genomic instability , a key hallmark of cancer , is driven in part by oncogene - induced dna replication stress ( drs ) .
specifically , in cancer cells , activated oncogenes induce dissociation of the replication machinery from the dna fork ( fork collapse ) , formation of dna double - strand breaks ( dsbs ) , and genomic instability ( gorgoulis et  al . , 2005 , bartkova et  al . , 2005 ,
, 2008 , negrini et  al . , 2010 , arlt et  al . , 2012 , hills and diffley , 2014 , macheret and halazonetis , 2015 ) .
prolonged exposure to chemical agents that interfere with dna replication can also lead to fork collapse ( branzei and foiani , 2010 , petermann et  al .
following fork collapse , dna replication can be completed by repair of the collapsed forks , by incoming replication forks , or by dormant origin firing ( branzei and foiani , 2010 , blow et  al . , 2011 , yeeles et  al . , 2013 , mayle et  al . , 2015 ) .
we previously described break - induced replication ( bir ) as a repair pathway for collapsed dna replication forks in cancer cells ( costantino et  al . , 2014 ) and ,
more recently , the scope of bir in human cells was expanded to include dna replication repair in prophase and alternative lengthening of telomeres ( minocherhomji et  al . , 2015 ,
bir has been studied extensively in budding yeast , as a homologous recombination ( hr)-based repair pathway for one - ended dna dsbs ( llorente et  al . , 2008 ,
notably , the d loop moves together with the replication fork , and dna replication is conservative ( donnianni and symington , 2013 , saini et  al .
these unique properties distinguish bir - initiated forks from origin - initiated forks and suggest the involvement of different proteins at these two types of forks .
indeed , pol32 , a nonessential subunit of budding yeast dna polymerase delta , is required for bir , but not for origin - initiated replication ( lydeard et  al . , 2007 ) .
mammalian pold3 , the ortholog of budding yeast pol32 , is also required for bir , as is pold4 , another subunit of mammalian dna polymerase delta that has no apparent ortholog in budding yeast ( costantino et  al . , 2014 ,
in addition to pol32 , bir in yeast requires rad52 ( llorente et  al . , 2008 ,
2008 ) . however , the role of rad52 in yeast is not specific to bir ; dna dsb repair by gene conversion ( a.k.a .
synthesis - dependent strand annealing ) and single - strand annealing also require rad52 , and yeast mutants lacking rad52 are very sensitive to dna damaging agents ( symington , 2002 , sugawara et  al . , 2003 ) .
rad52 is conserved at the amino acid level from yeast to human , but its function is apparently only partially conserved .
thus , human rad52 retains the strand - annealing activity ( kagawa et  al . , 2002 , singleton et  al . , 2002 ) ,
2015 ) . accordingly , whereas homozygous deletion of the brca2 gene in mice leads to embryonic lethality ( sharan et  al . , 1997 )
, rad52-knockout mice have a normal lifespan and no major phenotype , raising the question of what the physiological function of mammalian rad52 is ( rijkers et  al . , 1998 , yamaguchi - iwai et  al . , 1998 ) .
we previously performed an sirna screen to identify dna repair genes that are important for cell cycle progression when cyclin e is overexpressed ( costantino et  al . , 2014 ) .
here we performed a more focused screen centering on genes that function in hr , and we identified rad52 .
further characterization revealed that rad52 has a role in bir , making it a potential target for the development of cancer - specific therapies .
in an effort to identify genes that function in bir in human cells , we performed an sirna screen targeting about 70 genes that had previously been linked to dna dsb repair and hr ( table s1 ) .
the requirement of these genes in bir was examined using u2os cells that overexpress cyclin e in an inducible manner ( bartkova et  al . , 2005 ) .
in these well - characterized cells , cyclin e overexpression leads to drs , and the damaged replication forks are repaired to a significant degree by bir .
thus , when bir is inhibited  for example , by depleting pold3progression through the cell cycle is delayed ( costantino et  al . , 2014 ) .
to enable monitoring of cell cycle progression , the cells were pulsed consecutively with two thymidine analogs ( edu and brdu ; 1  hr pulse each with the two pulses separated by 6  hr ) and then examined by flow cytometry ( figure  1a and figure  s1a ) .
cells that remained in g1 during the 8  hr period would stain negatively for both edu and brdu , whereas cells that transitioned from g1 into s phase would stain negatively for edu and positively for brdu . as observed before ( costantino et  al . ,
2014 ) , the fraction of cells that remained in g1 over the 8  hr period decreased when cyclin e was overexpressed ( figure  1a , control sirna ; ne , normal cyclin e expression ; oe , cyclin e overexpression ) .
most of the sirnas tested did not affect the fraction of ne or oe cells that remained in g1 over the 8  hr period ( figure  1a and table s1 ) .
this included two sirnas that depleted the helicase pif1 , even though in budding yeast pif1 is required for bir ( wilson et  al . , 2013 ) .
a small number of sirnas preferentially enhanced the fraction of cells that remained in g1 when cyclin e was overexpressed .
these were the sirnas targeting slx4 , mus81 , smarcal1 , tipin , timeless , pold4 , and rad52 ( figures 1a and 1b ) .
slx4 , an adaptor protein that binds mus81 ; mus81 , a nuclease ; and smarcal1 , a helicase , remodel damaged replication forks ( btous et  al . , 2012 , pepe and west , 2014 , sarbajna et  al . , 2014 ) ; tipin and timeless are part of the replication fork protection complex ( chou and elledge , 2006 , errico and costanzo , 2012 ) , while pold4 functions in bir ( costantino et  al . , 2014 ) .
we decided to pursue the last hit ,  rad52 , which is dispensable for normal development in mice , but whose homolog in budding yeast is  important for all forms of hr , including bir . using crispr / cas9 ( jinek et  al . , 2012 ) , we generated rad52-knockout clones in the context of the u2os cells , in which cyclin e could be inducibly overexpressed .
clone 2 g contained two mutant alleles , whereas in clones 3c and 4a only one mutant allele was identified ( figure  s1b ) .
clone 3c retained a wild - type ( wt ) allele , whereas in clones 2 g and 4a no wt alleles could be identified .
consistent with the sequencing data , rad52 protein was undetectable in clones 2 g and 4a and barely detectable in clone 3c ( figure  1c ) .
importantly , all clones retained the capacity to regulate cyclin e levels in a tetracycline ( tet)-dependent manner ( figure  1c ) , and all of them displayed decreased progression from g1 into s phase when cyclin e was overexpressed ( figures 1d and s1c  s1f ) .
we next examined the intracellular localization of rad52 in cells that were either overexpressing cyclin e or exposed to chemical agents , such as hydroxyurea ( hu ) and camptothecin ( cpt ) , that induce drs . in about 20% of cells overexpressing cyclin e for 4  days , endogenous rad52 localized to drs foci , as marked by staining for rpa and atrip ( figures 2a , 2b , and s2a ) .
recruitment of rad52 to drs foci was also observed in about 50% of the cells exposed to hu for 24  hr but was mostly absent in cells exposed to hu for 2  hr ( figures 2c , 2d , and s2b ) .
these kinetics parallel the known effects of hu on dna replication forks ; short treatment of cells with hu leads to a reduction in the ribonucleotide pools and fork stalling , whereas forks collapse after prolonged exposure to hu ( petermann et  al . ,
exposure of cells to cpt , which induces covalent bonding of topoisomerase i to dna and , subsequently , fork collapse ( pommier , 2006 ) , also led to recruitment of rad52 to drs foci ; this was evident within 2  hr of exposure in some cells but was much more evident at 24  hr ( figures 2c and s2c ) . interestingly , in cells overexpressing cyclin e or exposed to either hu or cpt , rad51 foci were less prevalent than rad52 foci ( figures 2a and 2c ) ; however , the functional significance , if any , of this apparent difference remains to be investigated .
exposure of cells to hu for 24  hr was also associated with posttranslational modifications of the chromatin - bound fraction of rad52 , as detected by immunoblotting , whereas a high dose ( 9 gy ) of ionizing radiation did not elicit similar modifications ( figure  2e ) .
a subset of the rad52 posttranslational modifications induced in response to hu were atr dependent , since they were suppressed when the cells were treated with an atr inhibitor ( figure  2f ) .
furthermore , the modifications were sensitive to treatment of the chromatin extracts with lambda phosphatase ( figure  2f ) . taken together
if rad52 is important for bir , then its depletion should compromise the repair of collapsed forks and lead to a stronger dna damage response . to explore this possibility , we depleted rad52 by sirna and monitored h2ax phosphorylation both 2 and 24  hr after adding hu to the cells . at the 24  hr time point , the majority of the replication forks were collapsed , whereas at early time points , a large fraction of the forks are stalled and can resume replication upon hu withdrawal ( figure  s3a ) .
the effects of pold3 , mus81 , and rad51 depletion on h2ax phosphorylation were also examined ( figure  s3b ) .
two hours after exposure of the cells to hu , h2ax levels increased modestly and equally in the control , rad52- , pold3- , and mus81-depleted cells ( figures 3a and 3b ) .
after 24  hr exposure to hu , h2ax phosphorylation levels increased further , but the increase was much stronger in the cells depleted for rad52 or pold3 ( figures 3a and 3b ) .
codepleting rad52 and pold3 had the same effect as depleting only rad52 ( figure  3c ) .
these results are consistent with rad52 and pold3 functioning epistatically in repair of collapsed , but not stalled , replication forks .
interestingly , depletion of rad51 suppressed h2ax phosphorylation when compared to control sirna - treated cells at both the 2 and 24  hr time points , an observation that merits further study ( figures 3a and 3b ) . to examine whether rad52 facilitates restart of replication after fork collapse , we performed dna fiber analysis of cells exposed to hu for 6 or 24  hr .
however , we were concerned that new origin firing near a collapsed fork might be misinterpreted as fork restart if replication from the new origin proceeded all the way to the collapsed fork .
we therefore performed the assay in the presence of a cdc7 inhibitor that does not inhibit transcription ( menichincheri et  al . , 2010 , montagnoli et  al .
, 2010b ) after demonstrating that this inhibitor effectively suppressed new origin firing in cells released from a 24  hr hu replication block ( figures s3a and s3c ) .
to monitor fork restart , the cells were incubated with cldu for 1  hr , then incubated with hu for 6 or 24  hr in the presence of the cdc7 inhibitor ; finally , after release from the hu block , the cells were incubated with idu for 1  hr again in the presence of the cdc7 inhibitor to prevent new origin firing .
the 1  hr idu incubation period was found to be sufficient to observe restart of replication . in the parental u2os cells ,
replication restart was observed after exposure to hu for both 6 and 24  hr , although this was the case to a greater extent for the shorter incubation period ( figure  3d ) . in the rad52-knockout clone , replication restart
was significantly reduced , especially when the cells were exposed to hu for 24  hr ( figures 3d and s3d ) . since the cdc7 inhibitor prevented new origin firing
to further monitor the function of rad52 in bir , we employed a green fluorescent protein ( gfp)-based reporter assay in which bir - mediated repair of dna dsbs induced by the endonuclease i - scei leads to gfp fluorescence ( costantino et  al . , 2014 ) . since the initial description of this assay , we have generated a new ,  stably transfected cell clone expressing the gfp reporter plasmid that provides a better signal - to - noise ratio than the original clone .
depletion of rad52 by sirna in the new clone led to a significant suppression of gfp fluorescence , consistent with rad52 functioning in bir ( figure  3e ) .
depletion of pold3 and  pold4 also suppressed bir in this system , although not as efficiently as rad52 .
interestingly , codepletion of rad52 and pold3 or rad52 and pold4 suppressed bir , as efficiently as depletion of rad52 alone , further suggesting that pold3 , pold4 and rad52 function epistatically ( figure  3e ) .
since bir repairs collapsed forks in cells with oncogene - induced drs , its deletion should curtail cancer development and/or progression .
first , we examined tumor formation in apc mice rendered heterozygous for apc by constitutively expressing the cre recombinase under the control of a cmv promoter and enhancer .
in these mice , the entire coding sequence of the mutant apc allele is deleted by the cre recombinase ( cheung et  al . ,
2010 ) . in regard to rad52 , the mice either had two wt rad52 alleles ( rad52 ) or were homozygous for deletion of the rad52 gene ( rad52 ) . at 8  months of age , the mice , while still having no overt signs of disease , were sacrificed , and the presence of tumors in the entire small intestine was scored by histology .
deletion of rad52 did not affect the number of observed tumors ( a total of 46 versus 50 tumors in six rad52 and six rad52 mice , respectively ) , but resulted in smaller tumor sizes ( figure  4a ) .
deletion of rad52 also resulted in an increase in the fraction of tumor cells scoring positive for phosphorylated h2ax , consistent with an inability to repair collapsed replication forks ; however , this difference was only evident in the early stages of tumor development , when tumors were less than 1.5  mm in diameter ( figures 4b and s4 ) .
the effect of rad52 on cancer progression was further examined in apc heterozygous mice . in a wt rad52 background ,
the lifespan of these mice is significantly shorter than that of mice bearing the apc allele described above ( moser et  al . ,
indeed , lifespan of the apc heterozygous mice was significantly extended from a mean of 127  days for the rad52 mice to 178  days for the rad52 mice ( figure  4c ) .
the function of rad52 in dna repair is well established in yeast , but less well defined in higher eukaryotes and mammals ( symington , 2002 ) .
in fact , mice with homozygous deletion of the rad52 gene are viable and have no obvious phenotype ( rijkers et  al . , 1998 ) .
this could indicate that rad52 serves a backup function that is relevant only when the primary dna dsb repair pathways are inactivated or overwhelmed by excessive dna damage .
supporting this model , depletion of rad52 is synthetic lethal with brca2 deficiency in human cell lines ( feng et  al . , 2011 ,
lok and powell , 2012 ) . also , cells rely on rad52 for repair when extensive dna damage overwhelms the repair capacities of brca1 and 53bp1 ( ochs et  al . , 2016 ) . however , the findings reported here suggest that rad52 has more than a backup role in dna repair .
specifically , we propose that rad52 has a key role in bir repair of collapsed dna replication forks ( figure  4d ) .
endogenous rad52 localized to sites of drs in cells with collapsed dna  replication forks , depletion of rad52 led to increased levels of dna damage in cells exposed to hu for 24  hr , dna replication restart from collapsed forks was dependent on rad52 , and rad52 depletion suppressed repair of dna dsbs by bir in a gfp - based reporter assay . together with the known biochemical function of mammalian rad52 in strand annealing ( kagawa et  al .
2002 ) , these results argue that strand invasion by rad52 leads to dna structures that are conducive to initiation of dna replication after fork collapse . in yeast , rad52 is important for rad51-dependent and rad51-independent forms of hr . whereas repair of dna dsbs by gene conversion in yeast is rad51 dependent
, bir can be rad51 independent ( ira and haber , 2002 , payen et  al . , 2008 ) .
in this case , the strand annealing activity of rad52 is important , and indeed , it is easy to envision the presence of single - stranded template dna to which the invading strand can anneal at collapsed dna replication forks . by analogy , mammalian rad52 , with or without rad51 , may be involved in the strand - invasion step of bir , as is the case for its ortholog in yeast ( llorente et  al . , 2008 ,
a role for rad52 in bir can explain why its depletion in cells with drs leads to increased dna damage ( wray et  al .
, 2016 ; figures 3a and 3b ) , why the rad52 gene is amplified in human cancers , and why its inactivation curtails cancer development ( treuner et  al . , 2004 , cramer - morales et  al . , 2013 , lieberman et  al . , 2016 ; figure  4 ) .
bir also mediates dna repair synthesis in mitosis ( minocherhomji et  al . , 2015 ) , and it is noteworthy that rad52 is essential also in this context ( bhowmick et  al . , 2016 ) .
for the sirna screen , u2os cells engineered to overexpress cyclin e in an inducible ( tet - off system ) manner ( u2os - cyce cells ) were plated in the presence or absence of tet , and the next day they were transfected with sirna . then
, 3  days later , the cells were pulsed for 1  hr with edu ; 6  hr after that , they were pulsed for 1  hr with brdu and then processed for flow cytometry as described ( costantino et  al . , 2014 ) .
to generate rad52-knockout u2os - cyce cells , two crispr / cas9 constructs targeting exons 3 and 9 of the human rad52 gene , respectively , were used .
after transfection , single clones were expanded and characterized by dna sequencing of the targeted alleles and by immunoblotting for rad52 protein .
either u2os - cyce cells grown in the presence or absence of tet for 4  days or u2os parental cells treated with 2  mm hu or 2  m cpt were processed for immunofluorescence , as described ( costantino et  al . , 2014 ) .
to monitor h2ax levels by flow cytometry , u2os cells transfected with the indicated sirnas were treated with 2  mm hu for 0 , 2 , or 24  hr ; fixed in 70% ice - cold ethanol ; and stained using the flowcellect histone h2ax phosphorylation assay kit ( millipore ) .
u2os cells were pulse labeled with cldu for 1  hr , then treated with 2  mm hu and cdc7 inhibitor for 6 or 24  hr , and finally pulse labeled with idu for 1  hr in the presence of the cdc7 inhibitor .
dna fibers were spread on aps - coated coverslips and visualized using primary antibodies recognizing cldu or idu .
see the supplemental information for a full list of antibodies as well as a full list and detailed description of the methods used in this study .
proposed experiments , discussed the results , and contributed to the writing of the manuscript .
 summaryhuman cancers are characterized by the presence of  oncogene - induced dna replication stress ( drs ) , making them dependent on repair pathways such as break - induced replication ( bir ) for damaged dna replication forks . to better understand bir 
 , we performed a targeted sirna screen for genes whose depletion inhibited g1 to s phase progression when oncogenic cyclin e was overexpressed . 
 rad52 , a gene dispensable for normal development in mice , was among the top hits . in cells in which fork collapse was induced by oncogenes or chemicals , the rad52 protein localized to drs foci . 
 depletion of rad52 by sirna or knockout of the gene by crispr / cas9 compromised restart of collapsed forks and led to dna damage in cells experiencing drs . 
 furthermore , in cancer - prone , heterozygous apc mutant mice , homozygous deletion of the rad52 gene suppressed tumor growth and prolonged lifespan . 
 we therefore propose that mammalian rad52 facilitates repair of collapsed dna replication forks in cancer cells . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions