dna polymerases make and repair dna , thereby controlling both the stability and variability of genetic information .
whereas both replication and repair employ dna polymerases that maximize faithful dna synthesis , other tasks such as lesion bypass come at the price of lower fidelity and are performed by the so - called  sloppy  dna polymerases ( goodman and tippin , 2000 ) . in humans , 16 different dna - synthesizing enzymes are dedicated to replication , repair , damage tolerance , and variability of nuclear dna ( reviewed in hbscher et  al . , 2002 ) .
in stark contrast , only one dna polymerase has been described in mitochondria ( bolden et  al . , 1977 ) , dna polymerase gamma ( pol ) , which has long been presumed to mediate every dna synthesis reaction related to the replication and repair of mitochondrial dna ( mtdna ) .
none of the known 17 human dna polymerases can initiate dna synthesis , and , because rna polymerases can polymerize ribonucleotides from a single nucleoside 5-triphosphate ( ntp ) , rna is widely used to prime dna synthesis . in most cases , such rna primers are produced by specialized polymerases or  primases  ( frick and richardson , 2001 ) .
primases can be divided in two evolutionarily unrelated families : dnag - like ( bacteria ) and aep - like ( archaea and eukaryotes ) ( aravind et  al . , 1998 ; iyer et  al .
the human primase operating at nuclear dna is a heterodimer ( pri1 + pri2 ) wherein pri1 is the small catalytic subunit belonging to the aep family . during nuclear dna replication ,
the primase interacts with pol , forming a very stable complex ( pol-primase ) that triggers the initiation of dna synthesis .
the few primers made at ori sequences are further elongated by the leading - strand dna polymerase ( pol ) ; conversely , other primers are synthesized repeatedly , much like those used to initiate the synthesis of okazaki fragments in the antiparallel lagging strand . exceptionally , rna primers can be generated by the transcription activity of rna polymerases , as in plasmid cole1 ( itoh and tomizawa , 1980 ) and during the replication of mtdna ( chang and clayton , 1985 ; fust et  al . , 2010 ) .
the replication of mtdna ( a small circle of about 17 kb in human cells ) is a complex process involving multiple mechanisms ( holt and reyes , 2012 ) . at the replication fork
, the mitochondrial dna helicase twinkle opens the duplex , and the mitochondrial single - stranded binding protein ( mtssb ) stabilizes the unwound dna ( falkenberg et  al .
, 2007 ) , although the extent of coating of the lagging - strand template with mtssb may be minimal because of the incorporation of processed transcripts ( reyes et  al . , 2013 )
. mitochondrial rna polymerase ( polrmt ) primes at one major initiation site on each strand , orih and oril ( fust et  al .
, 2010 and the references therein ) , and primer elongation is performed by pol ( falkenberg et  al . , 2007 ) . however , minor replication products of coupled leading- and lagging - strand mtdna synthesis ( holt et  al . , 2000 )
would need frequent priming on the lagging strand . as long ago as 1985 , a primase activity distinct from polrmt was detected in human mitochondria , although the protein responsible has not yet been identified ( wong and clayton , 1985 ) . here ,
ccdc111 , a putative primase based on in  silico predictions ( iyer et  al . , 2005 ) , is shown to have both dna primase and dna polymerase activities ( and , thus , is renamed primpol ) and a striking capacity to tolerate dna damage .
primpol was shown to be present at both nuclear and mitochondrial dna compartments but is absent in mitochondria derived from a mouse primpol knockout ( ko ) model .
rnai experiments in human cells and analysis of primpol ko mouse embryonic fibroblasts ( mefs ) indicated that primpol plays a key role in mtdna synthesis .
a plausible hypothesis is that this primase / polymerase  facilitates replication restart after programmed or damage - induced fork arrest .
human gene ccdc111 ( also known as flj33167 ) , which is located on chromosome 4q35.1 and codes for coiled coil domain - containing ( ccdc111 ) protein , was predicted to be a member of the aep family ( iyer et  al . , 2005 ) .
ccdc111 contains the active - site motifs of aep primases and a putative zn finger similar to that of herpesvirus ul52 primase and other aep - like enzymes ( figures 1a and 1b ; figure  s1 available online ) .
iterative blast searches with human ccdc111 and putative homologs indicated the presence of ccdc111 in a broad range of unicellular and multicellular eukaryotes , including animals , plants , unicellular microalgae , protists , choanoflagellates , fungi , and picoeukaryotes such as ostreococcus ( the smallest known free - living eukaryote with a single chloroplast and a single mitochondrion ) .
alignment of putative ccdc111 homologs ( figure  s1 ) revealed 14 conserved regions , including the zn finger domain and the motifs a , b , and c shared with aep primases , nonhomologous end - joining primases , primpols , and herpes and poxvirus - like primases ( figures 1a , 1b , and s1 ) .
all members of the ccdc111 family have the motif a variant dxe ( figures 1b and s1 ) instead of the dxd motif present in most members of the aep superfamily .
the dxe variant is also found in the primpol of plasmid prn1 from sulfolobus islandicus , which has both primase and dna polymerase activities ( lipps et  al . , 2003 ) .
in agreement with in  silico predictions ( iyer et  al . , 2005 ) , purified human ccdc111 ( figure  s2a ) displayed rna primase activity on a single - stranded circular dna template ( m13 ssdna ) ( figure  1c , left ) .
interestingly , the protein was dependent on manganese for rna synthesis , yielding remarkably long primers ( > 100 nt ) that were not generated in the presence of magnesium ( figures s2b and s3a , left ) .
strikingly , a similar primase activity was observed when ntps were replaced by deoxynucleoside 5-triphosphates ( dntps ) ( figures 1c and s3a , right ) . in general , primases make rna primers , but the aep - related primases of archea and some bacteria are exceptions to this rule ( lao - sirieix et  al .
this unusual rna and dna primase activity is not present in escherichia coli and was demonstrated to be inherent to human ccdc111 by ablating the activity with a double mutation ( axa ) in the two potential metal ligands ( asp and glu , motif dxe ) at the predicted active site ( figure  1c ) , which was in agreement with enzymatic and structural studies of the primpol from plasmid prn1 ( lipps et  al . , 2004 ) .
next , we evaluated the primase activity on a template oligonucleotide in which a potential recognition sequence ( gtcc ) was flanked by thymine residues ( cavanaugh and kuchta , 2009 ) .
such a tract of pyrimidines is the preferred template context for several viral , prokaryotic , and eukaryotic rna primases ( frick and richardson , 2001 ) .
again , mn - activated synthesis of dna primers and their extensive elongation were observed with purified human ccdc111 ( figure  s2c ) .
ccdc111 primase recognized the 3 gtcc-5 sequence , preferentially forming initiating dinucleotides 5 a - dg-3 or 5 da - dg-3 ( figures 1d and 1e ) .
there was no difference in the efficiency of initiation when providing either a ribo- or a deoxynucleotide at the 5position ( figure  1d ) .
again , mutant axa was inactive in this assay , supporting the intrinsic nature of the primase activity attributable to ccdc111 protein . in respect to the second nucleotide , ccdc111 displayed a marked preference for a dntp ( figure  1e , left ) .
mn - activated formation of a ribo - deoxy - initiating dimer ( occurring at mn concentrations as low as 50  m ; figure  s3b ) is a hallmark of ccdc111 , given that several other dna synthesizing enzymes were unable to catalyze this reaction , and a conventional primase such as bacillus subtilis dnag displayed only a modest activity ( figure  1e , right ) .
unlike eukaryotic primases , archeal primases are able to carry out the initiation and extension of both rna and dna chains of up to 1 or 7 kb , respectively ( lao - sirieix and bell , 2004 ; lao - sirieix et  al . , 2005 ;
hence , these enzymes are both primases and polymerases , or primpols ( lipps et  al . , 2003 ) .
therefore , we tested whether , in addition to its dna and rna primase activity , ccdc111 possessed dna - dependent polymerase activity .
ccdc111 was able to extend a dna primer hybridized to m13 ssdna by polymerizing dntps ( figure  2a ) .
again , mn was the preferred metal ( figure  2a , compare panels 1 and 2 ) , allowing the nonprocessive elongation of dna chains up to 5 kb ( data not shown ) .
the enzyme also utilized ntps , albeit with a much lower efficiency ( figure  2a , panel 3 ) . as with the primase activity ,
the dna polymerase activity was abolished in the axa mutant ( figure  2a , panel 4 ) .
hence , the primase and polymerase activities of ccdc111 ( compared in figure  2b as a function of enzyme concentration ) appear to share the same active site . thus , in light of its identified activities , we propose that ccdc111 should be renamed primpol .
primpol efficiently catalyzed dna polymerization within a wide range of mn concentrations ( figure  s3c ) . even at the lowest mn concentration tested ( 10  m ) , primpol was more efficient than using a physiological concentration of mg ( 10  mm ) .
thus , mn is a more than 1,000-fold better metal activator than mg , suggesting that it is likely to be the physiological cofactor of primpol .
a similar behavior has been described for human pol ( blanca et  al . , 2003 ) .
independent of the metal cation , primpol behaved as a dna - instructed dna polymerase , preferentially inserting the complementary nucleotide dictated by the first available templating base ( figure  2c ) .
primpol provided a 1,000-fold bias toward correct watson - crick base pairs when extending a correctly - paired primer terminus ( figure  2d , top ) .
primpol could not excise a mismatched primer ( figure  2d , bottom ) , demonstrating the absence of a proofreading 3-5 exonuclease activity .
this is in agreement with the lack of exoi , exoii , and exoiii consensus motifs , which form an evolutionarily conserved 3-5exonuclease active site in several dna polymerase families ( bernad et  al . , 1989 ) .
however , the extension of a mismatched primer terminus by primpol is about 1,000-fold less efficient than the extension of a correctly paired primer terminus ( figure  2d ) . given that primpol is a dna primase / polymerase ( figures 1c1e , 2 , s2 , and s3 ) , it should logically operate in the nucleus and/or mitochondrion .
biochemical fractionation of hela cells revealed that primpol was distributed between the cytosol ( 47% ) , mitochondria ( 34% ) , and nuclear ( 19% ) compartments ( figure  3a ) . in order to determine whether the mitochondrial - associated primpol was inside the organelles , mitochondria from human embryonic kidney ( hek )
293 t cells were treated with trypsin and exposed to hypotonic buffer or digitonin ( figure  3b ) .
as expected , a mitochondrial protein located in the outer membrane ( tom20 ) proved sensitive to trypsin , as did the portion of an inner membrane protein facing the intermembrane space ( tim23 ) after rupturing of the outer membrane ( figure  3b ) , whereas matrix proteins ( tfam and hsp60 ) and primpol were resistant to trypsin in all cases ( figure  3b ) .
hence , the fraction of primpol associated with mitochondria is inferred to be in the matrix .
fractionation of detergent - solubilized mitochondria of hek 293 t cells on an iodixanol gradient ( ig ) separated the majority of primpol and rnase - h1 from mtdna and mtdna maintenance proteins such as twinkle and tfam ( figure  3c ) .
formaldehyde crosslinking increased the amount of primpol and rnase - h1 cofractionating with mtdna but not other mitochondrial proteins , such as vdac1 or etfb ( figure  3d ) .
these data suggest that primpol , like rnase - h1 , interacts with mtdna transiently and that this interaction may be disrupted by the conditions used to lyse mitochondria .
importantly , in the absence of crosslinking , ig sedimentation completely separated primpol from the mitochondrial rna polymerase polrmt ( figure  4a ) , thereby enabling the activities of the two enzymes to be assessed independently . when the different fractions from the igs were subjected to a dna primase assay with the use of oligo dt25 as template , those fractions enriched in mtdna ( 1012 ) yielded a mixed pattern of products ( figure  4a ) that can be attributed to the activities of pol alone or in conjunction with polrmt , terminal transferase(s ) , and poly - a polymerase ; in contrast , fractions enriched in primpol ( 1416 ) displayed a precise pattern of oligonucleotide products of 212 nt ( figure  4a , filled arrowhead , see also the zoomed - in area in figure  4a ) , whose relative abundance correlated with the amount of protein detected by immunoblotting and that could be further extended by klenow dna polymerase ( figure  4a , right ) . to establish
that primpol was responsible for the observed dna primase activity , we subjected mitochondrial fractions from mefs possessing ( + /+ ) or lacking ( / ) the primpol gene ( figure  s4 ) to the dna primase assay . in primpol
mef mitochondria , short dna products of 212 nt were detected in gradient fractions 15 and 16 ( figure  4b ) ; i.e. , the portion of the gradient where human primpol migrates ( figure  4a ) . no such products were detected in mitochondria from primpol cells ( figure  4c ; empty arrowhead , compare the zoomed - in areas in figures 4b and 4c ) .
in addition , selected mitochondrial fractions from hek 293 t cells were tested for the primpol - specific primase activity with oligo d(t20gtcct36 ) as a template , atp , and dgtp ( see the scheme in figure  4d ) .
fraction 15 of the ig ( where primpol is concentrated ) displayed the same initiation specificity and preference for mn as purified primpol , yielding the same products in the same ratios ( figure  4d ) , whereas ig fraction 11 ( containing pol and polrmt ) lacked such an activity . by virtue of its primase activity
, primpol might facilitate dna replication by restarting dna synthesis downstream of pauses or fork arrest due to damaged dna , as has been shown to occur in bacteria , yeast , and human cells ( heller and marians , 2006 ; lopes et  al .
on the other hand , the distributive pattern of dna polymerization and the lack of a proofreading exonuclease suggest that primpol might also perform translesion synthesis ( tls ) in order to facilitate elongation across a damaged template . considering its presence in mitochondria , tls by primpol
was tested on synthetic templates containing either an oxidized form of guanine ( 8-oxoguanine or 8oxog ) or an abasic ( ap ) site , given that these are the most frequent forms of damage occurring in mtdna as a consequence of oxidative stress ( berquist and wilson , 2012 ) . as a control , and as previously documented ( graziewicz et  al . , 2007 ) ,
pol dealt poorly with an 8oxog lesion in the template ( figure  s5a ) .
in contrast , primpol gave rise to similar elongation products with both damaged and undamaged templates ( figure  5a ) , indicating that it was not hindered by the presence of the 8oxog lesion . by using an oligonucleotide whose primer terminus was juxtaposed to the lesion ( standing start conditions ) , the efficiency and fidelity of nucleotide insertion opposite the 8oxog site was studied ( figure  5b ) . in agreement with an efficient tls capacity , the error - free insertion of dc opposite 8oxog by primpol paralleled that opposite an undamaged dg template , and the misinsertion of da opposite 8oxog was slightly more efficient ( 1.5-fold ) .
interestingly , primpol could also use cytidine 5-triphosphate ( ctp ) or atp to bypass 8oxog , although , in this case , the error - free insertion ( figure  5c ) was 10-fold more efficient than the error - prone one ( figure  5a ) .
mitochondrial pol was unable to bypass ap sites ( figure  s5b ) , given that its 3-5 exonuclease favors primer degradation near the site of the lesion .
conversely , human primpol efficiently polymerized across the ap site ( indicated with an x in figure  5c ) , although the accumulation of the  + 3 elongation product indicated some delay at this point .
primpol continued polymerization beyond the ap site in 80% of the molecules , which contrasts with the null bypass observed with pol. interestingly , the size of the longest elongation product ( + 16 ) was 1 nt shorter than that obtained on an equivalent template with no damage ( + 17 ) , suggesting that primpol does not insert a nucleotide opposite the ap site but skips the lesion , copying the next template base available ( figures 5c and 5d ) .
moreover , the size of the main ( + 11 ) and other minor elongated products ( + 14 ) suggested that primer extension was occurring by a different mechanism .
strikingly , the ap site was flanked by a 4 nt ( ctac ) repeat ( gray boxes in figure  5d ) .
given the difficulty to  copy  the ap site , the microhomology provides an opportunity for a partially extended ( + 3 ) primer terminus to be realigned ahead of the lesion .
further elongation of the relocated primer terminus would explain the most abundant  + 11 product obtained ( see figure  5d for additional details ) .
this peculiar capacity of primpol to bypass a lesion in the template probably reflects its features as a primase ( avidity for ssdna regions and flexibility to accept very limited primers [ even a single nucleotide ] ) .
thus , the marked ability of primpol to bypass oxidative stress dna lesions suggests that its dna polymerase activity may have an important role in dna damage tolerance during the replication of mtdna . in a first approach , we measured deoxynucleotide polymerization using circular m13 ssdna as a template , an assay which strictly requires a primase activity in order to allow the start of dna synthesis ( figure  5e ) ; by only providing primpol , some incorporation was observed ( figure  5e , lanes 2 and 5 ) , but incorporation was not observed when pol was independently provided ( figure  5e , lanes 3 and 6 )
. the simultaneous presence of both enzymes had a synergistic effect ( figure  5e , lanes 4 and  7 ) , particularly when a physiological concentration of gtp ( 100  m ) was provided in order to maximize the primase activity of primpol ( figure  5e , lane 7 ) .
moreover , under these optimized conditions ( figure  5f ) , much lower amounts of primpol ( 50  nm ; figure  5e , lane 5 ) or a very low concentration of manganese ions ( 10  m ; figure  5e , lane 9 ) were sufficient to detect the synergistic effect of primpol and pol , strongly suggesting their functional coupling .
very similar results were obtained when the synergy of primpol and pol was evaluated ( figure  5 g ) .
these experiments demonstrate that , during restart of dna synthesis , the dna primers made by primpol ( and most likely its tls products ) can be efficiently extended by the mitochondrial and nuclear replicative polymerases pol and pol , respectively ( figure  5h ) .
the presence of primpol in mitochondria suggested that it might play a role in mtdna replication and maintenance ; therefore , we investigated the effect of gene - silencing primpol on mtdna metabolism .
primpol small interfering rna ( sirna ) in human cells caused mtdna levels to decrease to half those of controls ( figure  6a ) , similar to twinkle sirna ( tyynismaa et  al . ,
2004 ) , and prevented the recovery of mtdna copy number after transient drug - induced mtdna depletion ( figure  6b ) .
both results suggest that mtdna replication is impaired when there is an abrupt loss of primpol .
we also analyzed mtdna replication intermediates , given that the perturbation of dna replication in mitochondria is typically associated with changes in their abundance and properties ( holt et  al .
although rna - containing intermediates ( yasukawa et  al . , 2006 ) increased after 48  hr of primpol sirna , they returned to normal levels at later time points ( figures s6a and s6b ) . because mtdna copy number was decreasing between 48 and 96  hr of primpol gene silencing , we inferred that replication was not progressing normally .
to determine the extent of mtdna replication after 72  hr of primpol gene silencing , we analyzed brdu incorporation into mtdna in double - stranded rna ( dsrna)-transfected cells after preincubation with aphidicolin , an inhibitor of nuclear dna replication .
almost all the control cells ( 96% ) incorporated brdu into mtdna after 24  hr of labeling , whereas the figure was 13% for primpol - depleted cells ( p  < 0.0001 ) ( figure  6c ) .
hence , primpol gene silencing in human cultured cells caused a profound arrest of mtdna synthesis . notwithstanding these results ,
the contribution of primpol to mtdna maintenance is redundant , given that it proved possible to derive a viable primpol mouse ( figure  s4 ) . although the mouse has adapted to the absence of primpol , mtdna replication was adversely affected in cells derived from the primpol ko mouse , given that the rate of restoring mtdna copy number after drug - induced transient depletion was 2.3-fold lower than in mouse cells with primpol ( figure  6d ) .
human ccdc111 ( or primpol ) possesses dna and rna primase and dna - dependent dna and rna polymerase activities ( figures 1 , 2 , s2 , and s3 ) , demonstrating its functional relationship to archeal primpol enzymes and its role as a unique human enzyme capable of de novo dna synthesis solely with dntps .
another remarkable feature of primpol is its capacity to tolerate lesions such as 8oxog and ap sites in dna .
some primpol is present in the nucleus , but a larger fraction is located inside mitochondria .
primpol gene silencing in human cultured cells has multiple adverse affects on mtdna , and mouse cells lacking primpol display inefficient mtdna replication .
however , given that a mouse lacking the gene is viable , primpol is not essential for mtdna maintenance .
polrmt , and not primpol , is believed to provide the primers for leading - strand mtdna synthesis at the heavy strand origin of replication in the major noncoding region and at the major site of second strand mtdna synthesis known as oril ( falkenberg et  al . , 2007 ) , and
this is compatible with primpol being a nonessential gene . instead , primpol might participate in priming at disparate sites when mtdna replication pauses or stalls due to damaged dna . in respect of damaged dna , primpol , as a tls dna polymerase ( figure  7b ) , could assist pol in  reading  template lesions ( e.g. , 8oxog ) , and it has the potential to bypass unreadable lesions ( e.g. , ap sites ) by realigning the stalled primer terminus to an alternative template position ahead of the lesion ( figure  7c ) . alternatively or additionally , primpol could exploit both its dna primase and dna polymerase activities in order to facilitate replication fork progression by acting as a specific tls primase in order to reinitiate replication at pause sites or downstream of lesions that block continuous synthesis by replicative dna polymerases ( figure  7d ) .
primpol would be very well suited for this task , given that using deoxynucleotides for priming would leave little or no rna to be further processed .
hence , the properties and behavior of primpol fit well with the proposed roles in mtdna metabolism .
finally , primpol might contribute to multipriming events ( figure  7a ) occurring on the lagging strand during coupled leading- and lagging - strand mtdna replication ( holt et  al .
it has only recently been recognized that leading - strand dna synthesis is not always continuous .
genotoxic damage has been linked to discontinuities in leading - strand synthesis that entail skipping the obstacle , resuming fork progression , and leaving a gap behind ( langston and odonnell , 2006 ; lopes et  al . , 2006 ; heller and marians , 2006 ; yeeles and marians , 2011 ) .
daughter - strand gap formation after dna damage induction has been detected in yeast and mammals , suggesting that repriming is a universal mechanism for bypassing dna lesions ( meneghini , 1976 ; prakash , 1981 ) . in this regard , we believe that primpol 's primase may play a direct role in replication restart during nuclear dna replication , further supported by the functional coupling shown here between primpol and human pol. we have recently found that primpol is required for repriming synthesis at stalled replication forks in the nucleus and its downregulation in cells produced persistent replication stress and  genome instability ( s.m
. , s. rodrguez - acebes , m.i.m .- j . , s.g .- g . , e.s.c . , l.b . , and j.m .
these findings give additional support to our hypothesis of a similar function for primpol in mitochondria , given that replicative stress is constitutive during mtdna replication , most likely due to a highly oxidative environment . in summary , we identify and characterize a dna primase / polymerase  in human cells endowed with the unique ability to initiate synthesis using dntps and a remarkable capacity for translesion synthesis .
our initial analysis of its function in mitochondria suggests that primpol plays a key role in dna metabolism , pointing to a greater level of complexity in mtdna replication than previously recognized and implying that pol is not the sole dna polymerase in mammalian mitochondria .
the presence of primpol in human mitochondria suggests that it represents an ancestral cellular response to genotoxic insults , including ap sites and oxidized bases , contributing to tolerance of the environmental damage inside mitochondria .
now , the challenge is to tease out its precise contribution to dna damage tolerance in the two dna - containing compartments of the cell .
the generation of primpol mice is described in detail in the supplemental information . all experiments with mice
were performed according to spanish and european regulations for the use and treatment of experimental animals and with the approval of the ethics committee of the centro de biologa molecular severo ochoa . the reaction ( 20  l ) was carried out in buffer r ( 50  mm tris - hcl [ ph 7.5 ] , 75  mm nacl , 1  mm mncl2 , 1  mm dithiothreitol , 2.5% glycerol , and 0.1mg / ml bsa ) and [ -p]ctp ( 16  nm ; 3,000 ci / mmol )  + atp , gtp , and utp ( 100  m ) or [ -p]dctp ( 16  nm ; 3,000 ci / mmol )  + datp , dgtp , and dttp ( 100  m ) in the presence of primpol ( 400  nm ) wild - type ( wt ) or mutant axa .
various concentrations of mgcl2 or mncl2 were used to define the range of metal activation .
after 60  min at 30c , reactions were stopped by the addition of formamide loading buffer ( 10  mm edta , 95% v / v formamide , and 0.3% w / v xylen - cyanol ) and loaded in 8  m urea - containing 20% polyacrylamide sequencing gels .
after electrophoresis , de novo synthesized polynucleotides were detected by autoradiography . to test the productive coupling of primpol with pol and pol ,
a similar assay was conducted in the presence of both mgcl2 and mncl2 along with 16  nm [ -p]dgtp and 100  m da , dt , and dctp . when indicated , the nucleotide mix was supplemented with 1  m dgtp or 100  m gtp .
human pol was kindly provided by zachary f. pursell ( tulane university school of medicine ) .
after incubation for 30  min at 37c , de novo synthesized products ( priming  + elongation ) , associated to the m13 ssdna template , were detected by autoradiography after electrophoresis on neutral agarose gels . as an alternative to m13 dna , 60-mer oligonucleotide 5-t36cctgt20 - 3 or a 29-mer 5-t15cctgt10 - 3 ,
both containing a putative herpes virus priming initiation site ( cavanaugh and kuchta , 2009 ) , were used as templates .
p]-labeled oligonucleotide primer ( 5-gttttcccagtcacgac-3 ) was hybridized to m13 ssdna . the reaction mixture ( 20  l ) contained buffer r , 1  mm mncl2 or 5  mm mgcl2 , 1.5  nm primed m13 ssdna , 1  m dntp or ntps , and 400  nm purified primpol , wt , or axa mutant . after the indicated incubation time at 30c , reactions were stopped and analyzed as described above .
when indicated , alternative templates were prepared by hybridization of a 5[p]-labeled 15-mer oligonucleotide primer ( 5-gatcacagtgagtac-3 ) to a 28-mer oligonucleotide template ( 5-agaagtgtatct(x)gtactcactgtgatc-3 where x stands for any of the four bases ) . the reaction mixture
( 20  l ) contained buffer r , 2.5  nm [ -p]-labeled template / primer dna , the specified dntp at the concentration indicated , and purified human primpol ( 200  nm ) .
when indicated , primpol was used at different concentrations . also when indicated , various concentrations of either mgcl2 or mncl2 were used to define the range of metal activation .
after 60  min of incubation at 30c , reactions were stopped and analyzed as described above .
elongation of a 3 terminally mismatched primer ( 16-mer , 5-gatcacagtgagtacc-3 ) hybridized to a 22-mer template ( 5-tctatcgtactcactgtgatc-3 ) was evaluated under the same conditions . for
running start  experiments , a 5 [ p]-labeled 12-mer oligonucleotide ( 5-gatcacagtgag-3 ) was hybridized to either the control ( undamaged ) 28-mer oligonucleotide ( 5-agaagtgtatctggtactcactgtgatc-3 ) template or an oligonucleotide template of the same sequence with the g in 13 position ( bold ) substituted with 8oxog ( obtained from eurogentec ) . for  standing start  experiments designed to evaluate the nucleotide inserted opposite 8oxog , a 5[p]-labeled 15-mer oligonucleotide ( 5-ctgcagctgatgcgc-3 ) was hybridized to either the control ( undamaged ) 34-mer oligonucleotide ( 5-gtacccggggatccgtacggcgcatcagctgcag-3 ) template or an oligonucleotide template of the same sequence with the g in 19 position ( bold ) substituted with 8oxog ( eurogentec ) .
alternatively , a 5 [ p]-labeled 13-mer oligonucleotide primer ( 5-cactgactgtatg-3 ) was hybridized to either control ( undamaged ) 30-mer oligonucleotide template ( 5-ctcgtcagcatcttcatcatacagtcagtg-3 ) or damaged templates ( 30- or 31-mer ) containing an ap site ( 5-ctcgtcagcatct(ap)catcatacagtcagtg-3 ) .
the synthetic ap nucleotide was obtained from linktech and used to customize oligonucleotide synthesis . the reaction mixture ( 20  l ) contained buffer r , 2.5  nm template / primer dna , dntps or ntps at the indicated concentration , and either purified human primpol ( 200  nm ) or human pol ( 20  nm , kindly provided by maria falkenberg s lab ) . in the latter case
after incubation for either 60  min at 30c ( primpol ) or 15  min at 37c ( pol ) , primer extension products were analyzed as described above .
hela cells were lysed and fractionated with a mitosciences cell fractionation kit ( ab109719 ) , which allowed the isolation of mitochondrial , cytoplasmic , and nuclear fractions .
two different markers were used to assess the purity of each fraction : cytoplasm ( mek2 and -tubulin ) , mitochondria ( atp synthase and hsp60 ) , and nucleus ( ctcf and histone h3 ) .
subcellular fractionation of hek 293 t cells was performed as described previously ( reyes et  al . , 2011 ) , and
sucrose - gradient purified mitochondria were left untreated or subjected to hypotonic or digitonin treatment ( ohsato et  al . , 2002 ) followed by incubation with 100  g / ml trypsin ( 30  min at room temperature [ rt ] ) and heat inactivation of trypsin at 95c for 5  min in 0.1% sds . for ig ,
sucrose - gradient - purified mitochondria ( 2  mg / ml ) were treated with 100  g / ml of trypsin at rt for 30  min .
n - dodecyl -  - maltoside and centrifuged ( 10  min at 1,000   gmax ) ; the supernatant was loaded on a 20%42.5% ig and centrifuged at 100,000   gmax for 14  hr . in some cases
, mitochondria were crosslinked with 1% formaldehyde for 30  min at rt prior to lysis .
when indicated , hek 293 t cells were treated with 100  ng / ml ethidium bromide for 72  hr or exposed to uv light ( 20 j / m ) and allowed to recover for 18  hr prior to mitochondrial extraction and further ig fractionation .
nucleic acid was extracted from a portion of each fraction of the gradient and after southern blotting and hybridized to a radiolabeled probe .
a complete list of the antibodies used in this study can be found in the supplemental information . for sirna experiments ,
hek 293 t and human osteosarcoma ( hos ) cells were transfected with lipofectamine rnaimax ( invitrogen ) and 10  nm stealth rna as described previously ( reyes et  al . ,
mitochondrial dna copy number was estimated by comparing the abundance of the mitochondrial cox2 gene to that of the nuclear app gene , as described previously ( tyynismaa et  al . , 2004 ) .
parental or primpol - silenced hos cells were incubated with 20  m aphidicolin for 3  hr in order to block nuclear dna replication before the addition of brdu ( 1:1,000 ; roche ) . after 24  hr
, brdu - labeled cells were stained with 60  nm mitotracker orange ( invitrogen ) , fixed , and sequentially incubated with anti - brdu and anti - mouse igg fluorescein according to the manufacturer s indications ( roche ) .
slides were mounted in dabco ( air products and chemicals ) supplemented with dapi ( 200  ng / ml ) and visualized in a zeiss lsm 510 confocal microscope .
 summarywe describe a second primase in human cells , primpol , which has the ability to start dna chains with deoxynucleotides unlike regular primases , which use exclusively ribonucleotides . moreover , primpol is also a dna polymerase tailored to bypass the most common oxidative lesions in dna , such as abasic sites and 8-oxoguanine . 
 subcellular fractionation and immunodetection studies indicated that primpol is present in both nuclear and mitochondrial dna compartments . 
 primpol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from primpol knockout mice . 
 primpol gene silencing or ablation in human and mouse cells impaired mitochondrial dna replication . 
 on the basis of the synergy observed with replicative dna polymerases pol and pol , primpol is proposed to facilitate replication fork progression by acting as a translesion dna polymerase or as a specific dna primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear dna replication . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions