Patent Application: US-94564598-A

Abstract:
fragments of fen1 that interact with pcna are disclosed , together with the use of such fragments or mimetics of fen1 in methods of screening for compounds useful in treating disorders in which pcna is implicated . in particular , substances which have the property of binding to pcna are disclosed , said substances comprising : a fragment of the fen1 protein containing a peptide of 89 amino acids from the c - terminal region or an active portion thereof ; or , a fragment of the fen1 protein containing the sequence motif qgrldxff ; or , a functional mimetic of said protein fragments ; where “ x ” is preferably the selected form the amino acids s , d or g .

Description:
the two hybrid screen and deletion analysis was carried out essentially as described previously ( warbrick et al ., 1995 , and international patent application pct / gb95 / 02583 ). further constructs were made by subcloning fragments from the human pcna open reading frame into pas2 . twenty amino acid peptides with 15 amino acid overlaps were synthesized ( chiron mimotopes , australia ) across the 89 amino acids from the c - terminal region of fen1 identified in the two hybrid screen as the pcna binding site on fen1 . these peptides were linked via residues sgsg ( seq id no : 3 ) at the amino acid terminus to biotin . 2 . 7 μg of each peptide was incubated with 10 μl streptavidin agarose beads ( sigma ) in phosphate buffered saline ( pbs ) for 1 hour at room temperature ( r . t .) then beads were washed extensively in pbs and recovered each time by centrifugation at 11 , 600 g . hela cell lysate was added to the washed beads to a final protein concentration of 1 mg / ml and incubated with the beads on ice for 1 hour . beads were extensively washed in pbs with 220 mm nacl , then bound proteins removed by boiling for 8 minutes in sds loading buffer with 0 . 2 m dtt . proteins were separated on 10 % sds - page then electrophoretically transferred to nitrocellulose . blots were blocked then incubated for 1 hour r . t . with pc 10 hybridoma supernatant ( waseem & amp ; lane , 1990 ) diluted 1 : 5 in dmem with 10 % fcs . after washing , secondary hrp - conjugated anti - mouse igg serum at 1 : 1000 in 2 % non - fat milk - pbs - 0 . 2 % tween was added for 1 hour at r . t . bound antibody was visualised using the ecl system according to the manufacturer &# 39 ; s instructions ( amersham plc .) elisa plates were coated overnight at 37 ° c . with 100 ng of streptavidin per well . plates were blocked with 5 % non - fat milk powder in pbs , washed extensively with pbs containing 0 . 2 % tween 20 ( pbs - t ) then 100 μl fenpbp — peptide 84 ( seq id no : 4 ) ( see fig2 )— at 10 μg / ml was added to each well . following 1 hour incubation at r . t . in a humid chamber , wells were washed extensively . approximately 3 . 5 μg total protein containing overexpressed recombinant human pcna was mixed with varying concentrations of a 9 amino acid peptide derived from p21pbp ( warbrick et al , 1995 ), of the sequence qtsmtdfyh ( seq id no : 2 ) ( purified by hplc and verified by mass spectrometry ) and applied to the wells for 1 hour at r . t . after extensive washing , anti - pcna polyclonal antibody 3009 ( cox et al ., manuscript in preparation ) was added at 1 : 1000 , followed by hrp - conjugated anti - rabbit igg serum at 1 : 1000 , and bound antibody was visualized by the tmb colormetric procedure ( harlow & amp ; lane , 1998 ). plated were read at 450 nm using a dynatech 5000 plate reader . the 89 amino acids from the c - terminal region of fen1 bind pcna in an interaction trap proteins that interact physically with human pcna were screened using a two hybrid interaction trap system that detects protein - protein interactions by reconstitution of a functional gal4 transcriptional activator in the yeast saccharomyces cerevisiae . a plasmid expressing human pcna in fusion with the dna binding domain of gal4 ( gal4as ) was used to screen plasmids expressing fusion constructs of molecules encoded by fragments from a human cdna library with the transcriptional activation domain of gal4 ( gal4act ). on dna sequencing , one class of clones isolated by this screen was found to encode fragments of the human protein fen1 ( harrington & amp ; lieber , 1994a ; harrington & amp ; lieber , 1994b ), also known as dnase iv ( robins et al ., 1994 ), rad2hs ( murray et al ., 1994 ) and mf1 ( waga et al ., 1994a ). the minimal fen1 fragment isolated in our screen ( fig1 ) encoded the 89 amino acids from the c - terminal region of the protein ( full length 380 aa , ( murray et al ., 1994 )), therefore localising the pcna binding site to these residues . pcna from drosophila melanogaster and schizosaccharomyces pombe also interacted in this system with human fen1 , suggesting strong evolutionary conservation of the interaction site on pcna . peptide mapping of sites on fen1 important for interaction with pcna to map precisely the residues of fen1 to which pcna binds , a series of biotinylated 20 amino acids peptides ( with 15 amino acid overlaps ) were synthesized representing the region of fen1 found to interact with pcna in the two hybrid screen ( fig1 ). fen1 peptides ( bound to streptavidin agarose beads ) were incubated with human hela cell extracts , then beads were precipitated and bound protein analysed on immunoblots probed with monoclonal anti - pcna antibody pc10 ( waseen & amp ; lane , 1990 ). the results showed that pcna was precipitated from hela cell extract with equal efficiency by peptides 84 ( seq id no : 4 ), 85 ( seq id no : 5 ), and 86 ( seq id no : 6 ) ( see fig2 ), strongly suggesting that the amino acids tqgrlddffk ( seq id no : 10 ) common to these three fen1 peptides are important for binding to pcna . comparison of the amount of pcna present in the undepleted extract and that precipitated by fen1 peptides 84 - 86 ( seq id nos : 4 , 5 , and 6 , respectively ) demonstrated quantitative removal of pcna protein from the whole cell lysate , indicating a high affinity of interaction between pcna and the fen1 peptides . the region of human fen1 recognised by pcna in the precipitation assay contains a motif highly conserved in the fen1 family . we examined whether human pcna could bind to his motif in the yeast homologues of fen1 , s . pombe rad2 and s . pombe rad27 / ykl510 . biotinylated peptides were synthesized according to the published sequences of s . pombe rad 2 ( murray et al ., 1994 ) and s . cerevisiae rad27 / ykl510 ( jacquier et al ., 1992 ) in the region homologues to that portion of fen1 shown to interact with pcna . these peptides 92 ( seq id no : 7 ) and 93 ( seq id no : 8 ) ( see fig2 ) were used in a precipitation reaction as described above . the pc10 immunoblot of proteins precipitated from hela cell extract by 20 amino acid peptides of this conserved box from s . pombe ( peptide 92 ) ( seq id no : 7 ) and s . cerevisiae ( peptide 93 ) ( seq id no : 8 ) showed that pcna binds to the conserved region in rad2 and rad27 with similar affinity to fen1 peptides 84 - 86 ( seq id nos : 4 , 5 and 6 , respectively ), demonstrating remarkable conservation of the binding motif . binding of pcna to the previously defined p21 cip1 - pcna binding peptide ( p21pbp — warbrick , 1995 ) was used as a positive control and streptavidin - agarose beads alone as the negative control . by comparison of the sequences of these pcna - interacting peptides from diverse species ( fig2 ), we can define a pcna binding site of qgrldxff ( seq id no : 1 ). fen1 is most closely homologous to rad2 of s . pombe and rad27 / ykl510 of s . cerevisiae , but it shares limited homology also with putative nucleotide excision repair - associated proteins rad13 ( s . pombe ), rad2 ( s . cerevisiae ) and , provocatively , the human xeroderma pigmentosum complementation group g protein . alignment of these sequences reveals similarity in the region that we find important for fen1 to bind to pcna , defining a novel pcna binding motif . remarkably , the residues of p21 cip1 that we have previously found to be critical for binding to pcna are very similar to those required by members of the fen1 family for pcna interaction . to define the region of pcna important for fen1 binding , we created a series of deletion constructs of pcna fused to gal4as and examined their ability to interact with fen1 plasmids in the two hybrid system . the results ( fig3 ) show that fen1 binds to a region between residues 100 and 150 of pcna . this region corresponds to the central loop of pcna between the two domains of each monomer , and includes also the structural motifs βi1 , βa2 and αa2 ( from the crystal structure of s . cerevisiae pcna , ( krishna et al ., 1994 )). remarkably , this region has also been implicated in the binding of the cyclin - kinase inhibitor p21 cip1 to pcna ( warbrick et al ., 1995 ) using n - terminal deletions of pcna . thus , we confirmed and extended the earlier observations of p21 cip1 - pcna interaction in the two hybrid system using the new series of deletion constructs ( fig4 ), and demonstrate that fen1 and p21 cip1 bind to the same region of pcna . since the region of pcna bound by fen1 is the same as that recognised by p21 cip1 , and the motif of fen1 required for pcna interaction appears homologous to the critical residues of p21pbp ( warbrick et al ., 1995 ), it is highly possible that fen1 and p21 cip1 might compete for binding to pcna . we therefore tested this hypothesis using pcna - interacting peptide 84 ( seq id no : 4 ) of fen1 ( dubbed fen1pbp for fen1 - pcna binding peptide ) immobilised in elisa wells . recombinant human pcna was added , together with increasing amounts of a non - biotinylated 9 - mer peptide of p21 cip1 containing the critical amino acids for pcna interaction qtsmtdfyh ( seq id no : 2 ), as defined by warbrick , et al . ( 1995 ). this short peptide of p21 cip1 clearly competes for pcna binding to fen1pbp ( fig4 ), with total displacement of pcna from immobilised fen1pbp at only 1 mm added p21 cip1 peptide . an irrelevant 20 - mer peptide was used to control from non - specific effects of peptides and the solvent dmso on pcna binding to fen1pbp . in this application , we disclose a direct interaction between two proteins implicated in dna replication and dna repair ; the flap endonuclease fen1 ( harrington & amp ; lieber , 1994a ; harrington & amp ; lieber , 1994b ) [ also known as dnase iv ( robins et al ., 1994 ), rad2hs ( murray et al ., 1994 ) and mf1 ( waga et al ., 1994 )] and the dna polymerase δ auxiliary sliding clamp , pcna ( bravo et al ., 1987 ; prelich et al ., 1987b ). deletion mapping in the two hybrid system permits us to define 89 amino acids of fen1 as containing the pcna binding site . analysis using peptides synthesized according to the shortest fen1 fragment isolated in the two hybrid screen then allowed us to map the pcna binding site to three overlapping fen1 - pcna binding peptides ( fen1 pbps ). comparison of sequence overlaps in these fen1 peptides , and those derived from fen1 homologues in s . cerevisiae ( ykl5 1i0 / rad27 ) and s . pombe ( rad2 ) defines the pcna binding region as 8 amino acids : qgrldxff ( seq id no : 1 ). this motif is partially conserved in another family of proteins probably required for nucleotide excision repair that share limited homology with fen1 , including the s . pombe rad13 gene product , s . cerevisiae rad2 and the human xeroderma pigmentosum xp - g protein . remarkably , residues critical for pcna binding are shared also with the region of the cyclin - kinase inhibitor , p21 cip1 , required for interaction with pcna ( warbrick et al ., 1995 ). accordingly , this motif may define a novel pcna binding consensus . a series of amino terminal and carboxy terminal deletions of pcna localised the fen1 binding site to the region between amino acids 100 - 150 , which include residues exposed in a loop between the two domains of each pcna monomer . although our data do not yet permit us to define the fen1 binding site at the level of individual amino acids , it is possible that interaction involves those residues of pcna most exposed in solution , ie those contained within the 17 amino acids of the loop . the interaction is conserved across evolution not only for fen1 homologues , but also for pcna , since pcna from d . melanogaster and s . pombe also bind to fragments of human fen1 in the two hybrid system . such a level of conservation suggests that this interaction may be critical for cellular dna replication and / or repair . we have already shown that this central loop of pcna may provide a binding site for p21 cip1 . since fen1 and p21 cip1 share amino acids essential for pcna binding , and appear to bind to the same site on pcna , they should competitively modulate pcna function , suggesting uses for these proteins in the treatment of cancer and other hyperproliferative disorders . pcna is fundamental to nuclear dna replication , acting as a sliding clamp that tethers the dna replication machinery to its template , enhancing the enzyme &# 39 ; s processivity . in addition , pcna is required for nucleotide excision repair ( shivji et al ., 1992 ). pcna may be recruited to these different roles in replication and repair by interaction with proteins specific to each process . examination of pcna &# 39 ; s partners is therefore a critical goal in the understanding of both dna repair and dna synthesis . this work demonstrates an interaction of pcna with fen1 , a structure - specific endonuclease . the demonstration of a direct pcna - fen1 interaction begs the question of the biological function and activity of such a complex . two hypotheses are consistent with our data : ( 1 ) that the pcna - fen1 complex functions in dna replication ; ( 2 ) that the complex is active in dna repair . does the known activity of fen1 support either hypothesis ? fen1 protein has been suggested to function in mammalian nucleotide excision repair , because of its homology to known repair gene products . it is a structure - specific flap endonuclease identified as the human homologue of the s . pombe rad2 gene product ( rad2hs , ( murray et al ., 1994 ), dnase iv , ( robins et al ., 1994 )). it specifically cuts dna flaps ending in a 5 ′ single stranded region , requiring the entire flap structure , but acting independently of flap length ( harrington & amp ; lieber , 1994a ). in addition , fen1 possesses 5 ′→ 3 ′ exonuclease activity specific for double stranded dna ( harrington & amp ; lieber , 1994a ). human and mouse fen1 share homology with structure - specific endonucleases ykl510 ( rad27 ) and rad2 from s . cerevisiae ( harrington & amp ; lieber , 1994b ), and human xeroderma pigmentosum complementation group g protein ( murray et al ., 1994 ; o &# 39 ; donovan et al ., 1994 ) also known as ercc5 , ( shiomi et al ., 1994 ). two uv - inducible upstream elements are present in the promoter of the s . cerevisiae rad2 gene , that show increased protein binding following genotoxic insult , characteristic of a dna repair gene ( siede & amp ; friedberg , 1992 ). interestingly , these two elements dre1 and dre2 may be differentially regulated through the cell cycle , since deletion of dre1 results in increased uv sensitivity only in g1 / s , while deletion of dre2 leads to greater sensitivity in g1 / s , s / g2 and in stationary phase ( siede & amp ; friedberg , 1992 ). structural and functional homology between fen1 and characterised repair proteins therefore supports the suggestion that fen1 is involved in mammalian nucleotide excision repair . several lines of evidence , however , point towards an additional role for fen1 in dna replication . it is essential for the maturation of replicated sv40 dna into covalently closed circles ( form i ), and possesses rnase h activity that may remove ribonucleotide primers from okazaki fragments ( waga et al ., 1994a ). the bovine homologue of fen1 has been detected in partially purified fractions of dna polymerase ε , and is required for complete dna synthesis on a synthetic dna substrate in a lagging strand reaction ( siegal et al ., 1992 ; turchii & amp ; bambara , 1993 ). although identified as radiation - sensitive mutations , the s . pombe and s . cerevisiae homologues of fen1 show mutant phenotypes consistent with a fault in dna replication ( eg . reagan et al ., 1995 ). provocatively , the upstream region of the s . cerevisiae rad27 gene contains two mlui boxes characteristic of genes whose products are required in s phase , and its transcript reaches maximal levels in g1 / s . these features of fen1 and its homologues are highly consistent with a role in s phase dna replication . two alternate roles of the protein in dna replication and repair are not incompatible , since pcna , for instance , functions in both processes . competition between fen1 and p21 cip1 for pcna binding may modulate function of the clamp protein , and though we do not wish to be bound by a particular theory , it is provocative to consider the idea that p21 cip1 binding to pcna may shift it from a replication competent form ( pcna - fen1 ) to a repair - competent form that cannot support dna replication . such an alteration in pcna function might provide a direct link between the cessation of dna replication following dna damage , and the onset of dna repair . consistent with this hypothesis , p21 cip1 is transcriptionally induced by the human tumour suppressor protein p53 ( el - deiry et al ., 1993 ) which is itself induced in response to genomic damage ( reviewed by cox & amp ; lane , 1995 ). coupled with higher levels of p21 cip1 following genomic damage , higher affinity of p21 cip1 than fen1 for pcna might well result in total displacement of fen1 from the complex . the results presented here have applications beyond the biological role of pcna and fen1 in dna replication and repair complexes . pcna is elevated in certain human tumours above the level of other cell cycle markers such as ki67 ( eg . knechel et al ., 1993 ), and we have already described a peptide of p21 cip1 , p21pbp , that may be useful in inhibiting pcna &# 39 ; s replication activity in hyperproliferating cells . development of new drugs that mimic the pcna - inhibitory activity of p21 cip1 requires a suitable screening procedure . thus , the work described herein in which a minimal peptide derived from p21pbp successfully competes with fen1pbp for binding to pcna , can be readily extended to provide a rapid and reproducible screen for small molecules that mimic p21pbp activity in pcna binding and possibly inhibition of dna synthesis . drugs defined by such a screen should prove useful for treatment of hyperproliferative diseases including cancer . bravo , r ., frank , r ., blundell , p . a . & amp ; macdonald - bravo , h . 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( 1994 ). differential effects by the p21 cdk inhibitor on pcna - dependent dna replication and repair . nature 371 , 534 - 537 . murray , j . m ., tarassoli , m ., al - harithy , r ., sheldrick , k . s ., lehman , a . r ., carr , a . m . & amp ; watts , f . z . ( 1994 ). structural and functional conservation of the human homologue of the schizosaccharomyces pombe rad2 gene , which is required for chromosome segregation and recovery from dna damage . mol . cell biol . 14 , 4878 - 4888 . o &# 39 ; donovan , a ., davies , a . a ., moggs , j . g ., west , s . c . & amp ; wood , r . d . ( 1994 ). xpg endonuclease makes the 3 ′ incision in human dna nucleotide excision repair . nature 371 , 432 - 435 . prelich , g ., kostura , m ., marshak , d . r ., mathews , m . b . & amp ; stillman , b . ( 1987a ). the cell - cycle regulated proliferating cell nuclear antigen is required for sv40 dna replication in vitro . nature 326 , 471 - 475 . prelich , g ., tan , c . k ., kostura , m ., mathews , m . b ., so , a . g ., downey , k . m . & amp ; stillman , b . ( 1987b ). functional identity of proliferating cell nuclear antigen and a dna polymerase delta auxiliary protein . nature 326 , 517 - 520 . reagan , m . s ., pittenger , c ., siede , w . & amp ; friedberg , e . c . ( 1995 ). characterisation of a mutant strain of saccharomyces cerevisiae with a deletion of the rad27 gene , a structural homolog of the rad2 nucleotide excision repair gene . j . bact . 177 , 364 - 371 . robins , p ., pappin , d . j ., wood , r . d . & amp ; lindahl , t . ( 1994 ). structural and functional homology between mammalian dnase iv and the 5 ′- nuclease domain of escherichia coli dna polymerase i . j . biol . chem . 269 , 28535 - 28538 . shiomi , t ., harada , y ., saito , n ., okuno , y . & amp ; yamaizumi , m . ( 1994 ). an ercc5 gene with homology to yeast rad2 is involved in group g xeroderma pigmentosum . mutat . res . 314 , 167 - 175 . shivji , m . k . k ., grey , s . j ., strausfeld , u . p ., wood , r . d . & amp ; blow , j . j . ( 1994 ). cip1 inhibits dna replication but not pcna - dependent nucleotide excision repair . curr . biol . 4 , 1062 - 1068 . shivji , m . k . k ., kenny , m . k . & amp ; wood , r . d . ( 1992 ). proliferating cell nuclear antigen is required for dna excision repair . cell 69 , 367 - 374 . siede , w . & amp ; friedberg , e . c . ( 1992 ). regulation of the yeast rad2 gene : dna damage - dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival . mol . gen . genet . 232 , 247 - 256 . siegal , g ., turchii , j . j ., myers , t . w . & amp ; bambara , r . a . ( 1992 ). a 5 ′ to 3 ′ exonuclease functionally interacts with calf polymerase e . proc . natl . acad . sci . 89 , 9377 - 9381 . turchii , j . j . & amp ; bambara , r . a . ( 1993 ). completion of mammlian lagging strand dna replciation using purified proteins . j . biol . chem . 268 , 15136 - 15141 . waga , s ., bauer , g . & amp ; stillman , b . ( 1994a ). reconstitution of complete sv40 dna replication with purified replication factors . j . biol . chem . 269 , 10923 - 10934 . waga , s ., hannon , g . j ., beach , d . & amp ; stillman , b . ( 1994b ). the p21 inhibitor of cyclin - dependent kinases controls dna replication by interaction with pcna [ see comments ]. nature 369 , 574 - 8 . warbrick , e ., lane , d . p ., glover , d . m . & amp ; cox , l . s . ( 1995 ). a small peptide inhibitor of dna replication defines the site of interaction between the cyclin - kinase inhibitor p21waf1 and proliferating cell nuclear antigen . current biology 5 , 275 - 282 . waseem , n ., labib , k ., nurse , p . & amp ; lane , d . p . ( 1992 ). isolation and analysis of the fission yeast gene encoding polymerase delta accessory protein pcna . embo j . 11 , 5111 - 5120 . waseem , n . h . & amp ; lane , d . p . ( 1990 ). monoclonal antibody analysis of the proliferating cell nuclear antigen ( pcna ). structural conservation and the detection of a nucleolar form . j . cell sci . 96 , 121 - 129 . ala ser lys thr ile pro gln gly arg leu asp ser phe phe lys pro