replication stress is a well - characterized tumor characteristic and has recently been considered as a potential new hallmark of cancer ( macheret and halazonetis , 2015 ) .
replication stress leads to the activation of the dna damage response ( ddr ) , which is associated with the early stages of cancer development ( bartkova et  al . , 2005 , gorgoulis et  al . ,
hypoxia ( low oxygen levels ) is one of the most physiologically relevant driving forces of replication stress and is characterized by an increased number of stalled replication forks and significantly reduced replication rates .
importantly , hypoxia - induced replication stress occurs in the absence of dna damage ( olcina et  al . ,
hypoxia - induced replication stress is one of the factors proposed to contribute to the selection pressure to lose key components of the ddr , including p53 ( gorgoulis et  al . , 2005 , hammond et  al . , 2007 , graeber et  al . , 1996 )
the degree of tumor hypoxia is an indicator of poor patient prognosis due to resistance to most current therapies and increased metastatic spread ( begg et  al . , 2011 ,
the importance of ribonucleotide reductase ( rnr ) lies in its unique ability to catalyze the rate - limiting step of the reduction of ribonucleotides ( ndps ) to the corresponding deoxyribonucleotides ( dndps ) , the precursors of dna ( kolberg et  al .
the large rrm1 dimer contains the catalytic site , whereas the smaller dimer ( rrm2 or rrm2b ) contains an oxygen - requiring di - iron tyrosyl - radical site , which is essential for catalysis ( kolberg et  al . , 2004 ,
rnr is highly regulated , as nucleotide imbalances can be detrimental to cell fate ( aye et  al . , 2015 ) .
elevated deoxyribonucleotide triphosphate ( dntp ) pools have been correlated with increased mutagenesis , while insufficient dntps cause replication stress and promote genomic instability ( bester et  al .
oxygen is essential for mammalian rnrs to oxidize the di - iron center found in the smaller subunit ( rrm2 or rrm2b ) ( huang et  al . , 2014 ) .
this generates the tyrosyl radical , which via a long - range pathway , transfers an electron to the catalytic site of rrm1 , enabling the reduction of ndps to dndps ( lundin et  al . , 2015 , kolberg et  al .
the mechanism by which iron is incorporated into the small subunit , and how oxygen activates the di - iron center for radical initiation , is still poorly understood and under intense investigation ( huang et  al .
the oxygen dependency of the small rnr subunit has led to the logical assumption that rnr is inactive in severely hypoxic conditions , something that has been verified for the rrm1/rrm2 ( r1/r2 ) version of the rnr enzyme ( brischwein et  al .
, 1997 , chimploy et  al . , 2000 , thelander et  al . , 1983 ,
reichard , 1993 , probst et  al . , 1989 , nordlund and reichard , 2006 ) .
in contrast , we demonstrate that rnr is  able to maintain dndp formation by switching subunits in hypoxic conditions ( < 0.1% o2 ) .
this switch is critical to preserve sufficient nucleotide levels for ongoing replication and avoid dna damage accumulation and apoptosis in hypoxia .
interestingly , we found that the rrm1/rrm2b ( r1/r2b ) version of the rnr enzyme retains activity in hypoxia due to adaptation of rrm2b to these conditions through two distinct mechanisms .
specifically , we determined that most of the tyrosyl radical in rrm2b remained stable when exposed to < 0.1% o2 , a property that was diminished after disruption of the helix b open conformation ( k37/k151 ) and the subsequent loss of the crosstalk between the two monomers .
furthermore , computational simulations suggest that y164 plays a role in the fine - tuning of the oxygen - entering frequency of rrm2b , meaning it is likely to be a better oxygen - sequestering agent than rrm2 . collectively , our results reveal a critical role for the rnr ( r1/r2b ) enzyme in hypoxic conditions in maintaining sufficient dntps for ongoing replication and therefore preventing dna damage , genomic instability , and subsequent loss of viability .
cells in hypoxic conditions ( < 0.1% o2 ) do not accumulate dna damage , although they are characterized by the presence of replication stress , which leads to the activation of the ddr ( figures s1a and s1b ) ( olcina et  al . , 2013 ) .
as predicted , hypoxia - induced replication stress leads to the accumulation of single - stranded dna ( ssdna ) , which can be detected through the visualization of replication protein a ( rpa ) foci in s - phase cells ( pires et  al . , 2010 ) .
however , the percentage of cells in hypoxia with rpa foci decreases after prolonged exposure to these conditions ( > 6  hr ) ( figure  1a ) .
therefore , our hypothesis is that although uncoupling of the helicases and polymerases occurs in hypoxia , replication can proceed . using dna fiber analysis
, we confirmed that dna replication is significantly slower in hypoxia ( 25% of normal rate ) but is not completely abrogated ( figure  1b ) .
dntp incorporation assays were carried out in rko cells exposed to hypoxia and demonstrated that , although the dntp pools were disrupted ( with the purines [ dgtp , datp ] being more affected than the pyrimidines [ dctp , dttp ] ) , there was nucleotide availability in hypoxic conditions ( figures 1c and 1d ) .
it has been suggested that pyrimidine levels persist due to compensatory activities of the pyrimidine salvage pathways or as a result of the deoxyuridine present in culture medium , which cells could uptake and phosphorylate , therefore contributing to pyrimidine levels , particularly dttp ( eriksson et  al .
interestingly , the pyrimidine salvage pathway has been reported to be more efficient than the purine pathway , suggesting an explanation for why purine levels are more sensitive to hydroxyurea ( hu ) and potentially hypoxia ( reichard , 1988 ) .
our data demonstrate sufficient nucleotide availability in hypoxia to  allow replication to proceed , albeit at a compromised rate ; thus raising the possibility that rnr is able to retain some activity in hypoxia .
expression analysis of the rnr complex showed that the rrm2b protein is significantly induced ( 2- to 3.5-fold , depending  on cell line ) in response to hypoxia in cell lines , including colorectal , osteosarcoma , glioblastoma , and esophageal cancer cells , whereas the rrm2 small subunit decreased over time and the rrm1 remained unchanged or showed a slight decrease ( figures 1e , 1f , and s1c  s1f ) .
as rko cells showed the highest level of rrm2b expression , we chose it as a model line for the majority of our experiments ( figure  s1 g ) .
rrm2b increased 4.6-fold after 24  hr in hypoxic conditions , whereas rrm2 and rrm1 mrna levels decreased 12.3- and 2.5-fold , respectively ( figures 1 g and s1h ) .
importantly , in  silico tgca ( the cancer genome atlas ) analysis of colorectal adenocarcinoma patient cohorts demonstrated that rrm2b expression correlates significantly with the  expression of a verified hypoxia signature ( figure  1h ) , suggesting that the oxygen - dependent overexpression of rrm2b also occurs in  vivo ( li et  al . , 2014 ) .
in contrast , rrm1 and rrm2 expression did not correlate with the same hypoxic signature ( figures s1i and s1j ) .
interestingly , overexpression and genetic alterations in rrm2b correlated with worse overall and disease - free survival in colorectal cancer patients ( figures s1k  s1n ) .
the transcription factor hif-1 ( hypoxia - inducible factor 1 ) is known to mediate significant gene expression changes in response to hypoxia and has roles in dna replication , dna repair , and respiration ( hubbi et  al .
, 2013 , fukuda et  al . , 2007 , crosby et  al . , 2009 ) .
therefore , we investigated if the induction of rrm2b in hypoxia was dependent on hif-1 by utilizing rko and rko cells exposed to hypoxia ( figures 2a , s2a , and s2b ) .
interestingly , both the mrna and the protein levels of rrm2b were induced in hypoxia irrespective of hif-1 status , in contrast to the well - documented hif-1 target glut1 .
next , using rko cells exposed to either 2% or  < 0.1% o2 , we investigated the oxygen dependency of the induction of rrm2b protein .
rrm2b was induced in response to the lower level of hypoxia ( < 0.1% o2 ) , where a robust p53 induction was also observed but did not increase in response to 2% o2 despite hif-1 stabilization ( figure  2b ) .
this finding is in agreement with our previous studies demonstrating that the lower level of hypoxia ( < 0.1% o2 ) induces replication stress and that this is the signal that initiates the ddr ( including p53 stabilization ) ( hammond et  al . , 2002 , olcina et  al . , 2013 ) .
rrm2b was first characterized as a p53-regulated rnr subunit ( p53r2 ) ( tanaka et  al . , 2000 ) . here ,
further analysis of the molecular pathways mediating rrm2b induction in hypoxia  demonstrated that the hypoxic overexpression of rrm2b occurs in a p53-dependent manner ( figures 2c , 2d , and s2c  s2h ) . to rule out the possibility of an indirect mechanism of induction of rrm2b by p53 ,
chromatin immunoprecipitation ( chip ) assays were carried out and demonstrated that p53 binds directly to the p53-response element at the rrm2b locus leading to transcriptional overexpression ( figures 2e , 2f , and s2i  s2k ) .
interestingly , although p53 expression was increased in response to the dna damaging agent adriamycin , this did not correlate with increased p53 binding to the p53-response element in rrm2b ( figures 2e and 2f ) .
most importantly , analysis of the tcga colorectal adenocarcinoma patient cohorts showed that rrm2b expression significantly correlated with a recently identified group of hypoxia - inducible p53-dependent genes ( leszczynska et  al . , 2015 ) , suggesting that hypoxia- and p53-dependent expression of rrm2b occurs in human cancers ( figure  2 g ) .
interestingly , in p53 null cell lines ( h1299 , hct116 ) a mild ( 1.3- to 1.7-fold ) increase in rrm2b protein levels was also observed in hypoxia ( figures 2c and 2h ) .
these findings suggest that additional post - translational p53-independent mechanisms exist for rrm2b stabilization and therefore the importance of rrm2b in hypoxic conditions . in order to investigate the biological significance of hypoxia - induced rrm2b
, we first verified that it forms a complex with the rrm1 subunit to reconstitute the r1/r2b holoenzyme in  < 0.1% o2 .
immunoprecipitation assays demonstrated that increased levels ( 5.3-fold ) of rrm2b protein were bound to the rrm1 subunit in hypoxia whereas the levels of rrm2 bound to rrm1 decreased by 1.8-fold ( figures 3a , s3a , and s3b ) .
next , we asked if the hypoxia - formed r1/r2b enzyme was functional . small interfering rna ( sirna)-mediated loss of rrm2b led to significantly lower intracellular dntp levels in hypoxia ( 50%55% less pyrimidines and 25%30% less purines  compared to the control [ sictl ] ) ( figures 3b and s3c ) .
in contrast , the loss of rrm2b did not significantly affect the dntp pools in normoxic conditions ( figure  s3d ) .
in addition , fluorescence - activated cell sorting ( facs ) analysis demonstrated that s - phase u2os cells lacking rrm2b incorporate 37.5% less brdu than the control - treated cells in hypoxia ( figures 3c and s3e ) .
these findings demonstrate that depletion of rrm2b in hypoxia leads to further disruption of the dntp pools and indicate that ongoing replication is disrupted .
to further investigate the hypoxic role of rrm2b , we used crispr / cas9 technology to construct a rrm2b knockout cell line ( rko ) ( figures s3f  s3i ) .
rrm2b - depleted rko cells ( both knockout and sirna treated ) showed a persistent formation of rpa foci during long ( 24  hr ) exposures to hypoxia , suggesting an accumulation of ssdna and failure to complete dna replication ( figures 3d and s4a  s4c ) . furthermore , cells with reduced levels of rrm2b formed 53bp1 foci indicating the presence of double - strand breaks ( dsbs ) specifically in hypoxia ( figures 3e , 3f , and s4d  s4f ) .
no evidence of elevated dna damage was observed in control cells or in cells lacking rrm2b in normoxia ; this was attributed to the rrm2-dependent supply of dntps when oxygen is abundant .
the increased dsbs observed in hypoxic cells with depleted rrm2b correlated with ( 1 ) a significant loss of viability measured by colony survival assay ( 77% fewer cells survived after 24  hr of hypoxia when rrm2b was silenced in comparison to sictl ) ( figure  3 g ) and ( 2 ) the induction of apoptosis as determined both morphologically and by induction of parp cleavage ( figures 3h and s4 g ) .
notably , xenograft tumors of rko and rko cells showed delayed tumor growth and increased radiosensitivity when rrm2b was lacking ( figures 3i and s4h ) .
additionally , a significant increase in apoptosis specifically in the hypoxic regions of the rrm2b tumors was observed ( figures 3j3l ) as determined by the presence of cleaved caspase-3 ( apoptosis ) in the pimonidazole ( pimo)-positive ( hypoxic ) regions .
increased apoptosis in the hypoxic fraction of the tumors offers a likely explanation for the increased radiosensitivity observed ( figure  3i ) .
the data presented so far suggest that the basal levels of dntps provided by r1/r2b ( as opposed to r1/r2 ) are sufficient for ongoing replication in hypoxic cells , preventing the collapse of replication forks that would ultimately lead to dsbs and loss of viability .
these data indicate that rrm2b contributes to the increased resistance of the most aggressive fraction of tumors ; therefore , we investigated the hypoxic adaptation of rrm2b by determining the enzymatic properties of both forms of rnr enzyme ( r1/r2 and r1/r2b ) in normoxia and hypoxia .
rrm1 , rrm2 , and rrm2b recombinant proteins were overexpressed and purified from e.  coli ( figures s5a and s5b ) , without additional iron or reductants , to prevent the variability associated with in  vitro assembly of the active cluster of the tyrosyl radical ( cotruvo and stubbe , 2011 ) .
we quantified the iron bound to rrm2 and rrm2b using inductively coupled plasma ( icp ) mass spectrometry and found comparable iron incorporation between the two recombinant proteins ( figure  s5c ) .
the level of iron incorporation was 10% of the level that has been reported in a fully loaded rrm2 , which would in turn predict lower enzymatic activity ( aye et  al .
the abilities of the two forms of the rnr enzyme ( r1/r2b and  r1/r2 ) to reduce cytidine diphosphate ( cdp ) ( substrate ) to deoxycytidine diphosphate ( dcdp ) ( product ) were assayed in normoxic and hypoxic conditions ( < 0.1% o2 ) .
0.1% o2 and normoxia with no statistically significant difference in the total amount of dcdp formed under either condition after 30  min ( figure  4a ) .
in fact , the r1/r2b enzyme continued converting cdp to dcdp for over 2  hr in hypoxia ( figures 4b and s5d ) .
in contrast , the r1/r2 enzyme did not sustain activity in hypoxia ( figure  4c ) and stopped producing dcdps after 15  min in < 0.1% o2 ( figures 4d and s5d ) .
importantly , we verified that the r1/r2 enzyme did not stop dcdp formation in hypoxia due to lack of available cdp ( figure  s5e ) . collectively , these data suggest that the compromised r1/r2 activity observed at < 0.1% o2 was a result of limited oxygen availability . it is important here to state that the oxygen does not affect the stability of the tyrosyl radical , and it is only required for tyrosyl radical formation ( stubbe , 2003 )
once assembled , the tyrosyl radical can catalyze multiple turnovers until regeneration is necessary , with mammalian r1/r2 being stable for 1525  min in  vitro ( cotruvo and stubbe , 2011 ) .
the key difference observed between r1/r2b and r1/r2 in hypoxia offers a mechanistic explanation for why the r1/r2b form of rnr is preferred in hypoxic conditions .
it should be noted that , as previously described , the overall activity of the rrm2b - containing rnr enzyme is significantly lower than that of the rrm2-containing rnr ( shao et  al . , 2004 )
( figures 4b and 4d ) . therefore , although our data suggest that r1/r2b is the preferred rnr enzyme in hypoxia , the levels of available nucleotides are still reduced compared to normoxia , thus explaining the continued replication stress in these conditions ( figure  1a ) . given the differences in normoxic and hypoxic activity between rrm2b and rrm2 , we compared the potential oxygen accessibility for the proteins using a modeling approach .
using the only available published structures of rrm2b ( pdb : 3hf1 ) and rrm2 ( pdb : 3vpn ) ( smith et  al . , 2009 ) , we investigated the theoretical differences and/or similarities in the dynamics of oxygen diffusion between the two proteins using classical molecular dynamics ( md ) simulations .
the intention of this study was to understand how molecular oxygen accesses the core of the protein and to probe whether any differences could be related to the activities of rrm2/rrm2b in normoxia versus hypoxia .
our analysis revealed three principal oxygen cavity tunnels ( t1t3 ) in both proteins , which could be employed as access points ( figures 4e , 4f , and s5f ) .
analysis of t1t3 suggested that rrm2b could act as a better oxygen - sequestering agent than rrm2 .
specifically , we found that the predicted oxygen - entering frequencies are greater for rrm2b ( 68% at monomer 1 and 22% at monomer 2 ) than for rrm2 ( 14% at monomer 1 and 0% at monomer 2 ) ( table 1 ) , which indicate differential oxygen - turnover susceptibilities between rrm2b and rrm2 .
t2 , and t3 ( figure  s5 g ) suggest that the origin of the selectivity between rrm2b and rrm2 for oxygen turnover resided primarily in tunnels t2 and t3 , meaning that the energy barriers for oxygen to cross t2 and t3 are higher for rrm2 than for rrm2b .
we then performed electron paramagnetic resonance ( epr ) spectroscopy to monitor the stability of the tyrosyl radical of the two proteins in hypoxic conditions ( figures 4 g and 4h ) .
strikingly , we observed that 66%   3.35% of the tyrosyl radical in rrm2b remained stable for 1  hr at 37c in < 0.1% o2 , while in the same conditions , only 43%   4% remained stable in rrm2 ( figure  4i ) .
collectively , our results provide compelling evidence that rrm2b is able to retain activity in hypoxia while the activity of rrm2 is compromised and therefore highlight a specific role for rrm2b in the hypoxic stress response . despite the fact that rrm2 and rrm2b share 83% sequence homology , there are distinct functional and structural differences ( shao et  al . , 2004 , smith et  al . , 2009 , zhou et  al . , 2010 , xue et  al . , 2006 , wang et  al . , 2009b ) .
the switch of c202 in rrm2 to y164 in rrm2b results in an open phenylalanine channel specifically in rrm2b ( smith et  al .
, 2009 ) , which is connected with oxygen t3 ( figures s5f and s5h ) .
it has been proposed that f183 in rrm2b versus y221 in rrm2 contributes to the differences in the enzymatic activity between the two proteins ( zhou et  al . , 2010 ) .
in addition , rrm2b has unique antioxidant capabilities , which are partially due to the y241 and y331 residues ( h279 and y369 in rrm2 ) ( xue et  al . , 2006 ) .
finally , rrm2b through k37 and k151 ( e76 and e190 in rrm2 ) exhibits greater crosstalk between its secondary structures , which stabilizes helix b in an open conformation ( smith et  al . , 2009 ) . based on these key differences between rrm2 and rrm2b ( figure  s6a ) and in order to determine the regions of rrm2b responsible for hypoxic adaptation , we constructed , overexpressed , and purified seven recombinant rrm2b mutants : y164c , y164f , f183y , k37e / k151e , y241h , y331f , and q127k ( figure  s6b ) .
the q127k mutant was generated as a negative control , as it diminishes enzymatic activity due to disruption of the stability of the radical cluster ( zhou et  al . , 2010 ) .
the seven purified mutants showed the same secondary structure , determined by circular dichroism , as the wild - type rrm2b recombinant protein ( figure  s6c ) .
the levels of iron incorporation were also similar for the mutant with the exception of q127k , as expected ( figure  s6d ) .
sustained reduction of cdp to dcdp was observed for 2  hr for most mutants , except y164c and k37e / k151e , which both appeared to stop reducing substrate after 30  min in hypoxia , reminiscent of r1/r2 ( figures s6e  s6k ) . for these two mutants , we then compared dcdp formation in normoxia and hypoxia and found that the ability to reduce cdp to dcdp in hypoxia was compromised in both the k37e / k151e and y164c mutants ( figures 5a and 5b ) .
these results indicate that these specific residues are critical to the role of rrm2b in hypoxia and that without them , rrm2b activity is not sustained , similar to rrm2 . to further probe the role of k37/k151 and y164
, we observed that while most of the rrm2b mutants behaved as the wt - rrm2b , which retains 60%79% of its tyrosyl radical in hypoxia ( figure  s7a ) , the k37e / k151e mutant retained only 28% of its tyrosyl radical in these conditions ( figures 5c and 5d ) . since k37/k151 residues affect helix b of rrm2b monomer 2 , the bending angle of the helices was estimated and showed that helix b in rrm2b is in an open conformation ( 2025 bending angle ) , whereas in rrm2 , it is closed ( 810 ) ( figure  s7b ) .
interestingly , md simulations for the k37e / k151e variant showed a closure of helix b after 300  ns from 20 to 8 bending angle , therefore resembling the closed conformation of rrm2 ( figures s7b and s7c ) .
although these are theoretical studies that provide only indications of the protein conformation , the distortion of helix b in monomer 2 of the k37e / k151e
mutant could suggest loss of the essential crosstalk between the two monomers , which may explain the loss of tyrosyl radical stability in hypoxia as demonstrated by our epr analysis .
additionally , md simulations showed a 4.8-fold lower oxygen  entering frequency for the y164c mutant in comparison to wt - rrm2b , while we did not observe any changes in the oxygen entering frequency for the k37e / k151e mutant ( table 2 ) .
this could be explained by disruption of the y164-f197 and y164-f198 contacts in monomer 1 of rrm2b , which could cause side - chain rearrangement of f95 in the y164c mutant .
this rearrangement may result in f95 moving closer to f197 , leading to closure of the rrm2b specific phenylalanine channel ( figures 5e and s5h ) , therefore explaining the lower oxygen - entering frequency in y164c mutant . finally , in order to demonstrate that our findings are relevant in  cells experiencing hypoxia and that the hypoxic adaptation  of  rrm2b depends on the identified residues ( y164 and  k37/k151 ) , we performed rescue experiments by transfecting our rko cells with the following constructs : prrm2b , prrm2b , prrm2b , and pcdna3.1 ( ctl ) .
as expected , when wt - rrm2b was reintroduced , a significant increase in the intracellular nucleotide levels was observed in hypoxia , whereas reintroduction of y164c and k37e / k151e showed significantly lower dntp levels compared to wt - rrm2b ( figures 5f , 5 g , s7d , and s7e ) .
additionally , by monitoring apoptosis , we observed that wt - rrm2b rescued the null phenotype from apoptosis , whereas loss of the key residues ( y164 and k37/k151 ) abrogated this rescue ( figures 5h and 5i ) , therefore validating our in  vitro experiments and theoretical studies .
overall , we demonstrate the critical importance of the hypoxic induction of rrm2b to mitigate replication stress and determine the molecular adaptations of rrm2b to hypoxia to support this function ( figure  5j ) .
we identified that in response to the physiological stress of hypoxia , rnr responds by isoform switching favoring rrm2b over rrm2 . in hypoxia the activity of rrm2 is severely compromised due to the lack of available oxygen , leading to replication stress .
rrm2b is able to retain its activity in hypoxia and is therefore induced to compensate and facilitate ongoing replication .
this property of rrm1/rrm2b , although not sufficient to resolve replication stress , does preserve replication fork integrity and prevent the accumulation of dna damage in hypoxia .
we verified that depletion of rrm2b results in lower dntp levels in hypoxic cells and that this has detrimental consequences for cell fate ( failure to complete dna replication , dna damage , and loss of viability ) .
importantly , loss of rrm2b in a xenograft model showed delayed tumor growth , increased radiosensitivity , and increased apoptosis specifically in hypoxic areas , further highlighting the biological importance of the hypoxic induction of rrm2b .
our data suggest that rrm2b is capable of retaining activity in  hypoxia through two mechanisms : ( 1 ) increased oxygen - entering frequency and ( 2 ) enhanced stability of the tyrosyl radical .
our md analyses suggested that y164 could increase the oxygen - entering frequency through oxygen tunnel t3 in monomer 1 by keeping f95 an optimum distance from f197 .
we also found that through the k37/k151 residues , the rrm2b protein retains 66%70% of its tyrosyl radical in hypoxia .
interestingly , monomer 2 of rrm2b showed a higher oxygen - entering frequency than rrm2 ( 22% and 0% , respectively ) , suggesting that the two monomers of the small rnr subunits could react differently in the overall production of the tyrosyl radical .
it is possible that monomer 2 contributes more to the tyrosyl radical in rrm2b than in rrm2 , possibly due to its helix b open conformation , and that this could be more relevant in hypoxic conditions . in support of this hypothesis ,
epr spectra of the double mutant , k37e / k151e , showed a marked reduction in the tyrosyl radical content in hypoxia , but not in normoxia .
we propose that the tyrosyl radical of monomer 2 is better protected in rrm2b , therefore contributing to the prolonged activity of the enzyme in hypoxic environments .
our proposed model , where both monomers contribute to tyrosyl radical formation , is in agreement with the established finding that the tyrosyl radical is equally distributed between each small subunit , suggesting the possibility of generation of two tyrosyl radicals per dimer ( cotruvo and stubbe , 2011 ) .
it is important to note that our md simulations were carried out with the only currently available crystal structure of human rrm2b protein ( pdb : 3hf1 ) , which has limitations in its active site configuration and specifically in the iron center .
however , we considered it unlikely that as a result of this , there would be large conformational changes in the global structure of the protein compared to its native state , at least to a degree that would influence our md analysis . indeed , this is supported by the results of our recombinant mutant analysis and , most importantly , our rescue experiments using our rrm2b knockout cell line .
collectively , this work highlights the importance of y164 , k37 , and k151 for rrm2b activity in hypoxia .
it is important to note that the recombinant proteins used in our biochemical assays were assembled without additional iron .
the biochemistry of rnr , especially the reconstitution of the tyrosyl radical , is extremely complex and not fully understood .
additional studies that also consider the relevant factors for accurate assembly are required and will likely further illuminate the role of rrm2b in both normoxia and hypoxia . to date ,
mammalian rrm2b has been primarily associated with dna repair and mitochondrial dna synthesis ( wang et  al .
however , it is probable that rrm2b has been evolutionarily maintained in order to be used as the hypoxic - specific rnr small subunit , especially as variants  of rrm2b protein have been found to be responsible for continued cell division in anoxia tolerant vertebrates ( sandvik et  al . , 2012 ) . here
, we propose that one of the principal functions of rrm2b is to act as the hypoxic - specific rnr subunit in order to be able to react promptly when this physiologically relevant stress occurs .
it is tempting to speculate that through antioxidant and catalase - like properties ( xue et  al . , 2006 ) , rrm2b might even play a role in increasing immediate oxygen availability .
interestingly , a number of previous reports have demonstrated that rrm2b is frequently affected by copy - number changes , typically showing gains , in a broad range of cancers ( chae et  al .
this has led to the suggestion that rrm2b is a tumor promoter ( aye et  al . , 2015 ) .
our study  demonstrates that increased rrm2b expression in hypoxia maintains replication and prevents dna damage , therefore providing a plausible explanation for why rrm2b is so often amplified in cancers .
our data suggest that targeting r1/r2b enzyme specifically in hypoxic tumor cells might be an effective therapeutic strategy .
rnr is a well - established therapeutic target , and a number of rnr inhibitors ( such as gemcitabine , clofarabine , hydroxyurea , and triapine ) are being used clinically ( manegold et  al . , 2000 , aye and
, 1998 , hehlmann et  al . , 1993 , nutting et  al . , 2009 ) .
interestingly , delivery of sirna against rrm2 by phosphorothionate oligodeoxynucleotides ( gti-2040 ) ( lee et  al . ,
2003 ) is in clinical trials for various solid tumors ( juhasz et  al . , 2006 , leighl et  al . , 2009 , oh and park , 2009 ) .
it is tempting to speculate that if this approach was modified to target rrm2b and was employed alongside patient stratification to identify those with significant tumor hypoxia , it may be an effective way to target the most aggressive and treatment - resistant fraction of tumors .
reagent or resourcesourceidentifierantibodiesgoat polyclonal anti - rrm1 ( t-16)santa cruzcat # sc-11733goat polyclonal anti - rrm2 ( n-18)santa cruzcat # sc-10844goat polyclonal anti - p53r2 ( n-16 ) ( rrm2b)santa cruzcat # sc-10840mouse monoclonal anti - chk1 ( g-4)santa cruzcat # sc-8408mouse monoclonal anti - p53 ( do-1)santa cruzcat # sc-126rabbit polyclonal anti - p53 ( fl-393)santa cruzcat # sc-6243mouse monoclonal anti--actin antibody ( ac-15)santa cruzcat # sc-69879mouse anti - human hif-1 clone 54bd transduction laboratoriescat # 610958mouse anti - brdu clone b44bd transduction laboratoriescat # 347580rat monoclonal anti brdu clone bu1/75 ( icr1)bio - radcat # obt0030cxmouse monoclonal anti - rrm2 clone 1e1bio - radcat # mca3434zrabbit polyclonal anti - kap-1bethyl / universal biologicalscat # a300 - 274rabbit polyclonal anti - phospho kap-1 ( s824)bethyl / universal biologicalscat # a300 - 767rabbit polyclonal anti - phospho kap-1 ( s473)biolegendcat # 644602 rrid : ab_2241094rabbit polyclonal anti-53bp1novus biologicalscat # nb100 - 904rabbit polyclonal anti - phospho - p53 ( ser15)cell signalingcat # 9284rat monoclonal anti - rpa32/rpa2 ( 4e4)cell signalingcat # 2208rabbit polyclonal anti - phospho - chk1 ( ser296)cell signalingcat # 2349rabbit polyclonal anti - phospho - chk1 ( ser317)cell signalingcat # 2344rabbit polyclonal anti - phospho - chk1 ( ser345)cell signalingcat # 2341rabbit polyclonal anti - parpcell signalingcat # 9542rabbit polyclonal anti - cleaved caspase-3 ( asp175)cell signalingcat # 9661anti - mouse igg , hrp - linkedcell signalingcat # 7076anti - rabbit igg , hrp - linkedcell signalingcat # 7074rabbit polyclonal anti - beta tubulin antibodyabcamcat # ab6046mouse monoclonal anti - pimonidazole clone 4.3.11.3)chemicon internationalcat # hp1 - 100alexa fluor 680 goat anti - mouse igg ( h+l)invitrogencat # a21057alexa fluor 680 goat anti - rabbit igg ( h+l)invitrogencat # a21076alexa fluor 680 donkey anti - goat igg ( h+l)invitrogencat # a21084irdye 800cw donkey anti - rabbit iggli - corcat # 926 - 32213irdye 800cw donkey anti - mouse iggli - corcat # 926 - 32212alexa fluor 488-conjugated goat anti - rabbit igginvitrogencat # a11070alexa fluor 488-conjugated donkey anti - rabbit igginvitrogencat # a21206alexa fluor 488-conjugated goat anti - mouse igginvitrogencat # a11017alexa fluor 488-conjugated donkey anti - goat igginvitrogencat # a11055alexa fluor 594-conjugated goat anti - rabbit igginvitrogencat # a11072alexa fluor 594-conjugated goat anti - rat igginvitrogencat # a11007alexa fluor 594-conjugated goat anti - mouse igginvitrogencat # a11020alexa fluor 594-conjugated donkey anti - goat igginvitrogencat # a11058alexa fluor 555-conjugated goat anti - rat igginvitrogencat # a21434alexa fluor 488-conjugated goat anti - mouse f(ab)2 fragmentinvitrogencat # a11017pierce recombinant protein a / gthermo fisher scientificcat # 21186bacterial and virus strainsbl21-gold(de3 ) competent cellsagilent technologiescat # 230132one shot top10 chemically competent e.  colithermo fisher scientificcat # c404010chemicals , peptides , and recombinant proteinshydroxyurea ( hu)sigmacat # h8627doxorubicin hydrochloride ( adriamycin)sigmacat # d15155-bromo-2-deoxyuridine ( brdu)sigmacat # b5002propidium iodide ( pi)sigmacat # s7109protease inhibitor cocktailrochecat # 04693159001cldu ( 5-chloro-2-deoxyuridine)sigmacat # c6891idu ( 5-iodo-2-deoxyuridine)sigmacat # i7125cytidine 5-diphosphocholine sodium salt dehydrate ( cdp)sigmacat # c9755protein g sepharose , fast flowsigmacat # p3296isopropyl -d-1-thiogalactopyranoside ( iptg)sigmacat # i6758deoxyadenosine 5-triphosphate , [ 8 - 3h(n ) ] ( datp - h3)perkin elmercat # net268250ucdeoxythymidine 5-triphosphate ( ( dttp ) tetrasodium salt ) ( dttp - h3)perkin elmercat # net520a250uctrizol reagentthermo fisher scientificcat # 15596018dharmafect 1dharmaconcat #
t-2001lipofectamine ltxthermo fisher scientificcat # 15338100prolong goldthermo fisher scientificcat # p36931critical commercial assayshypoxyprobe-1 kit for the detection of tissue hypoxiachemicon internationalcat # hp1 - 100real - time and dynamic monitoring of cell proliferation and viability for adherent cellsacea biosciences inc/ cambridge biosccat # 00380601050sybr green pcr master mixapplied biosystemscat # 4309155verso cdna synthesis kitthermo fisher scientificcat # ab1453bhistrap hp columnsge healthcarecat # 17 - 5248 - 02qiaprep spin miniprep kitqiagencat # 27106quickchange ii xl site - directed mutagenesis kitagilent technologiescat #
, 1998rkorrm2b/this papern / aexperimental models : organisms / strainsmice : female balb / c nude were used for xenograft experimentscharles river , ukoligonucleotidessirna - rrm2b ( sequence : ugaguuuguagcugacagauu)sigmapiao et  al .
, 2009sirna#2 - rrm2b ( sequence : ggaacaagcuuaaagcaga)ambion / life technologiess224156sirna - p53 ( sequence : guaaucuacugggacggaa)ambion / life technologiesleszczynska et  al . , 2015allstars negative control sirnaqiagensi03650318primers for rrm2b forward - chip ( sequence : cttgctgggaaatcttgacc)sigmatanaka et  al .
2007primers used for site - directed mutagenesis table s1n / an / aprimers used for dntp incorporation assay table s1n / an / aprimers used for quantitative pcr ( qpcr ) table s1n / an / arecombinant dnaplasmids used in this work are listed in table s2n / an / asoftware and algorithms7500 fast real - time pcr thermocycler was used with v2.0.5 softwareapplied biosystemshttp://www6.appliedbiosystems.com / support / software/7500/the software caver 3.0 was used for the analysis of the evolution of lateral fenestrations during the md simulationsn / an / athe particle mesh ewald ( pme ) algorithm was used for evaluation of electrostatics interactionsdarden et  al .
, 1993n / athe multi time step algorithm verlet - i / r - respa was used to integrate the equations of motiontuckerman et  al .
, 1992 , verlet , 1967n / athe pops ( parameter optimsed surfaces ) algorithm was used for calculation of the solvent - accessible surface area ( sasa ) of both proteins rrm2b and rrm2cavallo et  al .
, 2003 , fraternali and van gunsteren , 1996n / athe settle algorithm was used for constrained the lengths of covalent bonds involving hydrogen atomsmiyamoto and kollman , 1992n / aotherfor the analysis of rrm2b expression and genetic alterations in colorectal cancer datasets the cbioportal and prognoscanhttp://www.cbioportal.org/ ; http://www.abren.net/prognoscan/gao et  al . , 2013 ,
, 2009gene expression omnibus ; smith et  al . , 2010https://www.ncbi.nlm.nih.gov / geoaccession number gse17536icp - ms was performed with icp - ms trace element analysishttps://www.earth.ox.ac.uk / research / services / geochemical - analysis / icpms / n / a further information and requests for reagents may be directed to , and will be fulfilled by , the lead contact , dr . ester m. hammond ( ester.hammond@oncology.ox.ac.uk ) .
source of cell lines used in the study is reported in the reagent and resource table .
source of cell lines used in the study is reported in the reagent and resource table .
cells were grown in dmem with 10% fbs , in a standard humidified incubator at 37c and 5% co2 .
rko was constructed in this work using crispr - cas9 technology as previously described ( ran et  al . , 2013 ) .
rrm2b gene has three isoforms and in order to construct a full knock - out cell line two 20-bp target sgrna sequences were used targeting  exon 1 : ttcggcggagtctgcgcgat ( isoforms 1 and 3 ) and aaatgttattcgccgcggtc ( isoform 2 ) .
lipofectamine ltx ( invitrogen ) was used for plasmid transfections according to manufacturer s recommendations ( the plasmids used are listed in table s2 ) .
dharma - fect 1 reagent ( thermo fisher scientific ) was used for sirna knockdown according to manufacturer s instructions .
rko or u2os cells were transfected with the sirna sequences reported in the reagent and resource table in a final concentration of 50  nm .
hydroxyurea ( hu ) was used at a concentration of 2  mm for 6  hr .
hypoxia treatments were carried out in a bactron ii anaerobic chamber ( shell labs ) or an in  vivo2 400 ( baker ruskinn ) ( for oxygen concentrations at 2% ) . for experiments at 
oxygen concentrations were periodically verified using an oxylite probe ( oxford optronix , uk ) . for immunoblots
, cells were lysed in utb ( 9  m urea , 75  mm tris - hcl ph 7.5 and 0.15  m -mercaptoethanol ) and sonicated briefly .
the odyssey infrared imaging technology was used ( li - cor biosciences ) and the odyssey analysis system was used for quantification of the immunoblots . in each case , experiments were carried out in triplicate and a representative blot is shown unless otherwise stated .
the antibodies used in this study are listed in the reagent and resource table . for immunofluorescence
, cells were seeded on autoclaved cover glasses ( menzel - glaser ) . at the end of each experiment cells were fixed with 4% fixation buffer ( 4% ( w / v ) paraformaldehyde in pbs ) , lysed with 1% pbs - triton x-100 and then incubated with blocking buffer ( 2% ( w / v ) bsa in 0.1% pbs - triton x-100 ) .
incubation with the appropriate primary and then secondary antibody followed ( diluted in 2% ( w / v ) bsa - 0.1% pbs - triton x-100 ) ( antibodies used are listed in the reagent and resource table ) .
at least 100 cells were counted per condition . due to the presence of 53bp1 foci in the nuclei of unstressed cells , induction of dna damage
rna was prepared using trizol ( invitrogen / life technologies ) . for qpcr expression analysis cdna was reverse transcribed from total rna using verso kit ( thermo scientific ) .
qpcr was performed using sybr green pcr master mix kit ( applied biosystems ) in a 7500 fast real - time pcr thermocycler with v2.0.5 software ( applied biosystems ) .
mrna fold change was calculated using a 2 method in relation to the 18s reference gene .
facs analysis was performed as previously described ( olcina et  al . , 2013 ) .
u2os cells were treated with either rrm2b sirna or negative control sirna and were placed in normoxia or  < 0.1% o2 ( 3 hr ) .
samples were pulsed with brdu ( 20  m ) for 30  min before collection .
sub confluent cells ( 50%60% confluency ) were sequentially pulse labeled with 25  mm cldu and 250  mm idu for 20  min each and left at 21% o2 in a 37c incubator ( normoxic samples ) .
cells were washed once with fresh warm media before the addition of the second ( idu ) label .
for hypoxic treated samples , cells were first placed for 2  hr in the hypoxic chamber ( < 0.1% o2 ) and then treated with 25  mm cldu for the indicated times , followed by the addition of 250  mm idu for as long as needed for all samples to be in hypoxia for 6  hr in total . at the end of the treatment , labeled cells were lysed and spread with spreading buffer ( 200  mm tris - hcl , ph 7.4 , 50  mm edta , 0.5% sds ) in a tilting the slide .
dna fibers were then fixed with methanol / acetic acid ( 3:1 ratio ) and stained with rat anti - brdu monoclonal antibody ( for detection of cldu labeled tracts ) and mouse anti - brdu monoclonal antibody ( for detection of idu labeled tracts ) .
fibers were imaged using a bio - rad radiance or lsm780 ( carl zeiss microscopy ltd ) confocal microscope and analyzed using imagej software ( nih ) .
the length of the fiber tracts that had incorporated both labels ( cldu and idu ) was measured and replication rates were calculated with the following formula ( vcldu ( kb / min )  = [ ( x   0.132  m )   2.59 kb / m ] / t ( min ) , where x  = length of cldu ) . dntp pool determination in whole - cell extracts was carried out as previously described ( dangiolella et  al . , 2012 ) .
in brief , cells were seeded in glass dishes and treated either in  < 0.1% o2 ( for the indicated times ) or with 2  mm hu ( 6 hr ) .
the normoxic samples were processed immediately after the beginning of the hypoxic and/or hu treatment .
samples were then boiled , centrifuged and the supernatant was dried by centrifugal evaporation and finally dissolved in h2o . for the preparation of each primer mix a different set of primers
was used ( listed in table s1 ) supplemented with either [ 3h]datp or [ 3h]dttp ( perkinelmer ) . for dgtp and dctp determination 1  u of thermo sequenase dna polymerase ( ge healthcare )
was used ; for dttp determination 1.25  u klenow fragment ( thermo fisher scientific ) and for datp determination 0.625  u klenow fragment ( thermo fisher scientific ) was used .
reactions were incubated for 1  hr either at 48c ( for dgtp and dctp mixtures ) or at room temperature ( for dttp and datp mixtures ) . following incubation ,
the reaction mixture were spotted onto whatman de81 paper ( ge healthcare ) and let dry .
the papers were then washed with 5% na2hpo4 , followed by rinsing with distilled h2o and 100% etoh . after being dried ,
the radioactivity on the papers was measured in a ls 6500 multi - purpose scintillation counter ( beckman coultertm ) using 3.5  ml optiphase hisafe 3 ( perkinelmer ) counting fluid .
data present percentage of dntp incorporation compared to the positive control of each experiment . in experiments where both sirna and hypoxia treatment were used , a normalization to each normoxic control was preceded the final analysis .
briefly , rko cells were lysed in lysis buffer ( 100  mm nacl , 20  mm tris - hcl ph 7.4 , 5  mm mgcl2 , 0.5% np-40 ) with freshly added phosphatase / protease cocktail inhibitors ( roche ) .
the lysates were incubated with protein g sepharose beads ( sigma ) and the antibody of interest rrm1 ( santa cruz biotechnology : sc-11733 ) or a control igg antibody ( invitrogen : a10535 ) with agitation , at 4c overnight .
antibodies used were : rrm1 ( santa cruz biotechnology : sc-11733 ) , rrm2b ( santa cruz biotechnology : sc-10840 ) , rrm2 ( abd serotec , mca3434z ) and for the detection of rrm2 the pierce recombinant protein a / g ( thermo fisher scientific , 21186 ) secondary antibody was used .
chip in rko cells treated as indicated in figure legend was carried out as previously described ( leszczynska et  al . , 2015 ) .
for each sample 2  g of combined mouse p53 ( do-1 ; sc-126 ) and rabbit p53 ( fl-393 ; sc-6243 ) ( santa cruz ) antibodies were used for immunoprecipitation .
combined non - specific mouse ( 7076 ) and rabbit ( 7074 ) ( cell signaling ) were used as control iggs .
a no antibody control sample was also included in each experiment . bound fraction and input
were analyzed by qpcr using specific primer set for the rrm2b locus : cttgctgggaaatcttgacc ( tanaka et  al . , 2007 ) .
fold enrichment is expressed as a % of input and is normalized to total p53 in each sample .
rko cells were treated either with rrm2b sirna or negative control sirna and exposed to normoxia or hypoxia ( < 0.1% o2 for 24  hr ) . subsequently all cells were placed in normoxic incubator and left for 9  days to form colonies , which were visualized by crystal violet staining .
apoptosis assay were performed as previously described ( leszczynska et  al . , 2015 ) .
in brief , both adherent and floating cells were collected and fixed with 4% paraformaldehyde .
samples were then washed and the nuclei were stained with prolong gold mounting  medium with dapi ( thermo fisher scientific ) .
apoptosis was plotted as the percentage of cells with fragmented dna per field of view , with at least ten fields of view quantified per experiment .
the xcelligence real - time cell analyzer ( acea biosciences ) dp instrument equipped with an e - plate was used for proliferation assays .
the attachment , spreading and proliferation of the cells were monitored every 15  min for 60  hr . for quantification
all animal procedures were performed in accordance with current uk legislation and were approved by the university of oxford biomedical services ethical review committee , oxford , uk .
for the tumor growth curves ( xenografts ) , female balb / c nude mice or female athymic nude mice ( charles river , uk ) were randomized and injected subcutaneously with 3   10 rko or rko cells in 25% ( v / v ) matrigel and serum - free dmem .
tumor growth was monitored until they reached approximately 300 - 400  mm3 ( volume  = height x depth x width x  / 6 ) . for the irradiation experiment ,
the tumors in half of the animals were irradiated with 10  gy when tumor volumes reached 100  mm .
tumors were measured regularly , and tumor growth was plotted as a mean of tumor volumes   sem .
mice were injected with 50  mg / kg of pimonidazole ( pimo ) 2  hr before the end of the experiment .
tumors were split in half and either preserved in formalin for wax embedding or were snap frozen and embedded in optimal cutting temperature compound ( oct ) .
, tumors embedded in oct were sectioned ( 5  m ) and froze down at 20c .
sections were immediately fixed with 4% paraformaldehyde and then quenched with 50  mm nh4cl .
blocking was performed with 1% bsa in tbs , followed by incubation with mouse on mouse ( m.o.m . ) blocking reagent ( vector laboratories ) .
the sections were stained with pimo and cleaved caspase-3 primary antibodies , followed by secondary antibodies ( listed in the reagent and resource table ) and mounted with prolong gold mounting medium with dapi ( invitrogen / life technologies ) .
raw rna - seq data for 382 colorectal adenocarcinoma tumors were downloaded from the tcga project ( accessed through cbioportal : http://www.cbioportal.org/ ) . to examine tumor - associated hif - activity ( referred to as hypoxia signature ) , raw data for  each sequenced gene
were rescaled to set the median equal to 1 , and hif - activity was quantified by averaging the normalized  expression of 44 target genes , associated with hif activity ( encoding adm , gfbp3 , edn2 , pfkfb4 , flt1 , tfr2 , bnip3l , tgfa , bnip3 , pgk1 , egln1 , ldha , egln3 , cp , tgfb3 , pfkfb3 , hk1 , tfrc , edn1 , cdkn1a , ca9 , hmox1 , serpine1 , lox , ndrg1 , ca12 , pdk1 , vegfa , ero1l , rora , p4ha1 , mxi1 , slc2a1(glut1 ) , stc2 , mif , ddit4 , eno1 , cxcr4 , plod1 , p4ha2 , gapdh , pgam1 , tmem45a and pim1 ) ( li et  al . , 2014 ) .
log10 conversion of the hypoxia signature was plotted against log10 conversion of raw data for rrm1 , rrm2 and rrm2b .
two - tailed p value shown on each graph for each pearson and spearman r ( correlation coefficient ) . to examine tumor - associated p53-activity ( referred to as p53 group ) ,
raw data for each sequenced gene were rescaled to set the median equal to 1 , and p53-activity was quantified by averaging the normalized expression of 6 p53 target genes , associated with hypoxia - induced p53 activity ( encoding btg2 , cyfip2 , inpp5d , kank3 , phlda3 and sulf2 ) ( leszczynska et  al . , 2015 ,
log10 conversion of the p53 signature was plotted against log10 conversion of raw data for rrm2b ( also rescaled to set the median equal to 1 ) .
two - tailed p value shown on each graph for each pearson and spearman r ( correlation coefficient ) . for the analysis of rrm2b expression and genetic alterations in colorectal cancer datasets the cbioportal ( http://www.cbioportal.org/ ) and prognoscan ( http://www.abren.net/prognoscan/ ) tools were used on the 5th dec 2016 ( gao et  al . , 2013 , cerami et  al .
, 2012 , mizuno et  al . , 2009 ) . using cbioportal the tcga ( provisional )
dataset was analyzed ( rna seq v2 rsem ) for alterations , including mutations , putative copy - number alterations , mrna expression , mutations and survival probability ( 629 cancer patients ) .
for the prognoscan analysis , rrm2b expression was checked in all available colorectal cancer datasets and kaplan - meier graphs were extracted only for the statistically significant ( p  < 0.05 ) dependence between rrm2b expression ( probe 223342_at ) and survival probability .
this analysis used the publicly available dataset at gene expression omnibus ( https://www.ncbi.nlm.nih.gov/geo ) with the accession number gse17536 ( smith et  al . , 2010 ) .
the 6   his - tagged hrrm1 ( pet28a - rrm1 ) was kindly provided by prof joanne stubbe ( massachusetts institute of technology , cambridge , usa ) , expressed in e.coli bl21-gold ( de3 ) competent cells ( agilent technologies ) and as previously described ( ando et  al . ,
the 6   his - tagged hrrm2 and hrrm2b ( pet28a - rrm2 , pet28a - rrm2b ) were kindly provided by dr .
yun yen ( beckman research institute at city of hope , duarte , usa ) .
the proteins were expressed in e.coli bl21-gold ( de3 ) competent cells ( agilent technologies ) and purified as previously described ( shao et  al . , 2004 ) with minor modifications .
specifically , an overnight culture of the transformed bacteria was diluted 80-fold in 600  ml of 2   yt medium containing 50  g / ml kanamycin .
each culture was grown at 37c until od600 nm  = 0.7 - 0.9 and then induced by 1  mm isopropyl-1-thio--d - galactopyranoside ( iptg ) and incubated overnight at 18c .
cells were harvested by centrifugation and the pellets were lysed with appropriate amount ( 5  ml / gr of pellet ) lysis buffer ( 50  mm nah2po4 ph 7.0 ; 0.1% triton x-100 ; 10  mm -mercaptoethanol ; freshly added edta - free protease inhibitors and dnase i ) at 4c with vigorous agitation until the lysate was homogeneous .
the lysate was then sonicated ( 60% amplitude 30  s on / 30  s off ) and clarified by centrifugation at 48,000   g for 20  min at 4c .
soluble lysate was passed through a his - trap hp purification column ( ge healthcare ) , washed with at least 30-fold bed volume of wash buffer ( 50  mm nah2po4 ph 7.0 ; 800  mm nacl ; 50  mm imidazole ; 0.1% triton x-100 ; 10  mm -mercaptoethanol ) and finally eluted with elution buffer ( 50  mm nah2po4 ph 7.0 ; 300  mm nacl ; 125  mm imidazole ) .
the protein then underwent buffer exchange into 25  mm tris - hcl , ph 7.5 before being concentrated and stored at 80c .
the apo - forms of rrm2 and rrm2b recombinant proteins used in icp - ms experiments were overexpressed and purified as previously described ( wang et  al . , 2009a ) .
site - specific mutations in rrm2b ( y164c ; y164f ; f183y ; k31e / k151e ; y241h ; y331f and q127k ) were generated by pcr , using the pet28a - rrm2b as a template and the quickchange ii xl site - directed mutagenesis kit ( agilent technologies ) .
expression and purification of the hrrm2b mutated proteins were performed in the same way as for hrrm2b .
the enzyme concentration ( r1/r2b or r1/r2 ) was the same in all cases and the limited factor was the presence or not of oxygen ( normoxia versus hypoxia ) .
specifically , a final concentration of 1.25  m of purified rrm1 was incubated with 2.5  m of either rrm2 or rrm2b protein .
the reaction mixture contained 0.1  mm cdp , 50  mm hepes ( ph 7.5 ) , 6  mm dtt , 8  mm magnesium acetate , 2  mm atp and 1  mm nadph in a final volume of 400  l .
the rnr proteins purified were active without reassembling the iron center ( shao et  al .
( < 0.1% o2 ) the reaction mixture was assembled and the assay was carried out within an anaerobic chamber ( bactron , shel labs ) .
reactions were initiated by the addition of cdp , incubated at 37c and aliquots of 50  l were quenched by the addition of 10  l of 3% tricarboxylic acid over a time course of the reaction .
graphs present either percentage of dcdp formation when normoxia and hypoxia were compared ( data were normalized against the normoxic samples at 37c at 30  min where the dcdp formation was considered 100% ) or in m when the accumulation of dcdp formed in  < 0.1% o2 at a 0 - 120  min time course was examined .
cdp and dcdp levels following rnr activity assays were determined by ion - pairing hplc with uv detection ( 270  nm ) .
the column was an ace c 18 , 3  m , 125   3  mm ( hichrom ) maintained at 35c .
a gradient separation was achieved using 10% methanol , 10  mm kh2po4 , 10  mm tbaoh , ph 6.9 ( a ) and 30% methanol , 50  mm kh2po4 , 6  mm tbaoh , ph 7.0 ( b ) , with a linear gradient of 25  47% b over 8  min , 47 
80% b over 1  min , then returning to the started conditions 3  min .
the flow rate was 0.6  ml / min and the running time was 16.5  min .
circular dichroism ( cd ) spectra ( chirascan cd / fluorimeter spectrometer ( applied photophysics , uk ) of hrrm2b and the mutants ( 0.2  mg / ml ) were recorded in the wavelength range 260 to 185  nm in 100  mmol / l potassium phosphate buffer ( ph 7.5 ) . all measurements were carried out at 37c using a 0.1-cm path length quartz curette .
the data pitch was 0.1  nm with a 1-nm bandwidth at a scan speed of 1.0  nm / s .
all cd data were expressed as the mean residue ellipticity , [  ] , degcmdmol .
the thermo finnigan element 2 icp - ms was used for quantitation of iron content in the recombinant purified proteins ( 40  g of protein was used per 50  l of reaction ) .
calibrations were obtained using external standards ( a series of standards of known fe concentrations were prepared and analyzed to gain a calibration linear , prior to the measurement of the samples ) .
an external standard was diluted and measured from a dilution of high purity standards 10 ppm icp - ms-68 a standard .
all blanks , standards and samples were also spiked with 1  ng / g rh , so that any general instrument drift could be normalized .
dilutions were made using a 2% hno3 solution , prepared using in - house distilled nitric acid and 18.2 mohm di water .
all data results were first reported as elemental concentrations and then calculated in nm of fe per m of protein .
cw - epr spectra were collected in the center for advanced esr ( caesr ) in the inorganic chemistry laboratory at the university of oxford .
x - band measurements utilized a bruker - biospin micro emx spectrometer equipped with a premiumx microwave bridge , a cylindrical te011-mode resonator ( shqe - w ) , an esr-900 liquid helium cryostat , and an oxford instruments itc-503  s temperature controller . measurements were performed within the limit of resolution by temperature , 40k , and under non - saturating conditions .
the protein concentration for all epr samples was 200  m except for rrm2 protein , which was 70  m . the protein samples ( rrm2b , rrm2 or rrm2b mutants ) were diluted in 50  mm hepes , ph 7.5 and were incubated either at 37c for 5  min with oxygen present ( normoxia ) or at 37c for 60  min in a bactron ii anaerobic chamber ( < 0.1% o2 ) . at the end of each treatment 20% ( v / v ) glycerol was added for vitrification during the low - temperature recordings .
samples were then transferred to epr tubes and quick - frozen by immersion in liquid nitrogen .
spin quantitation was performed using the bruker spectrometer hardware spin calibration , given the input dimensions of the precision wilmad pq-706 tubes .
the results were verified with a 1  mm cu edta in 50  mm hepes ph 7.5 , 20% ( v / v ) glycerol that was measured under non - saturating conditions at 100 k with 50  w microwave power .
system setup : md simulations of o2 transport on hrrm2b ( pdb : 3hf1 ; resolution 2.60   ) and hrrm2 ( pdb : 3vpn ; resolution 2.25   ) were performed ( smith et  al . , 2009 ) .
the 3vpn x - ray structure is that of the  e106d variant of native rrm2 , and comprises residues m65 to m350 .
four simulations were performed in which 200 randomly chosen water molecules outside the solvated protein were replaced by 200 o2 molecules ( 0.5 m ) termed the 100% oxygen level simulation .
an additional two sets of simulations were performed to control for concentration effects ; the first set up has a tenfold reduction in oxygen concentration , termed 10% oxygen level simulation with effective concentration of 50  mm , for rrm2b and rrm2 .
the second set does not  include any oxygen molecules , termed 0% oxygen level simulation .
each system was solvated in a box of dimensions ( 80 , 80 , 92 )   using the  solvate plug - in of vmd ( humphrey et  al . , 1996 ) , with the total system size comprising 61,000 atoms . in accordance with the uniprot sequence alignment two mutants of native rrm2b
were prepared with the mutate plugin in vmd ( humphrey et  al . , 1996 ) , namely y164c and k37e / k151e .
calculations were done with namd 2.9 ( phillips et  al . , 2005 ) , using the charmm 27 protein force - field ( brooks et  al . , 2009 ) with the cmap correction ( buck et  al . , 2006 ) ,
together with the tip3p water model ( jorgensen et  al . , 1983 ) .
oxygen lennard - jones parameters were taken from charmm ( cohen et  al . , 2005 ,
the iron centers were modeled using charmm 27 force - field , with iron lennard - jones parameters o  = 0.00  kcal / mol and rmin/2  = 0.65   .
oxygen ( o2 ) was modeled using charmm 27 lennard - jones parameters from heme oxygen , o  = 0.12  kcal / mol and rmin/2  = 1.7   , as well as bond spring constants and bond spring lengths for o2 as 1.23   and 600  kcal / mol /  , respectively .
rrm2b contains the same diiron / dityrosyl cofactor that rrm2 does , and both rrm2b and rrm2 are biologically active as homodimers . in rrm2b , the active site iron coordination environment in monomer-1 is a mono - iron site , and monomer-2 has a di - iron site . in rrm2b , the active site iron coordination environment
after 1,000 steps of conjugate - gradient minimization , four classical molecular dynamics ( md ) simulations of the o2-containing solvated protein were carried out for 300  ns in the npt ensemble .
the high oxygen concentration was necessary to ensure sufficient sampling of protein cavities by o2 on the accessible timescale of current simulations .
the particle mesh ewald ( pme ) algorithm was used for evaluation of electrostatics interactions beyond 12   , with a pme grid spacing of 1   , and namd defaults for spline and  values ( darden et  al . , 1993 ) .
a cut - off at 12   was applied to non - bonded forces , both electrostatics and van der waals forces were smoothly switched off between the cut - off distance of 12   , and the switching distance of 10   , using the default namd switching function .
a verlet neighbor list with pairlist distance of 14   was used to only evaluate non - bonded neighboring forces within the pairlist distance ( verlet , 1967 ) .
the lengths of covalent bonds involving hydrogen atoms were constrained by the settle algorithm ( miyamoto and kollman , 1992 ) in order to be able to use a 2-fs time - step .
the multi time step algorithm verlet - i / r - respa ( tuckerman et  al .
, 1992 , verlet , 1967 ) was used to integrate the equations of motion .
non - bonded short - range forces were computed for each time step , while long - range electrostatic forces were updated every 2 time steps .
the pressure was kept at 1 atm by the nos - hoover langevin piston ( langevin , 1908 , nos , 1984a , nos , 1984b ) , with a damping time constant of 50  fs and a period of 100 fs .
the temperature was maintained at 300 k by coupling the system to a langevin thermostat , with a damping coefficient of 1 ps .
the standard mpi version 2.9 of the md simulation code namd was employed ( phillips et  al . , 2005 ) .
a free license can be obtained at http://www.ks.uiuc.edu / research / namd/. version 1.9 of visual molecular dynamics ( vmd ) was employed for analysis , with license obtained at http://www.ks.uiuc.edu / research / vmd/. the solvent - accessible surface area ( sasa ) of both proteins rrm2b and rrm2 was calculated using the pops ( parameter optimsed surfaces ) algorithm ( cavallo et  al . , 2003 , fraternali and van gunsteren , 1996 ) .
the bending angle of helices was estimated using bendix in vmd ( dahl et  al . , 2012 ) .
the diffusion - reaction model ( wang et  al . , 2013 ) presented in equation  ( 1 ) was adopted to describe the diffusion of o2 into the rnr active site:(1)enz+gaskoutkin[o2]enzgas .
this is a kinetic two - step model consisting of an initial diffusion step of the gas molecule , followed by a metal - gas molecule chemical reaction step ( wang et  al . , 2011 ,
wang and blumberger , 2012 ) . here , a classical force - field ( ff ) was employed , and only the diffusion and binding of o2 gas molecules to the rnr iron center were modeled:(2)rnr(fe)+o2k1k+1rnr(fe)o2 .
the simulation of o2 diffusing in the solvated rnr enzymes yields diffusion statistics that enable the construction of a probability density map of the o2-distribution within rnr ( rrm2b or rrm2 ) .
this distribution is then divided into a set of m discrete protein cluster states that correspond to the probability maxima .
first , a distance criterion is established to determine when an oxygen molecule has entered into the active site of hrrm2b and hrrm2 , defined as the difference between the center - of - mass ( com ) of the iron ion and the o2 molecule .
second , cut - off criteria of 8   , 9   and 10   were employed to generate statistics of the o2 entering .
an o2 molecule is counted as having entered the cluster if dcom ( o2-fe )  < c where c is 8 or 9   .
as previously described , o2 entry into wt rrm2b and rrm2 was identified to occur primarily through tunnels t1 ,
t2 and t3 . based on this assignment , equilibrium adaptive - biasing force ( abf ) md simulations ( chipot and pohorille , 2007 , darve and pohorille , 2001 , darve et  al . , 2008 , van gunsteren , 1989 ) were performed on oxygen entry into tunnels t1 , t2 and t3 in monomer-1 to determine relative differences in the thermodynamics of oxygen entry into rrm2b and rrm2 . in total , six abf simulations per rnr were performed , totalling 0.5  s of md simulation dynamics . each abf simulation was performed with a force bias on a single oxygen molecule meaning these simulations constitute single - molecule biased md simulations , using the collective variable  center - of - mass ( com ) distance between fe(iii ) and o2 , (rn)=d(o2fe(iii ) ) , with  ranging from 3   to 15 , in bins of width 0.25   .
each abf simulation obtains ergodic sampling by applying every 500 timesteps a biasing force fabf ( 3 ) onto the newtonian equations of motion , whose value is updated on - the - fly ( den otter , 2000 , den otter and briels , 1998 ) from the result , free - energy gradient along  , the potential of mean force ( pmf ) along  is obtained from numerical integration of the gradient.(3)fabf=ra=f()r(4)(dg()d)=u(rn)1in|j|=f(). tunnel t1 in monomer-1 extends for 30   and connects the two faces of the protein .
it was simulated as two separate tunnels of length 15   in monomer 1 , denoted tunnel t1 and t1 , and was compared to a single simulation of tunnel t1 in monomer 2 .
the software caver 3.0 was used for the analysis of the evolution of lateral fenestrations during the md simulations , using 100 evenly chosen snapshots from the md simulations , which is every 2  ns .
the outer surface of the protein is calculated by rolling a large spherical probe around the surface of the protein and then internal cavities are identified using a smaller spherical probe ( a radius of 0.8   was used here , and 12 probe spheres used ) .
all identified tunnels are grouped into clusters based on relative proximities and with an 8   cut off for each cluster node , meaning that tunnels differing from the node by more than 8   are excluded .
the values for shell radius and depth ( 15   ) influence the definition of the protein molecular surface .
the statistical significance of differences between datasets was determined assessed by using the following test .
student s t test ( two - tailed paired , except for dntp incorporation assays , where a two - tailed unpaired test was applied ) was used in all experiments where two means were compared .
one - way analysis of variance ( anova ) was used to compare several means and two - way anova was used for determination in a response that is affected by two factors ( one- or two - way anova analysis is indicated at the figure legends ) .
statistical significance was assumed if p  < 0.05 or lower and is noted in the figures .
error bars represent mean   standard error of the mean ( s.e.m . ) .
i.p.f . conceived , designed , performed , and interpreted the majority of the experiments and wrote the manuscript .
k.b.l . performed the xenografts , immunohistochemistry ( ihc ) , some experiments , and tcga analysis .
e.m.h . conceived the  project ; conceived , designed , and interpreted the majority of the experiments ; and wrote the manuscript .
 summarycells exposed to hypoxia experience replication stress but do not accumulate dna damage , suggesting sustained dna replication . 
 ribonucleotide reductase ( rnr ) is the only enzyme capable of de  novo synthesis of deoxyribonucleotide triphosphates ( dntps ) . 
 however , oxygen is an essential cofactor for mammalian rnr ( rrm1/rrm2 and rrm1/rrm2b ) , leading us to question the source of dntps in hypoxia . here 
 , we show that the rrm1/rrm2b enzyme is capable of retaining activity in hypoxia and therefore is favored over rrm1/rrm2 in order to preserve ongoing replication and avoid the accumulation of dna damage . 
 we found two distinct mechanisms by which rrm2b maintains hypoxic activity and identified responsible residues in rrm2b . 
 the importance of rrm2b in the response to tumor hypoxia is further illustrated by correlation of its expression with a hypoxic signature in patient samples and its roles in tumor growth and radioresistance . 
 our data provide mechanistic insight into rnr biology , highlighting rrm2b as a hypoxic - specific , anti - cancer therapeutic target . 
Introduction
Results
Discussion
STARMethods
Author Contributions