human cells show a different ddr during mitosis than in other cell cycle phases . during most of the cell cycle , after cells reach the point of no return , however , dsbs on mitotic chromosomes do not trigger cell - cycle delay or arrest ; and the cells rather proceed through mitosis even if they contain unrepaired dsbs or fragmented chromosomes.(32 ) indeed , the sensitivity to ionizing radiation is higher in mitotic cells than in interphase cells.(33,34 ) moreover , dsb introduction into mitotic cells by etoposide treatment induces massive chromosome aberrations in the next cell cycle in a cancer cell line ( fig . thus , dsbs during mitosis are very toxic to cells because of the induction of severe genomic instability . effect of etoposide - induced double - strand breaks ( dsbs ) during mitosis on genomic instability of mitotic chromosomes . representative images of chromosome spreads from etoposide - treated ( + etoposide ) and non - treated ( etoposide ) in hct116 human colon cancer cells arrested in mitosis . during mitosis , dsbs activate pikks to induce phosphorylation of h2ax , and mdc1 as well as the mrn complex are recruited to the dsb sites , as seen during interphases ( fig . however , the recruitment of rnf8 , rnf168 , 53bp1 , and brca1 to dsb is largely suppressed during mitosis ( fig . 1b , d , right).(30,33 ) the mechanism of the suppression and its purpose were not well understood . two groups recently showed that localization of rnf8 and 53bp1 to dsbs is inhibited during mitosis.(35,36 ) the first work done by orthwein et al . ( 2014 ) showed that , in human cells , rnf8 and 53bp1 are phosphorylated during mitosis by cdk1 and that inhibition of the phosphorylation restores their localization to dsb sites ( fig . orthwein et al . also identified t198 on rnf8 as a cdk1 phosphorylation site and showed that rnf8-t198a , a phosphorylation - defective protein , can localize to mitotic chromatin after dsb introduction . rnf8 prepared from mitotic extracts can not bind to mdc1 in vitro , but rnf8 prepared from cdk1 activity - inhibited cells interacts with mdc1 in vitro . they also found that t1609 and s1618 on 53bp1 are phosphorylated during mitosis ( fig . 3a ) and that 53bp1-t1609a / s1618a double mutant protein can localize to mitotic chromatin and restores dsb repair in cells expressing rnf8-t198a . their study confirmed that t1609 is phosphorylated by cdk1 , whereas s1618 is a target of plk1 in vitro . these show that there are at least two distinct mechanisms to regulate the recruitment of these ddr effector proteins.(35 ) domain structure of 53bp1 and comparison of cdk1 and plk1 sites among xrcc4 orthologs . ( b ) domain structure of xrcc4 and conservation of phosphorylation sites among various species . s326 in the c - terminus of human xrcc4 ( hs ) is a potential cdk1 phosphorylation site . the gray and black boxes show xlf and dna ligase iv binding sites , respectively . phosphorylation of cdk1 or plk1 sites are shown in mouse ( ms ) , chicken ( gg ) , zebrafish ( de ) , and budding yeast ( sc ) . dna ligase iv and xlf binding sites in mouse , chicken , and zebrafish were predicted by aligning with the dna ligase iv and xlf binding sites of human xrcc4 using t - coffee software . ( 2014 ) indicated that t1609 and s1618 of 53bp1 are hyperphosphorylated in the absence of pp4c phosphatase in human cell lines.(36,37 ) they analyzed the function of pp4c phosphatase during ddr and found that these sites are phosphorylated specifically during mitosis.(36 ) these mitosis - specific phosphorylation sites are located in the ubiquitin - dependent recruitment motif of 53bp1 , a recognition site of ubiquitinated h2a , which is required for the localization of the protein to dsb sites.(1517 ) both of the reports(35,36 ) showed that dna damage during mitosis in cells expressing phosphorylation - defective 53bp1-t1609a / s1618a mutant , which restores its localization to dsb sites , leads to increased micronuclei formation . this suggests that restoration of dsb repair during mitosis causes defects in proper chromosome segregation and that inhibition of dsb repair during mitosis serves to maintain genomic stability . in addition to micronuclei formation , orthwein et al . showed that restoration of accumulation of rnf8 and 53bp1 to damaged mitotic chromosomes increases telomere fusions , which seems to be mediated by dsb repair such as c - nhej.(35 ) therefore , suppression of dsb repair by phosphorylation of rnf8 or 53bp1 by cdk1 or plk1 may protect the genome during mitosis . in particular , suppression of rnf8- and 53bp1-dependent dsb repair during mitosis may prevent telomere fusions . two groups showed that inhibition of the dna - pkcs suppresses micronuclei formation induced by mitotic dsbs in 53bp1-t1609a / s1618a - expressing or 53bp1-t1609a / s1618a rnf8-t198a - expressing cells , suggesting and that the core c - nhej complex plays a role in the segregation defects.(35,36 ) recently , we showed another dsb repair suppression mechanism by modulating the c - nhej core complex during mitosis.(38 ) dsb repair is largely suppressed during mitosis , but a substantial level of repair still occurs.(38 ) c - nhej causes formation of anaphase bridges , which are bridge - like dna structures that span daughter chromosomes and frequently induce genomic instability through inappropriate chromosome segregation.(39,40 ) in our study , severe chromosome aberrations such as dicentric and fragmented chromosomes are induced by mitosis - specific dsb introduction by transient treatment with etoposide ( fig . 2).(38 ) this observation raises a possibility that anaphase bridges are caused by dicentric chromosome formation by interchromosomal connections between telomeres or other chromosomal loci . these results suggest that there is a process to connect sister chromatids or individual chromosomes in ddr during mitosis . dna ligase iv - dependent c - nhej contributes to dicentric chromosome formation through telomere fusion in cells with dysfunctional telomeres,(41 ) which act as dsb ends.(42 ) therefore , we hypothesized that c - nhej is involved in the formation of anaphase bridges . both xrcc4 and xlf are regulatory subunits of the dna ligase iv complex and are essential for its activity.(4345 ) anaphase bridge formation is reduced after xrcc4 knockdown , suggesting that c - nhej contributes to the formation of some of these bridges during mitosis.(38 ) knockdown of the hr - specific factor xrcc3 has almost no effect on anaphase bridge formation , indicating that c - nhej is more critical than hr for the formation of anaphase bridges in mitotic cells . our studies strongly suggest that inappropriate use of the c - nhej pathway causes chromosome bridges , which lead to chromosome aberrations from mitotic dsbs . a phosphoproteomics study revealed that many sites are phosphorylated during mitosis , including those on xrcc4,(46 ) so we evaluated cell - cycle regulation of xrcc4 phosphorylation.(38 ) this mitosis - specific phosphorylation of xrcc4 contributes to the suppression of anaphase bridge formation after induction of dsbs during mitosis.(38 ) the mitosis - specific phosphorylation of xrcc4 depends on both cdk1 and plk1 activities.(38 ) s326 is a putative cdk phosphorylation site , and s326a substitution substantially reduces mitosis - specific phosphorylation of xrcc4 ( fig . 3b).(38 ) interestingly , the s326 phosphorylation residue overlaps with the polo box recognition motif.(47 ) both s256 and s326 are phosphorylated during mitosis.(46 ) because s256 resides in a putative plk1 phosphorylation site ( fig . 3b ) , cdk - dependent phosphorylation of s326 might prime the mitosis - specific phosphorylation of xrcc4 by plk1 . although the ortholog of xrcc4 has not yet been identified in nematode or fission yeast , most of the dna ligase iv subunits have been identified in various species ( table 1 ) . we previously analyzed cell - cycle regulation of lif1p , which is the s. cerevisiae ortholog of xrcc4.(4850 ) lif1p is phosphorylated by cdks from s phase through mitosis and this phosphorylation plays a role in suppressing c - nhej in s to m phases through the pathway dependent on sae2 , the s. cerevisiae ortholog of ctip.(49 ) these findings prompted us to assess the conservation of the phosphorylation sites among xrcc4 orthologs . although the locations of the phosphorylation sites are different , both s. cerevisiae lif1p and human xrcc4 have one putative target site for cdk1 and multiple sites for plk1 ( fig . both the cdk1 and plk1 sites near the c - terminus were conserved between human and mouse ( fig . 3b ) . in chicken , there are two cdk1 sites near the c - terminus and one plk1 site . however , in zebrafish , although the cdk1 site near the c - terminus was not conserved , there are four cdk1 sites in other locations . three of them are located downstream of the dna ligase iv binding site and overlap with plk1 sites ( fig . all of the xrcc4 orthologs have cdk1 or plk1 phosphorylation sites downstream region of the dna ligase iv interaction domain . thus , the function of mitosis - specific phosphorylation of xrcc4 is likely to be conserved among many species . we showed that xrcc4 phosphorylation - defective mutant ( xrcc4-s326a ) restores rapid repair of mitotic dsbs associated with more anaphase bridge formation.(38 ) this suggests that mitotic xrcc4 phosphorylation is involved in suppressing c - nhej to prevent chromosome instability in human cells . ctip promotes dsb end resection in hr and a - nhej.(51 ) because anaphase bridge formation is increased in ctip - depleted cells,(38 ) ctip may have a function to suppress anaphase bridges , possibly through the a - nhej pathway . brca1 , which is important for recruitment of ctip to dsb sites , does not localize to mitotic dsb sites.(33 ) however , only the function of ctip in hr , but not in a - nhej , is dependent on interaction with brca1.(52 ) moreover , in xenopus m - phase extract , ctip can bind mitotic chromatin.(53 ) thus , ctip may function as an a - nhej factor in suppression of genomic instability during mitosis . alternatively , ctip - dependent end resection may have functions to suppress anaphase bridge formation . conservation of dna ligase iv complex subunits among species although dna ligase iv ( catalytic subunit ) localizes to mitotic chromosomes , xrcc4 does not,(54 ) suggesting that the dna ligase iv complex function during mitosis is different from those in other cell cycles . because complex formation of the dna ligase iv with xrcc4 is believed to be essential for its activity , an activity of the dna ligase iv complex may be modified during mitosis . we confirmed the difference in the localization of dna ligase iv and xrcc4 during mitosis.(38 ) like wild - type protein , xrcc4 phosphorylation - defective mutant protein also fails to localize to mitotic chromosomes , showing that the phosphorylation of xrcc4 is not responsible for inhibiting the localization to chromosomes during mitosis . on the basis of these findings , we propose that there may be two mechanisms to suppress c - nhej : the reduced recruitment of c - nhej - specific factors to mitotic chromosomes , and mitosis - specific phosphorylation of xrcc4 by modulating the function of the dna ligase iv complex . taken together , these results suggest that xrcc4 , a key regulatory subunit of the dna ligase iv complex , is required not only for c - nhej in interphase but also for suppression of c - nhej during mitosis to prevent genomic instability in human cells . cells prevent carryover of dna lesions at the g2/m checkpoint because dna damage as well as the dna repair process are toxic during mitosis . as described above , there are several types of dsb repair suppression systems that occur during mitosis . however , mitotic cells do not undergo the second level of ddr : neither 53bp1 nor brca1 localizes to dsb sites . mitosis - specific phosphorylation of 53bp1 and rnf8 prevents their localization to dsb sites.(35,36 ) because 53bp1 and brca1 localization to dsb sites is important for nhej and hr , respectively , dsb repair pathways should be largely suppressed on mitotic chromosomes ( fig . we found that the third level of ddr is also suppressed or modified by mitosis - specific phosphorylation of xrcc4 , a component of the core c - nhej complex.(38 ) even if dsb repair pathways are largely suppressed by mitosis - specific phosphorylation of 53bp1 and rnf8 , considerable levels of dsb repair still occur and cause genomic instability during mitosis . phosphorylation of xrcc4 during mitosis slows dsb repair.(38 ) thus , xrcc4 phosphorylation has some functions to modulate dna ligase iv complex activity ( fig . in addition , xrcc4 fails to localize to mitotic chromatin.(54 ) the failure of xrcc4 to localize to mitotic chromosomes also may modify the composition of the dna ligase iv complex . ctip , but not rad51 , is recruited to mitotic chromatin.(53 ) this also suggests that there is the third level of suppression mechanisms in hr and that ctip has some functions to prevent genomic instability without the requirement of brca1 localization . ( a ) double - strand break ( dsb ) induces histone h2ax phosphorylation ( h2ax ) by atm . ( b ) cdk1 and plk1 phosphorylate rnf8 and 53bp1 to inhibit 53bp1 and brca1 localization of dsb sites . ( c ) cdk1 and plk1 phosphorylate xrcc4 , a regulatory subunit of the dna ligase iv complex , to suppress canonical non - homologous end joining ( c - nhej ) activity . ctip - dependent alternative non - homologous end joining ( a - nhej ) may prevent anaphase bridge formation . cells use multiple safeguards to prevent genomic instability by suppression or modification of dsb repair activities during mitosis , through the activation of mitotic kinases , cdk1 and plk1 . the molecular mechanisms of mitosis - specific phosphorylation and the change in localization of the dna ligase iv component xrcc4 are still unknown . analyzing how dna ligase iv activity in mitotic cells differs at the molecular level from during other cell - cycle phases is critical to understand the mechanisms of these controls . another c - nhej component , dna - pkcs also phosphorylated by plk1 , is dephosphorylated by pp6 and is required for accurate chromosome segregation.(55 ) it is also interesting to analyze how those c - nhej factors are involved in accurate mitosis.(56 ) active dsb repair during mitosis affects chromosome segregation , which often results in apoptosis , aneuploidy , or other chromosome aberrations . thus , studies of dsb repair control during mitosis are important to understand the origin of genomic instability , which causes cell tumorigenesis . in addition , activation of c - nhej during mitosis causes severe damage to growing cells like cancer cells , but the activation of nhej by itself would not be detrimental to interphase cells . this property would be useful for the development of anticancer drugs in the future .
double - strand breaks ( dsbs ) are one of the severest types of dna damage . 
 unrepaired dsbs easily induce cell death and chromosome aberrations . to maintain genomic stability , cells have checkpoint and dsb repair systems to respond to dna damage throughout most of the cell cycle . 
 the failure of this process often results in apoptosis or genomic instability , such as aneuploidy , deletion , or translocation . 
 therefore , dsb repair is essential for maintenance of genomic stability . during mitosis 
 , however , cells seem to suppress the dna damage response and proceed to the next g1 phase , even if there are unrepaired dsbs . the biological significance of this suppression is not known . in this review , we summarize recent studies of mitotic dsb repair and discuss the mechanisms of suppression of dsb repair during mitosis . 
 dsb repair , which maintains genomic integrity in other phases of the cell cycle , is rather toxic to cells during mitosis , often resulting in chromosome missegregation and aberration . 
 cells have multiple safeguards to prevent genomic instability during mitosis : inhibition of 53bp1 or brca1 localization to dsb sites , which is important to promote non - homologous end joining or homologous recombination , respectively , and also modulation of the non - homologous end joining core complex to inhibit dsb repair . 
 we discuss how dsbs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability .