaired primarily by the base excision repair  pathway  <cit> .
the first step in ber is carried out by a dna glycosylase, which cleaves the damaged or mismatched base.
the resulting apurinic/apyrimidinic  site is processed by ap-endonuclease  <dig> , allowing the downstream ber enzymes to complete the dna repair process  <cit> .
muty homolog  excises adenines from a/go mismatches and thus reduces g:c to t:a mutations .
mutations in the human myh  gene can lead to colorectal cancer   <cit> .
ber is coordinated with other cellular processes in eukaryotic cells  <cit> .
eukaryotic myh contains unique motifs to mediate interactions with partner proteins involved in dna replication, mismatch repair, and dna damage response .
we have shown that the interdomain connector  located between the n- and c-terminal domains of hmyh is uniquely oriented  <cit>  to interact with ape <dig>  <cit>  and hus <dig>  <cit> , a subunit of the heterotrimeric rad9–rad1–hus <dig>  checkpoint clamp.
ape <dig> is essential for cell viability  <cit>  and participates in many aspects of dna metabolism and telomere maintenance .
in dna repair, ape <dig> cleaves the phosphodiester bond 5′ to an ap site and removes various forms of 3′-blocked lesions at dna strand breaks  <cit> .
because ap sites are mutagenic and cytotoxic  <cit> , they must be recognized by ape <dig> immediately after the action of a dna glycosylase.
a “passing-the-baton” model has been proposed for ber  <cit> , consistent with findings that ape <dig> stimulates many dna glycosylases .
ape <dig> forms a stable complex with myh and 9-1- <dig>  <cit> .
besides serving as a damage sensor, 9-1- <dig> is involved in many dna metabolisms including ber .
intriguingly, 9-1- <dig> interacts with and stimulates the activity of almost every enzyme in the ber pathway and has been proposed to serve as a platform to coordinate ber.
sirt <dig> is a member of nad+-dependent histone/protein deacetylase family  and also has mono-adp-ribosyltransferase and protein lysine fatty acyl removal activities  <cit> .
sirt <dig> plays a role in stress response, dna repair, telomere integrity, retrotransposition, and metabolic homeostasis .
sirt <dig> knockout mice display a shortened lifespan associated with impaired dna repair  <cit> .
moreover, sirt <dig> depletion leads to telomere dysfunction and premature cellular senescence  <cit> .
during the course of aging and in response to dna damage, sirt <dig> is depleted from l <dig> retrotransposon loci, allowing their activation  <cit> .
sirt <dig> has been implicated in ber  <cit> .
it has been demonstrated that sirt <dig> can activate parp <dig>  <cit>  and is a partner of thymine dna glycosylase  <cit> .
xu et al.
<cit>  recently reported that sirt <dig> regulates ber in a parp1-depdendent manner.
however, direct physical and functional interactions between sirt <dig> and ber enzymes remained to be elucidated.
here, we provide evidence for a direct role of sirt <dig> in ber and dna damage response .
we show that mouse sirt <dig>  interacts with myh, ape <dig>  and 9-1- <dig> and stimulates myh and ape <dig> activities.
our data demonstrate that sirt <dig>  ape <dig>  and hus <dig> bind to hmyh without competition.
instead, one myh partner enhances the association of the other two partners to the myh complex.
by using novel systems for confining oxidative dna damage to defined human genomic regions, we show that myh and sirt <dig> are efficiently recruited to dna damage sites within transcriptionally active chromatin, but not within inactive chromatin in human cells.
in addition, we show that the number of myh nuclear foci, frequently found to localize on telomeres, increase in sirt6−/− mouse embryonic fibroblast cells.
a further increase in telomere localization of myh foci was found in the presence of oxidative stress.
thus, sirt <dig> represents an interesting connection between chromatin remodeling and myh-directed ber.
RESULTS
sirt <dig> interacts with myh and ape1
to examine whether sirt <dig> plays a direct role in genomic integrity through ber, we analyzed the physical and functional interactions between sirt <dig> and two major ber enzymes .
first, association between sirt <dig> and myh was demonstrated by co-immunoprecipitation  .
human cell extracts were subjected to immunoprecipitation with hmyh antibody followed by western blot analysis with anti hmyh or hsirt <dig> antibodies.
both hmyh and hsirt <dig> were found in the pellet with hmyh antibody but not with control igg.
it is interesting to note that hmyh primarily interacts with the upper band of sirt <dig> .
we suspect the upper band may be a modified form of sirt <dig> because sirt <dig> can undergo auto mono-adp-ribosylation  <cit> .
conversely, human cell extracts were subjected to immunoprecipitation with hsirt <dig> antibody followed by western analysis with hsirt <dig> or hmyh antibodies.
in this assay, both bands of hsirt <dig> interacted with sirt <dig> antibody.
human myh was co-immunoprecipitated with hsirt <dig> antibody .
using the similar approach, association between hsirt <dig> and hape <dig> was established by co-ip .
as observed in hmyh-sirt <dig> interaction, hape <dig> primarily interacts with the upper band of sirt <dig> .
thus, hsirt <dig> associates with hmyh and hape <dig> figure  <dig> sirt <dig> interacts with myh, ape <dig>  and rad9–rad1–hus <dig>  a sirt <dig> can be co-immunoprecipitated by hmyh antibody from hela extracts.
immunoprecipitation  was performed with hmyh antibody and detected on the western blot using hsirt <dig> or hmyh antibody .
lane  <dig> contains 10% of input cell extracts .
lane  <dig> is a negative control in which the immunoprecipitation was performed with igg.
b interaction between hmyh and hsirt <dig> is enhanced following h2o <dig> treatment.
myh was co-immunoprecipitated by sirt <dig> antibody from extracts prepared from untreated hela cells  or from cells treated with  <dig>  mm h2o <dig> for 1 h and recovered for 6 h .
western blots were detected by hmyh or hsirt <dig> antibody.
control lanes are similar to those described in .
c sirt <dig> and ape <dig> co-immunoprecipitated from hela extracts.
immunoprecipitation was performed with hape <dig> antibody and western blotting was performed with hape <dig> or hsirt <dig> antibody.
control lanes are similar to a. d interaction between hape <dig> and hsirt <dig> is enhanced following h2o <dig> treatment.
ape <dig> was co-immunoprecipitated by sirt <dig> antibody from extracts prepared from untreated  and h2o <dig> treated  hela cells.
western blots were detected by hape <dig> or hsir <dig> antibody.
e, f immobilized gst-hmyh  and gst-hape <dig> , respectively, were used to pull down flag-msirt <dig> .
lane  <dig> contains 10% of input msirt <dig> protein.
lane  <dig> used gst-beads alone.
flag-msirt <dig> was detected by an anti-flag antibody.
g immobilized gst, gst-tagged intact hmyh, myhΔc <dig>  myhΔc <dig>  myhΔc3m, and myhΔc <dig>  were used to precipitate flag-msirt <dig>  the hmyh constructs are depicted.
to investigate the effect of dna damage on the interactions between sirt <dig> and ber enzymes, we performed co-ip experiments with extracts from hela cells treated with  <dig>  mm h2o <dig> for 1 h and recovered for 6 h. interestingly, the interactions of hmyh and hape <dig> with hsirt <dig> were enhanced after h2o <dig> treatment .
this result indicates that hsirt <dig> interactions with ber enzymes are enhanced following oxidative treatment.
to show direct physical interactions between sirt <dig> and ber enzymes, we performed gst-pull-down assays in the presence of ethidium bromide to eliminate the effect of nucleic acid on protein–protein interactions.
due to technical difficulties for purifying human enzymes, we purified mouse sirt <dig>  and mouse myh  that is  <dig> and 77% identical to hsirt <dig> and hmyh, respectively.
this high conservation suggests that interactions between hmyh/mmyh <dig> and hsirt6/msirt <dig> may be interchangeable between human and mouse components.
both msirt <dig> and mmyh were purified to more than 90% homogeneity as judged by coomassie blue staining and western blotting .
our data indicate that msirt <dig> could be pulled down by gst-hmyh .
similarly, interaction between msirt <dig> and hape <dig> was established by gst-pull-down assays .
thus, our results show that sirt <dig> directly interacts with these two ber enzymes.
the interdomain connector of myh is important for interactions with sirt6
to determine the regions of hmyh protein engaged in the physical interaction with msirt <dig>  we generated three hmyh deletion constructs fused to gst .
the purified proteins were analyzed by sds–polyacrylamide gel electrophoresis as shown in figure s2a in additional file  <dig>  compared to intact myh , only myh  had a reduced interaction with msirt <dig>  while myh  and myh  had similar binding to msirt <dig> .
our results indicate that residues 316– <dig> of hmyh are critical for msirt6-hmyh interaction.
interestingly, residues 295– <dig>  constituting the interdomain connector  of hmyh  <cit> , are also required for ape <dig> and hus <dig> interactions  <cit> .
we have shown that valine at position  <dig>  of hmyh is important for hus <dig> interaction but is dispensable for interaction with ape <dig>  <cit> .
to test whether v <dig> of hmyh is important for msirt <dig> interaction, we analyzed the binding of msirt <dig> with gst-tagged hmyh containing a v315a mutation.
the result  demonstrates that the v315a mutant of hmyh substantially attenuated its interaction with msirt <dig>  taken together, the idc region of myh is critical for association with hus <dig>  ape <dig>  and sirt <dig> and v <dig> of hmyh is important for sirt <dig> and hus <dig>  but not for ape <dig>  interactions.
sirt <dig> enhances the activities of myh and ape1
to determine the functional output of sirt <dig> binding to myh and ape <dig>  we measured myh and ape <dig> enzymatic activities in the presence of sirt <dig>  in these assays, we kept the ratios of mmyh and hape <dig> to dna lower than  <dig>  in order to observe better stimulation effect.
figure 2a shows that purified msirt <dig> could enhance mmyh <dig> glycosylase activity on fam-labeled a/go-dna.
quantification results  showed that at a sirt6/myh ratio of  <dig>  msirt <dig> significantly enhanced mmyh activity by threefold .
human ape <dig> has very robust ap endonuclease activity and weak 3′-phosphodiesterase activity  <cit> .
we observed that msirt <dig> moderately stimulated both activities of hape <dig> .
mouse sirt <dig> stimulated the ap endonuclease activity of hape <dig> on fam-labeled thf/g-dna by twofold at a sirt6/ape <dig> ratio of  <dig>   .
please note that the concentrations of ape <dig> and sirt <dig> are  <dig>  and  <dig>  fold lower than the dna concentration under this reaction condition.
because the weak 3′-phosphodiesterase activity of ape <dig> could not be detected using thf/g-dna substrate, we used a u/g-containing dna labeled with fam at the 3′-end.
in this case, ape <dig> does not cleave at the 5′ to the uracil, allowing the weak 3′-phosphodiesterase activity of ape <dig> to be detected.
the phosphodiester bond between 3′-fam and dna could be cleaved by hape <dig> and this activity was enhanced by msirt <dig> .
at a sirt6/ape <dig> ratio of  <dig>  msirt <dig> could enhance the phosphodiesterase activity of hape <dig> by twofold  .
under this reaction condition, the concentrations of ape <dig> and sirt <dig> are  <dig>  and fivefold lower than the dna concentration.
it is noteworthy that stimulation of myh glycosylase activity by sirt <dig> reaches saturation with increasing sirt <dig> , no saturation is observed for sirt <dig> enhancement of the hape <dig> activities  in the tested sirt6/ape <dig> ratios.
it may need higher sirt6/ape <dig> ratios to reach saturation.
as a comparison, it has been shown that hmyh can stimulate hape <dig> endonuclease activity by  <dig> -fold with  <dig> -fold molar excess of hmyh over hape <dig>  <cit> .
thus, sirt <dig> stimulates enzymatic activities of hmyh and ape <dig> in vitro.figure  <dig> sirt <dig> stimulates myh and ape <dig> activities.
a msirt <dig> enhances mmyh glycosylase activity.
dna substrates used are shown with arrows indicating the cleavage sites.
lane  <dig> 5′-fam-labeled a/go-containing dna.
lane  <dig> 5 nm a/go-dna was incubated with  <dig>  nm mmyh.
lanes 3– <dig> are similar to lane  <dig> but with added  <dig>   <dig>   <dig>   <dig>   <dig>  and 32 nm msirt <dig>  respectively.
lane  <dig> a/go-dna was incubated with 32 nm msirt <dig> without mmyh.
arrows mark the intact dna substrate  and the cleavage product .
percentage  of product generated is shown below each lane.
c msirt <dig> enhances the ap endonuclease activity of hape <dig>  lane  <dig> 3′-fam-labeled tetrahydrofuran /g-dna.
lane  <dig> 20 nm thf/g-dna was incubated with  <dig>  nm hape <dig>  lanes 3– <dig> are similar to lane  <dig> but with added  <dig>   <dig>   <dig>  and 8 nm msirt <dig>  respectively.
lane  <dig> thf/g-dna was incubated with 8 nm msirt <dig> without ape <dig>  e msirt <dig> enhances the phosphodiesterase activity of hape <dig> that cleaves the 3′fam from a u/g-containing dna.
lane  <dig> 3′-fam-labeled u/g-dna.
lane  <dig> 20 nm u/g-dna was incubated with  <dig>  nm hape <dig>  lanes 3– <dig> are similar to lane  <dig> but with added  <dig>   <dig>   <dig>   <dig>  and 16 nm msirt <dig>  respectively.
lane  <dig> u/g-dna was incubated with 16 nm msirt <dig> without ape <dig>  the product is a free fam.
b, d, and f, quantitative analyses of the fold of stimulation from results as in a, c, and e, respectively.
error bars indicate sd; n =  <dig>
as shown above, both hmyh and hape <dig> primarily interacts with the upper band of sirt <dig>  because sirt <dig> can undergo auto mono-adp-ribosylation  <cit> , it is possible that the upper band of sirt <dig> is a modified form of sirt <dig>  to determine whether mono-adp-ribosyltransferase activity of sirt <dig> is important for the stimulation of ber repair, we assayed sirt6g60a mutant which is defective in this activity  <cit> .
sirt6g60a mutant had a significantly reduced ability to stimulate myh glycosylase and ape <dig> endonuclease activities  as compared to wild-type sirt <dig>  suggesting that mono-adp-ribosyltransferase activity is important for their functional interactions.
sirt <dig> interacts with 9-1-1
because myh and ape <dig> interact with the 9-1- <dig> complex  <cit> , we tested whether sirt <dig> had any interaction with the 9-1- <dig> complex.
equal molar of gst-tagged hus <dig>  rad <dig>  and rad <dig> proteins  were separately immobilized on beads to pull down msirt <dig>  as shown in figure 3a, msirt <dig> bound strongly to gst-rad <dig>  and gst-rad <dig> , and weakly to gst-hus <dig> .
thus, sirt <dig> binds to the 9-1- <dig> complex asymmetrically.
sirt <dig> binds weakly to the hus <dig> subunit, while hmyh and hape <dig> bind preferentially to the hus <dig> subunit  <cit> .
the unique structure of hus <dig> may contribute to this asymmetry in protein–protein interactions.
association between hsirt <dig> and hhus <dig> in vivo was established by co-ip .
the interaction of hsirt <dig> with hrad <dig> was enhanced after h2o <dig> treatment .
thus, hsirt <dig> interactions with 9-1- <dig>  myh, and ape <dig> are all enhanced following oxidative stress.
these results are consistent with a role of sirt <dig> in dna damage response  <cit> .figure  <dig> msirt <dig> interacts with 9-1- <dig> and hmyhq324h does not interact with hhus <dig>  but retains interaction with hape <dig> and msirt <dig>  a immobilized gst, gst-tagged hrad <dig>  hhus <dig>  and hrad <dig>  were used to pull-down flag-msirt <dig>  the procedures are similar to those described in figure 1g.
lane  <dig> contains 10% of input msirt <dig> protein.
b interaction between hsirt <dig> and hrad <dig> is enhanced following h2o <dig> treatment.
rad <dig> was co-immunoprecipitated by sirt <dig> antibody from extracts prepared from untreated  and h2o <dig> treated  hela cells.
western blots were detected by hrad <dig> or hsirt <dig> antibody.
lanes  <dig> and  <dig> contain 10% of input cell extracts .
lanes  <dig> and  <dig> are negative control in which the immunoprecipitation was performed with igg.
c immobilized gst, gst-hmyh, and gst-hmyhq324h  were used to precipitate hape <dig>  msirt <dig>  and hhus <dig>  lanes  <dig> in upper, middle, and lower panels contain 10% of input hape <dig>  20% of input msirt <dig>  and 10% of input hhus <dig>  respectively.
western blotting was performed with hape <dig>  flag, or his antibodies.
d quantitative analyses of bound proteins on gst-tagged myh constructs from three experiments.
the western blots were quantified by the imagequant software .
relative interaction was calculated using input in lane  <dig> as standard.
open bars input references, stripped bars bound proteins on gst-tagged wild-type myh beads, filled bars bound proteins on gst-tagged mutant myh beads.
the hmyhq324h  mutant found in map patients has been reported to attenuate its interaction with hhus <dig> and hrad <dig> by  <dig> and 50%, respectively, in comparison with wild-type hmyh  <cit> .
to examine whether q <dig> is important for sirt <dig> and ape <dig> interaction, we analyzed the binding of msirt <dig> and hape <dig> with gst-tagged hmyhq324h.
we showed that gst-hmyhq324h had no interaction with hhus <dig>  but its interactions with hape <dig> and sirt <dig> were only slightly reduced .
thus, although hus <dig>  ape <dig>  and sirt <dig> bind to the idc region of myh, the binding is mediated by different sequence motifs.
ape <dig>  hus <dig>  and sirt <dig> do not compete for myh association
next, we examined whether sirt <dig>  ape <dig>  and hus <dig> compete or stimulate each other for binding to myh.
we have shown that hus <dig> enhances the myh/ape <dig> complex formation  <cit> .
using the similar approaches, we performed gst pull-down assays of msirt <dig> with immobilized gst-hmyh  in the presence of increasing amounts of hape <dig> .
we observed that hape <dig> enhanced hmyh/msirt <dig> interaction .
there is a threefold stimulation of msirt <dig> binding to myh when a tenfold excess of ape <dig> was added .
when we performed gst pull-down assays of msirt <dig> with immobilized hmyhv315a mutant  in the presence of increasing amounts of hape <dig>  neither enhancement nor inhibition was observed .
thus, the enhancement of the myh/sirt <dig> complex by ape <dig> requires a stable interaction between sirt <dig> and myh.figure  <dig> association of sirt <dig> with myh is stabilized by ape <dig> and hus <dig>  a–c myh/sirt <dig> complex formation is enhanced by ape <dig>  gst-hmyh-wt  or gst-hmyhv315a   were used to pull-down msirt <dig> in the presence of hape <dig>  lanes  <dig> in both  and  contain 10% of input msirt <dig> protein.
lanes  <dig> in both  and  are gst alone.
western blots were detected by flag antibody.
c quantitation of the relative amount of sirt <dig> in the precipitates from three experiments as in a and b. at an 1: <dig>  and 1: <dig>  molar ratio of msirt6:hape <dig>  the amount of msirt <dig> pulled down by gst-hmyh-wt increases, but remains the same by gst-hmyhv315a.
d–f myh/ape <dig> complex formation is enhanced by sirt <dig>  reactions are similar to a–c.
gst-hmyh-wt  or gst-hmyhv315a   were used to pull-down hape <dig> in the presence of msirt <dig>  western blots were detected by ape <dig> antibody.
g–i myh/sirt <dig> complex formation is enhanced by hus <dig>  reactions are similar to a–c.
gst-hmyh-wt  or gst-hmyhq324h    was used to pull-down msirt <dig> in the presence of hus <dig>  western blots were detected by flag antibody.
j myh/sirt <dig> complex formation is enhanced by ape <dig> and hus <dig>  reactions are similar to a. gst-hmyh-wt was used to pull-down msirt <dig> in the combination of ape <dig> and hus <dig>  western blots were detected by flag antibody.
k quantitation of the relative amount of sirt <dig> in the precipitates from three experiments as in j. the quantitation in c, f, i, and k is similar to that described for figure 3c.
inversely, we performed gst pull-down assays of hape <dig> with immobilized hmyh in the presence of increasing amounts of msirt <dig>  msirt <dig> also enhanced hmyh/hape <dig> interaction .
when we performed gst pull-down assays of hape <dig> with immobilized hmyhv315a mutant in the presence of increasing amounts of msirt <dig>  neither enhancement nor inhibition was observed .
in another gst pull-down assay, hhus <dig> enhanced hmyh/msirt <dig> interaction  but did not enhance hmyhq324h/msirt <dig> interaction  .
moreover, when ape <dig> and hus <dig> were added to the pull-down assay of msirt <dig> with immobilized hmyh, both stabilized the myh/sirt <dig> complex .
taken together, our data suggest the formation of a functional dna repair complex constituting of myh, ape <dig>  9-1- <dig>  and sirt <dig>  our results also show that the enhancement of the myh interaction with any one partner by a second protein partner requires a stable interaction between myh and these proteins.
human myh and sirt <dig> are efficiently recruited to confined oxidative dna damage within transcriptionally active chromatin, but not in inactive chromatin
it has been reported that sirt <dig> responds to dna double-strand breaks  <cit>  and sirt <dig> might be important for the regulation of the chromatin states at the sites of damage.
to investigate ber in vivo within chromatin, we have developed novel human systems for confining oxidative dna damage to defined genomic regions within either transcriptionally inactive chromatin or active chromatin  <cit> .
in this approach, local dna damage is induced by activating site-specific killerred  protein  .
kr protein was fused to a tet-repressor  or a transcription activator  and was recruited to a defined genome site in human osteosarcoma u2os cells via the interaction between tetr and integrated tetracycline responsive elements    <cit> .
transcription is suppressed by tetr repressor alone but is activated in the presence of vp <dig>  after activation of the kr by fluorescent light, local oxidative damage is induced within transcriptionally inactive  or active  chromatin.
therefore, we applied the kr systems to examine the damage response of green fluorescence protein -tagged myh and gfp-sirt <dig> following oxidative damage.
without dna damage, gfp-myh and gfp-sirt <dig> were not enriched at sites with ta-mcherry, which serve as negative controls .
after light activation of the kr protein, both the frequency and intensity of 8-oxog production and γh2ax foci at the sites of tetr-kr and ta-kr were similar  <cit> , indicating that comparable amounts of dna damage were produced by tetr-kr and ta-kr.
interestingly, gfp-tagged hmyh and hsirt <dig> were only recruited to damage sites within transcriptionally active chromatin  , but not to damaged sites located within inactive chromatin  .
for quantification, we analyzed  <dig> cells from each group.
after kr activation, over 90% of cells expressing gfp-myh or gfp-sirt <dig> showed the colocalization of gfp-myh foci or gfp-sirt <dig> foci with ta-kr.
in contrast, none of the gfp-myh or gfp-sirt <dig> expressing cell showed foci at sites of tetr-kr.
our data suggest that myh and sirt <dig> act together to repair oxidative dna damages within transcriptionally active chromatin.figure  <dig> hmyh and hsirt <dig> are recruited to oxidatively damaged sites located within transcriptionally active chromatin , but not within inactive chromatin  in human u2os tre cells.
a scheme of tetr- and ta-tagged kr expression in the u2os tre cell  <cit> .
to induce ros-mediated damage at a specific locus in the human genome, we fused kr to the tetracycline repressor  to induce ros damage in a 90-kb tre array  in u2os cells.
we also fused kr to the transcription activator tetr + vp <dig>  to examine damage response at active chromatin.
gfp-hmyh or gfp-hsirt <dig> was co-transfected into the cells to analyze the recruitment of these proteins to the oxidative dna damage sites.
b, c gfp-myh and gfp-sirt <dig> are not enriched at sites with ta-mcherry in undamaged cells.
d, e damage response of gfp-myh and gfp-sirt <dig> to the site of ta-kr after light activation.
f, g no recruitment of gfp-myh and gfp-sirt <dig> to the site of tetr-kr after light activation.
analyses of about  <dig> cells in each kr activated group indicated that over 90% of cells showed the colocalization of gfp-myh foci or gfp-sirt <dig> foci with ta-kr, in contract, none of the myh or sirt <dig> expressing cell showed foci at sites of tetr-kr.
myh foci induced by oxidative stress and sirt <dig> depletion are frequently localized on mouse telomeres
telomeres contain highly c:g rich repetitive dna sequences and specific protein factors at the ends of chromosomes.
these structures are highly susceptible to oxidative damage  <cit>  and maintaining their integrity requires efficient ber .
it has been shown that human sirt <dig>  ape <dig>  and 9-1- <dig> are associated with telomeres and are essential for telomere stability  <cit> .
we have shown that schizosaccharomyces pombe myh <dig> is enriched on telomeres  <cit> .
to examine whether mammalian myh is co-localize at telomeres, we performed myh immunostaining along with telomere fluorescence in situ hybridization   <cit> .
we compared normal  and sirt <dig> knockout  mouse embryonic fibroblast  cells following oxidative dna damage by h2o <dig> treatment.
h2o <dig> treatment to sirt6+/+  cells substantially induced nuclear mmyh foci formation  with 35% of myh foci localized to telomeres .
surprisingly, we observed that myh foci increased in the sirt6−/− cells  even without oxidative stress .
this may be consistent with a previous finding that sirt <dig> knockdown leads to increased γ-h2ax foci at telomeres in human cells  <cit>  indicating that deficiency of sirt <dig> leads to telomere dysfunction.
h2o <dig> treatment to sirt6−/− cells slightly increased the number of mmyh foci  with about 45% localized on telomeres .
these data suggest that myh may play a role in repairing oxidative dna damage at telomeres.figure  <dig> myh foci induced by oxidative stress and sirt <dig> depletion are frequently localized on telomeres.
a, b normal  mef cells; c, d
sirt6
−/−  mef cells.
b, d cells were treated with  <dig>  mm h2o <dig> for 1 h and recovered for 4 h. telomere fish  in red, immunostained mmyh in green, dapi-stained dna in blue.
arrows indicate yellow mmyh foci localized on telomeres.
e quantitation of the number of mmyh foci per nucleus.
f quantitation of percentage of mmyh foci on telomeres .
experiments were repeated twice and more than  <dig> images were analyzed.
error bars indicate sd; n ≥  <dig>  the data of wild-type cell without oxidative stress is not included because the number of mmyh foci is very few .
two stars indicate that p values are smaller than  <dig> .
discussion
sirt <dig> functions as an adp-ribosyltransferase and protein deacetylase, and can remove the fatty acyl modification from proteins  <cit> , thus affecting many cellular functions including genomic stability.
it has been shown that sirt <dig> is involved in dna double-strand break repair through interacting with several factors.
sirt <dig> promotes dna double-strand break repair by interacting with dna-dependent protein kinase  and recruiting dna-pk to chromatin at double-strand breaks  <cit> .
sirt <dig> mono-adp-ribosylates parp <dig>  and stimulates the poly-adp-ribosyltransferase activity of parp <dig>  <cit>  while sirt <dig> deacetylates ctip   <cit> .
in addition, sirt <dig> recruits the chromatin remodeling factor snf2h to double-strand breaks which, in turn is required for proper recruitment of downstream ddr factors and efficient dna repair  <cit> .
sirt <dig> has been suggested to be involved in ber based on the phenotypes of sirt <dig> knockout mouse cells  <cit> .
three recent publications support sirt6′s role in ber.
polyakova et al.
<cit>  used yeast two-hybrid analyses to identify thymine dna glycosylase as a partner of sirt6; however, the authors did not further examine their direct physical and functional interactions.
mao et al.
<cit>  reported that sirt <dig> activates parp <dig> and xu et al.
<cit>  recently reported that sirt <dig> regulates ber in a parp1-dependent manner.
while those reports show the participation of sirt <dig> in ber, our results provide a detailed mechanism of sirt <dig> function in ber.
in this study, we provide the first evidence that sirt <dig> interacts with myh, ape <dig>  and the 9-1- <dig> complex and these interactions are enhanced following oxidative stress.
sirt <dig> stimulates the enzymatic activities of myh and ape <dig> in vitro in the absence of nad+.
these findings suggest that sirt <dig> does not modify myh or ape <dig> in our in vitro assays.
consistent with our observation, xu et al.
<cit>  stated that they did not identify ber proteins which could be deacetylated by sirt <dig>  however, it remains to be tested whether sirt <dig> regulates these interacting partners in vivo through protein modification.
interestingly, the sirt6-myh interaction involves the inter-domain connector of hmyh that is also important for association with ape <dig>  <cit>  and hus <dig>  <cit> .
however, sirt <dig>  ape <dig>  and hus <dig> bind myh through overlapping but different sequence motifs.
the hmyh v315a mutation attenuates its interaction with hhus <dig>  <cit>  and sirt <dig>  but not with hape <dig>  <cit>  while the hmyh q324h mutation abolishes its interaction with hhus <dig>  <cit>  but not with hape <dig> and sirt <dig> .
by gst pull-down assays in the presence of more than two myh protein partners, we did not detect any competition between ape <dig>  hus <dig>  or sirt <dig> binding to myh.
rather, one myh partner enhances the association of the other two to myh .
these data suggest that sirt <dig>  hus <dig>  and ape <dig> may form a complex with myh.
how these three partner proteins interact with myh within this short ~50-residue region remains to be determined.
the structure of the idc  of hmyh has been shown to adopt a stabilized conformation projecting away from the catalytic domain  <cit>  and may be suitable to form a docking scaffold for 9-1- <dig>  ape <dig>  and sirt <dig>
the ability of hus <dig> to stabilize the myh/ape1/sirt <dig> complex supports the model that 9-1- <dig> serves as a platform to coordinate ber  <cit>  and maximize repair efficiency.
we observed that the enhancement synergy of the myh interaction with its partners is lost with hmyhv315a and hmyhq324h mutants.
hmyhq324h variant, found in map patients, is defective in interacting with 9-1- <dig>  <cit> .
the phenotypes of hmyhq324h variant, as observed in myh knockout mouse embryo fibroblasts, are associated with increased go levels, hypersensitivity to oxidants, and accumulation of the cell population in the s phase of the cell cycle  <cit> .
some of these repair-defective phenotypes of hmyhq324h may be attributed by its inefficient recruitment of sirt <dig> or ape <dig> to the damage sites.
although sirt <dig> physically interacts with myh and ape <dig>  msirt <dig> stimulation on mmyh and hape <dig> activities is very subtle.
at first glance, the effects of these functional interactions appear minor, as the glycosylase activity of mmyh increases only threefold in the presence of 30-fold excess of msirt <dig>  even worse, the ape <dig> activities increase only twofold in the presence of 400- <dig> -fold excess of msirt <dig>  however, these in vitro reactions were performed with enzyme concentrations far lower than dna concentrations.
it seems unlikely that sirt <dig> would attain intracellular concentrations that would be  <dig> -fold higher than ape <dig> in mammalian cells.
because ap endonuclease activity of hape <dig> is very robust, it may not require further stimulation by other factors.
however, this mild stimulation on ber enzymes has been found in several cases.
for example, we have shown that myh can be enhanced by fourfold with tenfold molar excess of 9-1- <dig>  <cit>  and can be stimulated by fourfold with 125-fold molar excess of ape <dig>  <cit> .
in addition, ape <dig> activity can be moderately stimulated by myh with  <dig> -fold molar excess of myh over ape <dig>  <cit> .
although the interactions of myh with sirt <dig>  ape <dig>  and 9-1- <dig> produce modest stimulation on myh catalytic activity in vitro, these interactions may be physiologically significant.
we have demonstrated that a catalytically active spmyh1i261a/e262q mutant, which corresponds to the hmyhv315a/e316q mutant, cannot reduce the mutation frequency of myh1Δ cells  <cit> .
in addition, expression of a peptide consisting of the idc of spmyh <dig> that interferes with the interactions between spmyh <dig> and interacting proteins in s. pombe cells makes cells more sensitive to h2o <dig>  <cit> .
such a regulatory network of weak protein interactions may offer the ber pathway sufficient flexibility to coordinate with dna replication, dna damage response, and other dna repair pathways.
interestingly, myh and ape <dig> prefer to bind to the upper band of sirt <dig> in co-immunoprecipitation analyses.
although the nature of the upper band of sirt <dig> is unknown, we suspect it may be a modified form of sirt <dig>  it has been shown that sirt <dig> can undergo auto mono-adp-ribosylation which may contribute to the self-regulation of sirt <dig> function  <cit> .
we also observed that mono-adp-ribosylation defective sirt6g60a mutant could not efficiently stimulate myh glycosylase and ape <dig> endonuclease activities as compared to wild-type sirt <dig>  this suggests that auto mono-adp-ribosylation is important for sirt <dig> function in ber.
thus, we suggested that the modified form of sirt <dig> participates in ber.
we favor a model that sirt <dig> in the complex with myh, ape <dig>  and 9-1- <dig> at sites of dna damage may undergo auto mono-adp-ribosylation leading to enhanced chromatin remodeling and optimal dna repair efficiency .figure  <dig> a model for sirt <dig> interaction with myh, ape <dig>  and rad9-rad1-hus <dig> to repair dna damages on transcriptionally active chromatin and telomeres.
the n- and c-terminal domains  of myh are connected with the interdomain connector .
sirt <dig>  ape <dig>  and rad9-rad1-hus <dig> bind overlapping but different sequence motifs on idc region of myh.
the model suggests that sirt <dig> in the complex with myh, ape <dig>  and 9-1- <dig> at sites of dna damage may undergo auto mono-adp-ribosylation leading to enhanced chromatin remodeling and optimal dna repair efficiency.
hmyh and hsirt <dig> are efficiently recruited to kr-induced confined oxidative dna damage within transcriptionally active chromatin, but not the dna damage within inactive chromatin.
this property of hmyh is unique because other dna glycosylases such as nth <dig>  neil <dig>  neil <dig>  and mbd <dig> are recruited to both transcriptionally active chromatin and inactive chromatin  <cit> .
the preferred recruitment at sites of dna damage within active chromatin is also found with fen <dig> and pcna  <cit> , consistant with the reports that myh interacts with pcna in the long-patch ber pathway  <cit> .
however, the determinants for long-patch ber pathway occurs at transcriptionally active chromatin are not clear.
because myh needs to recognize both the mismatched adenine and go, transcriptionally inactive heterochromatin may be not accessible to myh even in the presence of sirt <dig>  sirt <dig> has been shown to interact with parp <dig>  <cit>  and to regulate ber in a parp1-depdendent manner  <cit> , however, parp <dig> is recruited to dna damage within transcriptionally inactive chromatin more efficiently than in active chromatin  <cit> .
although this differential response to kr-induced oxidative damage is not fully understood, this may imply that sirt <dig> stimulation of parp <dig> activity at ta-kr-bound sites may be subtle and that parp <dig> may play minimal role in myh-mediated long-patch ber.
the parp1-depdendent ber analyzed by plasmid reactivation assay used by xu et al.
<cit>  may not involve myh.
telomeric dna are highly susceptible to oxidative damage compared to other regions of the chromatin  <cit> .
oxidative damage to telomeric dna accelerates telomere shortening and requires efficient dna repair to maintain its integrity .
in addition, chromatin at telomeres contains hypoacetylated histones  <cit>  and requires remodeling to give the repair machinery full access to sites of dna damage.
it has been shown that human sirt <dig>  ape <dig>  and 9-1- <dig> are associated with telomeres and are essential for telomere stability  <cit> .
similar to s. pombe myh <dig>  <cit> , for the first time, we show that mouse myh is associated with damaged telomeres.
it has been shown that γ-h2ax foci increase at telomeres in sirt <dig> knockdown cells  <cit> .
this telomere dysfunction is consistent with increased myh foci at telomeres in sirt <dig> knockout cells without oxidative damage .
the interaction of 9-1- <dig> with sirt <dig> and ber enzymes ensures that dna repair, chromatin remodeling, and ddr are coordinated.
our findings indicate that sirt <dig> interacts with myh, ape <dig>  and 9-1- <dig> to maintain genomic integrity of telomeres and transcriptionally active chromatin, but not transcriptionally inactive chromatin .
our model suggests that sirt <dig> in the complex with myh, ape <dig>  and 9-1- <dig> at sites of dna damage may undergo auto mono-adp-ribosylation leading to enhanced chromatin remodeling and optimal dna repair efficiency.
the similar response of sirt <dig> and myh to oxidative dna damage within transcriptionally active chromatin suggests that sirt <dig> may alter the chromatin structure and facilitate dna repair.
because myh needs to recognize both the mismatched adenine and the go located on the other strand, the dna glycosylase activity of myh is expected to be strongly prohibited on nucleosome-bound mismatches.
thus, myh repair may require substantial nucleosome remodeling to expose the mismatch for efficient repair.
it has been reported that sirt <dig> recruits the chromatin remodeler snf2h to double-strand breaks and focally deacetylates histone h3k <dig>  <cit> .
it will be interesting to see whether sirt <dig> recruits snf2h to oxidatively damaged telomeres to enhance ber.
CONCLUSIONS
the results presented in this study demonstrate that sirt <dig> has a direct role in ber by forming a complex with and stimulating myh and ape <dig>  our finding that sirt <dig> interacts with the 9-1- <dig> complex suggests sirt <dig> may have a role in ddr and is consistent with the finding that the lack of sirt <dig> profoundly impacts upon downstream recruitment of dna repair factors  <cit> .
our data demonstrate that sirt <dig>  ape <dig>  and hus <dig> bind to the idc region of hmyh without competition.
we demonstrate that hmyh and hsirt <dig> are efficiently recruited to confined oxidative dna damage in transcriptionally active chromatin in human cells and that mmyh foci induced by oxidative stress and msirt <dig> depletion are frequently localized on mouse telomeres.
overall, our findings suggest that sirt <dig> forms a complex with myh, ape <dig>  and 9-1- <dig> to maintain genomic and telomeric integrity.
sirt <dig> interactions with myh, ape <dig>  and 9-1- <dig> fit well with the observed marked genomic instability of sirt <dig> deficient cells and aging phenotypes of sirt <dig> knockout mice  <cit> .
taken together, our data highlight a potential role of chromatin remodeling in dna repair and ddr.

 reactive oxygen species and radiation cause dna strand breaks and base lesions, thus affecting genomic integrity. particularly, the c-g-rich telomeres are highly susceptible to oxidative damage. a frequent and highly mutagenic oxidative lesion is 8-oxo- -dihydroguanine, which mispairs with adenine during dna replication, resulting in g:c to t:a mutations. oxidative dna lesions are repaired primarily by the base excision repair pathway. the first step in ber is carried out by a dna glycosylase, which cleaves the damaged or mismatched base. the resulting apurinic/apyrimidinic site is processed by ap-endonuclease, allowing the downstream ber enzymes to complete the dna repair process. muty homolog excises adenines from a/go mismatches and thus reduces g:c to t:a mutations. mutations in the human myh gene can lead to colorectal cancer. ber is coordinated with other cellular processes in eukaryotic cells. eukaryotic myh contains unique motifs to mediate interactions with partner proteins involved in dna replication, mismatch repair, and dna damage response. we have shown that the interdomain connector located between the n- and c-terminal domains of hmyh is uniquely oriented to interact with ape and hus, a subunit of the heterotrimeric rad9–rad1–hus checkpoint clamp. ape is essential for cell viability and participates in many aspects of dna metabolism and telomere maintenance. in dna repair, ape cleaves the phosphodiester bond 5′ to an ap site and removes various forms of 3′-blocked lesions at dna strand breaks. because ap sites are mutagenic and cytotoxic, they must be recognized by ape immediately after the action of a dna glycosylase. a “passing-the-baton” model has been proposed for ber, consistent with findings that ape stimulates many dna glycosylases. ape forms a stable complex with myh and 9-1-. besides serving as a damage sensor, 9-1- is involved in many dna metabolisms including ber. intriguingly, 9-1- interacts with and stimulates the activity of almost every enzyme in the ber pathway and has been proposed to serve as a platform to coordinate ber. sirt is a member of nad+-dependent histone/protein deacetylase family and also has mono-adp-ribosyltransferase and protein lysine fatty acyl removal activities. sirt plays a role in stress response, dna repair, telomere integrity, retrotransposition, and metabolic homeostasis. sirt knockout mice display a shortened lifespan associated with impaired dna repair. moreover, sirt depletion leads to telomere dysfunction and premature cellular senescence. during the course of aging and in response to dna damage, sirt is depleted from l retrotransposon loci, allowing their activation. sirt has been implicated in ber. it has been demonstrated that sirt can activate parp and is a partner of thymine dna glycosylase. xu et al. recently reported that sirt regulates ber in a parp1-depdendent manner. however, direct physical and functional interactions between sirt and ber enzymes remained to be elucidated. here, we provide evidence for a direct role of sirt in ber and dna damage response. we show that mouse sirt interacts with myh, ape and 9-1- and stimulates myh and ape activities. our data demonstrate that sirt ape and hus bind to hmyh without competition. instead, one myh partner enhances the association of the other two partners to the myh complex. by using novel systems for confining oxidative dna damage to defined human genomic regions, we show that myh and sirt are efficiently recruited to dna damage sites within transcriptionally active chromatin, but not within inactive chromatin in human cells. in addition, we show that the number of myh nuclear foci, frequently found to localize on telomeres, increase in sirt6−/− mouse embryonic fibroblast cells. a further increase in telomere localization of myh foci was found in the presence of oxidative stress. thus, sirt represents an interesting connection between chromatin remodeling and myh-directed ber.  sirt interacts with myh and ape1 to examine whether sirt plays a direct role in genomic integrity through ber, we analyzed the physical and functional interactions between sirt and two major ber enzymes. first, association between sirt and myh was demonstrated by co-immunoprecipitation. human cell extracts were subjected to immunoprecipitation with hmyh antibody followed by western blot analysis with anti hmyh or hsirt antibodies. both hmyh and hsirt were found in the pellet with hmyh antibody but not with control igg. it is interesting to note that hmyh primarily interacts with the upper band of sirt. we suspect the upper band may be a modified form of sirt because sirt can undergo auto mono-adp-ribosylation. conversely, human cell extracts were subjected to immunoprecipitation with hsirt antibody followed by western analysis with hsirt or hmyh antibodies. in this assay, both bands of hsirt interacted with sirt antibody. human myh was co-immunoprecipitated with hsirt antibody. using the similar approach, association between hsirt and hape was established by co-ip. as observed in hmyh-sirt interaction, hape primarily interacts with the upper band of sirt. thus, hsirt associates with hmyh and hape figure  sirt interacts with myh, ape and rad9–rad1–hus a sirt can be co-immunoprecipitated by hmyh antibody from hela extracts. immunoprecipitation was performed with hmyh antibody and detected on the western blot using hsirt or hmyh antibody. lane contains 10% of input cell extracts. lane is a negative control in which the immunoprecipitation was performed with igg. b interaction between hmyh and hsirt is enhanced following h2o treatment. myh was co-immunoprecipitated by sirt antibody from extracts prepared from untreated hela cells or from cells treated with  mm h2o for 1 h and recovered for 6 h. western blots were detected by hmyh or hsirt antibody. control lanes are similar to those described in. c sirt and ape co-immunoprecipitated from hela extracts. immunoprecipitation was performed with hape antibody and western blotting was performed with hape or hsirt antibody. control lanes are similar to a. d interaction between hape and hsirt is enhanced following h2o treatment. ape was co-immunoprecipitated by sirt antibody from extracts prepared from untreated and h2o treated hela cells. western blots were detected by hape or hsir antibody. e, f immobilized gst-hmyh and gst-hape, respectively, were used to pull down flag-msirt. lane contains 10% of input msirt protein. lane used gst-beads alone. flag-msirt was detected by an anti-flag antibody. g immobilized gst, gst-tagged intact hmyh, myhΔc myhΔc myhΔc3m, and myhΔc were used to precipitate flag-msirt the hmyh constructs are depicted. to investigate the effect of dna damage on the interactions between sirt and ber enzymes, we performed co-ip experiments with extracts from hela cells treated with  mm h2o for 1 h and recovered for 6 h. interestingly, the interactions of hmyh and hape with hsirt were enhanced after h2o treatment. this result indicates that hsirt interactions with ber enzymes are enhanced following oxidative treatment. to show direct physical interactions between sirt and ber enzymes, we performed gst-pull-down assays in the presence of ethidium bromide to eliminate the effect of nucleic acid on protein–protein interactions. due to technical difficulties for purifying human enzymes, we purified mouse sirt and mouse myh that is and 77% identical to hsirt and hmyh, respectively. this high conservation suggests that interactions between hmyh/mmyh and hsirt6/msirt may be interchangeable between human and mouse components. both msirt and mmyh were purified to more than 90% homogeneity as judged by coomassie blue staining and western blotting. our data indicate that msirt could be pulled down by gst-hmyh. similarly, interaction between msirt and hape was established by gst-pull-down assays. thus, our results show that sirt directly interacts with these two ber enzymes. the interdomain connector of myh is important for interactions with sirt6 to determine the regions of hmyh protein engaged in the physical interaction with msirt we generated three hmyh deletion constructs fused to gst. the purified proteins were analyzed by sds–polyacrylamide gel electrophoresis as shown in figure s2a in additional file compared to intact myh, only myh had a reduced interaction with msirt while myh and myh had similar binding to msirt. our results indicate that residues 316– of hmyh are critical for msirt6-hmyh interaction. interestingly, residues 295– constituting the interdomain connector of hmyh, are also required for ape and hus interactions. we have shown that valine at position of hmyh is important for hus interaction but is dispensable for interaction with ape. to test whether v of hmyh is important for msirt interaction, we analyzed the binding of msirt with gst-tagged hmyh containing a v315a mutation. the result demonstrates that the v315a mutant of hmyh substantially attenuated its interaction with msirt taken together, the idc region of myh is critical for association with hus ape and sirt and v of hmyh is important for sirt and hus but not for ape interactions. sirt enhances the activities of myh and ape1 to determine the functional output of sirt binding to myh and ape we measured myh and ape enzymatic activities in the presence of sirt in these assays, we kept the ratios of mmyh and hape to dna lower than in order to observe better stimulation effect. figure 2a shows that purified msirt could enhance mmyh glycosylase activity on fam-labeled a/go-dna. quantification results showed that at a sirt6/myh ratio of msirt significantly enhanced mmyh activity by threefold. human ape has very robust ap endonuclease activity and weak 3′-phosphodiesterase activity. we observed that msirt moderately stimulated both activities of hape. mouse sirt stimulated the ap endonuclease activity of hape on fam-labeled thf/g-dna by twofold at a sirt6/ape ratio of. please note that the concentrations of ape and sirt are and fold lower than the dna concentration under this reaction condition. because the weak 3′-phosphodiesterase activity of ape could not be detected using thf/g-dna substrate, we used a u/g-containing dna labeled with fam at the 3′-end. in this case, ape does not cleave at the 5′ to the uracil, allowing the weak 3′-phosphodiesterase activity of ape to be detected. the phosphodiester bond between 3′-fam and dna could be cleaved by hape and this activity was enhanced by msirt. at a sirt6/ape ratio of msirt could enhance the phosphodiesterase activity of hape by twofold. under this reaction condition, the concentrations of ape and sirt are and fivefold lower than the dna concentration. it is noteworthy that stimulation of myh glycosylase activity by sirt reaches saturation with increasing sirt, no saturation is observed for sirt enhancement of the hape activities in the tested sirt6/ape ratios. it may need higher sirt6/ape ratios to reach saturation. as a comparison, it has been shown that hmyh can stimulate hape endonuclease activity by -fold with -fold molar excess of hmyh over hape. thus, sirt stimulates enzymatic activities of hmyh and ape in vitro.figure  sirt stimulates myh and ape activities. a msirt enhances mmyh glycosylase activity. dna substrates used are shown with arrows indicating the cleavage sites. lane 5′-fam-labeled a/go-containing dna. lane 5 nm a/go-dna was incubated with  nm mmyh. lanes 3– are similar to lane but with added and 32 nm msirt respectively. lane a/go-dna was incubated with 32 nm msirt without mmyh. arrows mark the intact dna substrate and the cleavage product. percentage of product generated is shown below each lane. c msirt enhances the ap endonuclease activity of hape lane 3′-fam-labeled tetrahydrofuran /g-dna. lane 20 nm thf/g-dna was incubated with  nm hape lanes 3– are similar to lane but with added and 8 nm msirt respectively. lane thf/g-dna was incubated with 8 nm msirt without ape e msirt enhances the phosphodiesterase activity of hape that cleaves the 3′fam from a u/g-containing dna. lane 3′-fam-labeled u/g-dna. lane 20 nm u/g-dna was incubated with  nm hape lanes 3– are similar to lane but with added and 16 nm msirt respectively. lane u/g-dna was incubated with 16 nm msirt without ape the product is a free fam. b, d, and f, quantitative analyses of the fold of stimulation from results as in a, c, and e, respectively. error bars indicate sd; n =  as shown above, both hmyh and hape primarily interacts with the upper band of sirt because sirt can undergo auto mono-adp-ribosylation, it is possible that the upper band of sirt is a modified form of sirt to determine whether mono-adp-ribosyltransferase activity of sirt is important for the stimulation of ber repair, we assayed sirt6g60a mutant which is defective in this activity. sirt6g60a mutant had a significantly reduced ability to stimulate myh glycosylase and ape endonuclease activities as compared to wild-type sirt suggesting that mono-adp-ribosyltransferase activity is important for their functional interactions. sirt interacts with 9-1-1 because myh and ape interact with the 9-1- complex, we tested whether sirt had any interaction with the 9-1- complex. equal molar of gst-tagged hus rad and rad proteins were separately immobilized on beads to pull down msirt as shown in figure 3a, msirt bound strongly to gst-rad and gst-rad, and weakly to gst-hus. thus, sirt binds to the 9-1- complex asymmetrically. sirt binds weakly to the hus subunit, while hmyh and hape bind preferentially to the hus subunit. the unique structure of hus may contribute to this asymmetry in protein–protein interactions. association between hsirt and hhus in vivo was established by co-ip. the interaction of hsirt with hrad was enhanced after h2o treatment. thus, hsirt interactions with 9-1- myh, and ape are all enhanced following oxidative stress. these results are consistent with a role of sirt in dna damage response.figure  msirt interacts with 9-1- and hmyhq324h does not interact with hhus but retains interaction with hape and msirt a immobilized gst, gst-tagged hrad hhus and hrad were used to pull-down flag-msirt the procedures are similar to those described in figure 1g. lane contains 10% of input msirt protein. b interaction between hsirt and hrad is enhanced following h2o treatment. rad was co-immunoprecipitated by sirt antibody from extracts prepared from untreated and h2o treated hela cells. western blots were detected by hrad or hsirt antibody. lanes and contain 10% of input cell extracts. lanes and are negative control in which the immunoprecipitation was performed with igg. c immobilized gst, gst-hmyh, and gst-hmyhq324h were used to precipitate hape msirt and hhus lanes in upper, middle, and lower panels contain 10% of input hape 20% of input msirt and 10% of input hhus respectively. western blotting was performed with hape flag, or his antibodies. d quantitative analyses of bound proteins on gst-tagged myh constructs from three experiments. the western blots were quantified by the imagequant software. relative interaction was calculated using input in lane as standard. open bars input references, stripped bars bound proteins on gst-tagged wild-type myh beads, filled bars bound proteins on gst-tagged mutant myh beads. the hmyhq324h mutant found in map patients has been reported to attenuate its interaction with hhus and hrad by and 50%, respectively, in comparison with wild-type hmyh. to examine whether q is important for sirt and ape interaction, we analyzed the binding of msirt and hape with gst-tagged hmyhq324h. we showed that gst-hmyhq324h had no interaction with hhus but its interactions with hape and sirt were only slightly reduced. thus, although hus ape and sirt bind to the idc region of myh, the binding is mediated by different sequence motifs. ape hus and sirt do not compete for myh association next, we examined whether sirt ape and hus compete or stimulate each other for binding to myh. we have shown that hus enhances the myh/ape complex formation. using the similar approaches, we performed gst pull-down assays of msirt with immobilized gst-hmyh in the presence of increasing amounts of hape. we observed that hape enhanced hmyh/msirt interaction. there is a threefold stimulation of msirt binding to myh when a tenfold excess of ape was added. when we performed gst pull-down assays of msirt with immobilized hmyhv315a mutant in the presence of increasing amounts of hape neither enhancement nor inhibition was observed. thus, the enhancement of the myh/sirt complex by ape requires a stable interaction between sirt and myh.figure  association of sirt with myh is stabilized by ape and hus a–c myh/sirt complex formation is enhanced by ape gst-hmyh-wt or gst-hmyhv315a were used to pull-down msirt in the presence of hape lanes in both and contain 10% of input msirt protein. lanes in both and are gst alone. western blots were detected by flag antibody. c quantitation of the relative amount of sirt in the precipitates from three experiments as in a and b. at an 1: and 1: molar ratio of msirt6:hape the amount of msirt pulled down by gst-hmyh-wt increases, but remains the same by gst-hmyhv315a. d–f myh/ape complex formation is enhanced by sirt reactions are similar to a–c. gst-hmyh-wt or gst-hmyhv315a were used to pull-down hape in the presence of msirt western blots were detected by ape antibody. g–i myh/sirt complex formation is enhanced by hus reactions are similar to a–c. gst-hmyh-wt or gst-hmyhq324h was used to pull-down msirt in the presence of hus western blots were detected by flag antibody. j myh/sirt complex formation is enhanced by ape and hus reactions are similar to a. gst-hmyh-wt was used to pull-down msirt in the combination of ape and hus western blots were detected by flag antibody. k quantitation of the relative amount of sirt in the precipitates from three experiments as in j. the quantitation in c, f, i, and k is similar to that described for figure 3c. inversely, we performed gst pull-down assays of hape with immobilized hmyh in the presence of increasing amounts of msirt msirt also enhanced hmyh/hape interaction. when we performed gst pull-down assays of hape with immobilized hmyhv315a mutant in the presence of increasing amounts of msirt neither enhancement nor inhibition was observed. in another gst pull-down assay, hhus enhanced hmyh/msirt interaction but did not enhance hmyhq324h/msirt interaction. moreover, when ape and hus were added to the pull-down assay of msirt with immobilized hmyh, both stabilized the myh/sirt complex. taken together, our data suggest the formation of a functional dna repair complex constituting of myh, ape 9-1- and sirt our results also show that the enhancement of the myh interaction with any one partner by a second protein partner requires a stable interaction between myh and these proteins. human myh and sirt are efficiently recruited to confined oxidative dna damage within transcriptionally active chromatin, but not in inactive chromatin it has been reported that sirt responds to dna double-strand breaks and sirt might be important for the regulation of the chromatin states at the sites of damage. to investigate ber in vivo within chromatin, we have developed novel human systems for confining oxidative dna damage to defined genomic regions within either transcriptionally inactive chromatin or active chromatin. in this approach, local dna damage is induced by activating site-specific killerred protein. kr protein was fused to a tet-repressor or a transcription activator and was recruited to a defined genome site in human osteosarcoma u2os cells via the interaction between tetr and integrated tetracycline responsive elements. transcription is suppressed by tetr repressor alone but is activated in the presence of vp after activation of the kr by fluorescent light, local oxidative damage is induced within transcriptionally inactive or active chromatin. therefore, we applied the kr systems to examine the damage response of green fluorescence protein -tagged myh and gfp-sirt following oxidative damage. without dna damage, gfp-myh and gfp-sirt were not enriched at sites with ta-mcherry, which serve as negative controls. after light activation of the kr protein, both the frequency and intensity of 8-oxog production and γh2ax foci at the sites of tetr-kr and ta-kr were similar, indicating that comparable amounts of dna damage were produced by tetr-kr and ta-kr. interestingly, gfp-tagged hmyh and hsirt were only recruited to damage sites within transcriptionally active chromatin, but not to damaged sites located within inactive chromatin. for quantification, we analyzed cells from each group. after kr activation, over 90% of cells expressing gfp-myh or gfp-sirt showed the colocalization of gfp-myh foci or gfp-sirt foci with ta-kr. in contrast, none of the gfp-myh or gfp-sirt expressing cell showed foci at sites of tetr-kr. our data suggest that myh and sirt act together to repair oxidative dna damages within transcriptionally active chromatin.figure  hmyh and hsirt are recruited to oxidatively damaged sites located within transcriptionally active chromatin, but not within inactive chromatin in human u2os tre cells. a scheme of tetr- and ta-tagged kr expression in the u2os tre cell. to induce ros-mediated damage at a specific locus in the human genome, we fused kr to the tetracycline repressor to induce ros damage in a 90-kb tre array in u2os cells. we also fused kr to the transcription activator tetr + vp to examine damage response at active chromatin. gfp-hmyh or gfp-hsirt was co-transfected into the cells to analyze the recruitment of these proteins to the oxidative dna damage sites. b, c gfp-myh and gfp-sirt are not enriched at sites with ta-mcherry in undamaged cells. d, e damage response of gfp-myh and gfp-sirt to the site of ta-kr after light activation. f, g no recruitment of gfp-myh and gfp-sirt to the site of tetr-kr after light activation. analyses of about cells in each kr activated group indicated that over 90% of cells showed the colocalization of gfp-myh foci or gfp-sirt foci with ta-kr, in contract, none of the myh or sirt expressing cell showed foci at sites of tetr-kr. myh foci induced by oxidative stress and sirt depletion are frequently localized on mouse telomeres telomeres contain highly c:g rich repetitive dna sequences and specific protein factors at the ends of chromosomes. these structures are highly susceptible to oxidative damage and maintaining their integrity requires efficient ber. it has been shown that human sirt ape and 9-1- are associated with telomeres and are essential for telomere stability. we have shown that schizosaccharomyces pombe myh is enriched on telomeres. to examine whether mammalian myh is co-localize at telomeres, we performed myh immunostaining along with telomere fluorescence in situ hybridization. we compared normal and sirt knockout mouse embryonic fibroblast cells following oxidative dna damage by h2o treatment. h2o treatment to sirt6+/+ cells substantially induced nuclear mmyh foci formation with 35% of myh foci localized to telomeres. surprisingly, we observed that myh foci increased in the sirt6−/− cells even without oxidative stress. this may be consistent with a previous finding that sirt knockdown leads to increased γ-h2ax foci at telomeres in human cells indicating that deficiency of sirt leads to telomere dysfunction. h2o treatment to sirt6−/− cells slightly increased the number of mmyh foci with about 45% localized on telomeres. these data suggest that myh may play a role in repairing oxidative dna damage at telomeres.figure  myh foci induced by oxidative stress and sirt depletion are frequently localized on telomeres. a, b normal mef cells; c, d sirt6 −/− mef cells. b, d cells were treated with  mm h2o for 1 h and recovered for 4 h. telomere fish in red, immunostained mmyh in green, dapi-stained dna in blue. arrows indicate yellow mmyh foci localized on telomeres. e quantitation of the number of mmyh foci per nucleus. f quantitation of percentage of mmyh foci on telomeres. experiments were repeated twice and more than images were analyzed. error bars indicate sd; n ≥  the data of wild-type cell without oxidative stress is not included because the number of mmyh foci is very few. two stars indicate that p values are smaller than. discussion sirt functions as an adp-ribosyltransferase and protein deacetylase, and can remove the fatty acyl modification from proteins, thus affecting many cellular functions including genomic stability. it has been shown that sirt is involved in dna double-strand break repair through interacting with several factors. sirt promotes dna double-strand break repair by interacting with dna-dependent protein kinase and recruiting dna-pk to chromatin at double-strand breaks. sirt mono-adp-ribosylates parp and stimulates the poly-adp-ribosyltransferase activity of parp while sirt deacetylates ctip. in addition, sirt recruits the chromatin remodeling factor snf2h to double-strand breaks which, in turn is required for proper recruitment of downstream ddr factors and efficient dna repair. sirt has been suggested to be involved in ber based on the phenotypes of sirt knockout mouse cells. three recent publications support sirt6′s role in ber. polyakova et al. used yeast two-hybrid analyses to identify thymine dna glycosylase as a partner of sirt6; however, the authors did not further examine their direct physical and functional interactions. mao et al. reported that sirt activates parp and xu et al. recently reported that sirt regulates ber in a parp1-dependent manner. while those reports show the participation of sirt in ber, our results provide a detailed mechanism of sirt function in ber. in this study, we provide the first evidence that sirt interacts with myh, ape and the 9-1- complex and these interactions are enhanced following oxidative stress. sirt stimulates the enzymatic activities of myh and ape in vitro in the absence of nad+. these findings suggest that sirt does not modify myh or ape in our in vitro assays. consistent with our observation, xu et al. stated that they did not identify ber proteins which could be deacetylated by sirt however, it remains to be tested whether sirt regulates these interacting partners in vivo through protein modification. interestingly, the sirt6-myh interaction involves the inter-domain connector of hmyh that is also important for association with ape and hus. however, sirt ape and hus bind myh through overlapping but different sequence motifs. the hmyh v315a mutation attenuates its interaction with hhus and sirt but not with hape while the hmyh q324h mutation abolishes its interaction with hhus but not with hape and sirt. by gst pull-down assays in the presence of more than two myh protein partners, we did not detect any competition between ape hus or sirt binding to myh. rather, one myh partner enhances the association of the other two to myh. these data suggest that sirt hus and ape may form a complex with myh. how these three partner proteins interact with myh within this short ~50-residue region remains to be determined. the structure of the idc of hmyh has been shown to adopt a stabilized conformation projecting away from the catalytic domain and may be suitable to form a docking scaffold for 9-1- ape and sirt the ability of hus to stabilize the myh/ape1/sirt complex supports the model that 9-1- serves as a platform to coordinate ber and maximize repair efficiency. we observed that the enhancement synergy of the myh interaction with its partners is lost with hmyhv315a and hmyhq324h mutants. hmyhq324h variant, found in map patients, is defective in interacting with 9-1-. the phenotypes of hmyhq324h variant, as observed in myh knockout mouse embryo fibroblasts, are associated with increased go levels, hypersensitivity to oxidants, and accumulation of the cell population in the s phase of the cell cycle. some of these repair-defective phenotypes of hmyhq324h may be attributed by its inefficient recruitment of sirt or ape to the damage sites. although sirt physically interacts with myh and ape msirt stimulation on mmyh and hape activities is very subtle. at first glance, the effects of these functional interactions appear minor, as the glycosylase activity of mmyh increases only threefold in the presence of 30-fold excess of msirt even worse, the ape activities increase only twofold in the presence of 400- -fold excess of msirt however, these in vitro reactions were performed with enzyme concentrations far lower than dna concentrations. it seems unlikely that sirt would attain intracellular concentrations that would be -fold higher than ape in mammalian cells. because ap endonuclease activity of hape is very robust, it may not require further stimulation by other factors. however, this mild stimulation on ber enzymes has been found in several cases. for example, we have shown that myh can be enhanced by fourfold with tenfold molar excess of 9-1- and can be stimulated by fourfold with 125-fold molar excess of ape. in addition, ape activity can be moderately stimulated by myh with -fold molar excess of myh over ape. although the interactions of myh with sirt ape and 9-1- produce modest stimulation on myh catalytic activity in vitro, these interactions may be physiologically significant. we have demonstrated that a catalytically active spmyh1i261a/e262q mutant, which corresponds to the hmyhv315a/e316q mutant, cannot reduce the mutation frequency of myh1Δ cells. in addition, expression of a peptide consisting of the idc of spmyh that interferes with the interactions between spmyh and interacting proteins in s. pombe cells makes cells more sensitive to h2o. such a regulatory network of weak protein interactions may offer the ber pathway sufficient flexibility to coordinate with dna replication, dna damage response, and other dna repair pathways. interestingly, myh and ape prefer to bind to the upper band of sirt in co-immunoprecipitation analyses. although the nature of the upper band of sirt is unknown, we suspect it may be a modified form of sirt it has been shown that sirt can undergo auto mono-adp-ribosylation which may contribute to the self-regulation of sirt function. we also observed that mono-adp-ribosylation defective sirt6g60a mutant could not efficiently stimulate myh glycosylase and ape endonuclease activities as compared to wild-type sirt this suggests that auto mono-adp-ribosylation is important for sirt function in ber. thus, we suggested that the modified form of sirt participates in ber. we favor a model that sirt in the complex with myh, ape and 9-1- at sites of dna damage may undergo auto mono-adp-ribosylation leading to enhanced chromatin remodeling and optimal dna repair efficiency.figure  a model for sirt interaction with myh, ape and rad9-rad1-hus to repair dna damages on transcriptionally active chromatin and telomeres. the n- and c-terminal domains of myh are connected with the interdomain connector. sirt ape and rad9-rad1-hus bind overlapping but different sequence motifs on idc region of myh. the model suggests that sirt in the complex with myh, ape and 9-1- at sites of dna damage may undergo auto mono-adp-ribosylation leading to enhanced chromatin remodeling and optimal dna repair efficiency. hmyh and hsirt are efficiently recruited to kr-induced confined oxidative dna damage within transcriptionally active chromatin, but not the dna damage within inactive chromatin. this property of hmyh is unique because other dna glycosylases such as nth neil neil and mbd are recruited to both transcriptionally active chromatin and inactive chromatin. the preferred recruitment at sites of dna damage within active chromatin is also found with fen and pcna, consistant with the reports that myh interacts with pcna in the long-patch ber pathway. however, the determinants for long-patch ber pathway occurs at transcriptionally active chromatin are not clear. because myh needs to recognize both the mismatched adenine and go, transcriptionally inactive heterochromatin may be not accessible to myh even in the presence of sirt sirt has been shown to interact with parp and to regulate ber in a parp1-depdendent manner, however, parp is recruited to dna damage within transcriptionally inactive chromatin more efficiently than in active chromatin. although this differential response to kr-induced oxidative damage is not fully understood, this may imply that sirt stimulation of parp activity at ta-kr-bound sites may be subtle and that parp may play minimal role in myh-mediated long-patch ber. the parp1-depdendent ber analyzed by plasmid reactivation assay used by xu et al. may not involve myh. telomeric dna are highly susceptible to oxidative damage compared to other regions of the chromatin. oxidative damage to telomeric dna accelerates telomere shortening and requires efficient dna repair to maintain its integrity. in addition, chromatin at telomeres contains hypoacetylated histones and requires remodeling to give the repair machinery full access to sites of dna damage. it has been shown that human sirt ape and 9-1- are associated with telomeres and are essential for telomere stability. similar to s. pombe myh, for the first time, we show that mouse myh is associated with damaged telomeres. it has been shown that γ-h2ax foci increase at telomeres in sirt knockdown cells. this telomere dysfunction is consistent with increased myh foci at telomeres in sirt knockout cells without oxidative damage. the interaction of 9-1- with sirt and ber enzymes ensures that dna repair, chromatin remodeling, and ddr are coordinated. our findings indicate that sirt interacts with myh, ape and 9-1- to maintain genomic integrity of telomeres and transcriptionally active chromatin, but not transcriptionally inactive chromatin. our model suggests that sirt in the complex with myh, ape and 9-1- at sites of dna damage may undergo auto mono-adp-ribosylation leading to enhanced chromatin remodeling and optimal dna repair efficiency. the similar response of sirt and myh to oxidative dna damage within transcriptionally active chromatin suggests that sirt may alter the chromatin structure and facilitate dna repair. because myh needs to recognize both the mismatched adenine and the go located on the other strand, the dna glycosylase activity of myh is expected to be strongly prohibited on nucleosome-bound mismatches. thus, myh repair may require substantial nucleosome remodeling to expose the mismatch for efficient repair. it has been reported that sirt recruits the chromatin remodeler snf2h to double-strand breaks and focally deacetylates histone h3k. it will be interesting to see whether sirt recruits snf2h to oxidatively damaged telomeres to enhance ber.  the results presented in this study demonstrate that sirt has a direct role in ber by forming a complex with and stimulating myh and ape our finding that sirt interacts with the 9-1- complex suggests sirt may have a role in ddr and is consistent with the finding that the lack of sirt profoundly impacts upon downstream recruitment of dna repair factors. our data demonstrate that sirt ape and hus bind to the idc region of hmyh without competition. we demonstrate that hmyh and hsirt are efficiently recruited to confined oxidative dna damage in transcriptionally active chromatin in human cells and that mmyh foci induced by oxidative stress and msirt depletion are frequently localized on mouse telomeres. overall, our findings suggest that sirt forms a complex with myh, ape and 9-1- to maintain genomic and telomeric integrity. sirt interactions with myh, ape and 9-1- fit well with the observed marked genomic instability of sirt deficient cells and aging phenotypes of sirt knockout mice. taken together, our data highlight a potential role of chromatin remodeling in dna repair and ddr. 
ber is coordinated with the checkpoint machinery and requires chromatin remodeling for efficient repair. rather, one myh partner enhances the association of the other two partners to myh. rad9–rad1–hus checkpoint clamp promotes cell cycle checkpoint signaling and dna repair. dna repairsirtuin muty homologape1checkpoint clamprad9/rad1/hus1telomeresprotein–protein interactionissue-copyright-statement© the author 2015 in addition, myh foci induced by oxidative stress and sirt depletion are frequently localized on mouse telomeres. here we investigate the direct physical and functional interactions between sirt and ber enzymes. within human cells, myh and sirt are efficiently recruited to confined oxidative dna damage sites within transcriptionally active chromatin, but not within repressive chromatin. sirt is involved in dna double-strand break repair and has been implicated in ber. the interdomain connector of myh is important for interactions with sirt ape and 9–1– mutagenesis studies indicate that sirt ape and hus bind overlapping but different sequence motifs on myh. although sirt ape and 9-1- bind to the interdomain connector of myh, they do not compete for myh association. our findings indicate that sirt forms a complex with myh, ape and 9-1- to maintain genomic and telomeric integrity in mammalian cells. myh glycosylase and ape are two base excision repair enzymes involved in mutation avoidance from oxidative dna damage. these interactions are enhanced following oxidative stress. sirt a member of the nad+-dependent histone/protein deacetylase family, regulates genomic stability, metabolism, and lifespan.
 sirt a member of the nad+-dependent histone/protein deacetylase family, regulates genomic stability, metabolism, and lifespan. myh glycosylase and ape are two base excision repair enzymes involved in mutation avoidance from oxidative dna damage. rad9–rad1–hus checkpoint clamp promotes cell cycle checkpoint signaling and dna repair. ber is coordinated with the checkpoint machinery and requires chromatin remodeling for efficient repair. sirt is involved in dna double-strand break repair and has been implicated in ber. here we investigate the direct physical and functional interactions between sirt and ber enzymes.  we show that sirt interacts with and stimulates myh glycosylase and ape in addition, sirt interacts with the 9-1- checkpoint clamp. these interactions are enhanced following oxidative stress. the interdomain connector of myh is important for interactions with sirt ape and 9–1– mutagenesis studies indicate that sirt ape and hus bind overlapping but different sequence motifs on myh. however, there is no competition of ape hus or sirt binding to myh. rather, one myh partner enhances the association of the other two partners to myh. moreover, ape and hus act together to stabilize the myh/sirt complex. within human cells, myh and sirt are efficiently recruited to confined oxidative dna damage sites within transcriptionally active chromatin, but not within repressive chromatin. in addition, myh foci induced by oxidative stress and sirt depletion are frequently localized on mouse telomeres.  although sirt ape and 9-1- bind to the interdomain connector of myh, they do not compete for myh association. our findings indicate that sirt forms a complex with myh, ape and 9-1- to maintain genomic and telomeric integrity in mammalian cells. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords dna repairsirtuin muty homologape1checkpoint clamprad9/rad1/hus1telomeresprotein–protein interactionissue-copyright-statement© the author 2015 