inactivation of the mouse polh gene was performed by a conditional knock - out strategy that introduces loxp sites flanking exon 4 , an exon containing the de polymerase motifs conserved in all y - family polymerases and indispensable for pol catalytic function ( fig . 1 and references 15 , 24 ) .
this strategy was designed with the aim of crossing the animals obtained with mice harboring other dna repair defects , allowing the follow - up of the b cell lineage in case of lethality of their combined deficiencies .
deletion of exon 4 was performed here in the targeted embryonic stem ( es ) cell clone , leaving in place a single loxp site and , therefore , minimizing disturbance around the gene .
the xpo5 gene , encoding exportin-5 involved in the nuclear export of micrornas , indeed initiates 200 bp upstream from the first polh exon in opposite transcriptional orientation ; affecting expression of this gene is , therefore , likely to result in an early embryonic lethal phenotype ( 25 ) .
( a ) scheme of the mouse polh gene encoding dna polymerase . ( closed boxes ) coding exons ; ( open boxes ) noncoding regions .
homologous recombination of the gene - targeting construct results in the introduction of loxp sites on both sides of exon 4 .
( c ) screening of mouse genotypes : deletion of exon 4 is monitored by a pcr around this exon , using primers shown in b. ( d ) pol gene expression in control and gene - targeted mice : amplification of pol transcripts is performed using primers in exons 2 and 6 , on twofold consecutive dilutions of rt products from total spleen rna .
the amplified product in pol animals corresponds to the splicing of exons 35 , which generates an out - of - frame sequence .
the heterozygotes obtained by breeding chimeras with c57bl/6 mice were crossed together and screened for the presence of the stop codon mutation born by the poli gene from the 129/ola genetic background of the es cells .
wild - type , homozygous pol-deficient , and double pol-pol mutant mice were selected for analysis .
deletion of exon 4 of pol resulted at the transcriptional level in the direct splicing of exons 35 , an out - of - frame junction that introduces a stop codon 11 amino acids downstream in exon 5 and generates a truncated pol protein of 102 amino acids ( fig .
1 d ) . apart from deletion of exon 4 , such a truncated protein would lack domains involved in nuclear localization and repair foci formation ( 19 ) .
wild - type , pol-deficient , and double pol-pol mutant mice were analyzed for the mutation pattern of their ig locus .
different types of sequences were analyzed : intronic sequences flanking rearranged jh4 sequences (  jh4 intronic sequences  ) and a 560-bp region upstream from the repeats of the s core sequence ( hereafter referred to as  pre - switch  ) , both from peyer 's patch pna b cells ( table i ) .
somatic mutations in jh4 intronic sequences and s core upstream sequences from normal and mutant mice no b cell anomalies were observed in either type of polymerase - deficient animals .
ig sequences from pol pna b cells displayed the same specific pattern as those obtained from memory b cells of xp - v patients ; i.e. , a marked reduction of mutations at a / t base pairs : 86.1% mutations at g / c base pairs for the jh4 intron and 88.6% for the pre - switch sequence ( vs. 89.3 and 89.8% for the corresponding ig gene sequences in xp - v patients ; figs . 2 and 3 , table ii , and reference 17 ) .
mutation frequencies were somewhat lower for pol-deficient animals compared with wild type when jh4 intronic sequences are considered , but similar for the pre - switch sequences .
such a quantitative dissociation has been described for msh6- and for ung / msh2-deficient animals ( 5 , 13 ) .
distribution of mutations in the vhdjh4 flanking sequence (  jh4 intronic region  ) of peyer 's patches pna b cells from pol- and pol-pol-deficient mice . ( a ) pattern of nucleotide substitution in control mice ( two animals ) , pol-deficient mice ( three animals ) , pol- and pol-deficient mice ( two animals ) , and from all mutant animals together .
data are given as percent substitution after correction for base composition ( 490 bp starting from the exon / intron border : a , 26.3% ; c , 14.9% ; g , 27.4% ; t , 31.4% ) .
the distribution of sequences harboring a given number of mutations relative to the total number of mutated sequences is represented for the three mouse genotypes .
distribution of mutations in the s core upstream region ( pre - switch ) of peyer 's patches pna b cells from pol- and pol-pol-deficient mice .
( a ) pattern of nucleotide substitution from the same control and mutant animals described in fig .
base composition : 560 bp ; a , 33.2% ; c , 16.4% ; g , 27.5% ; t , 22.9% .
same representation as in fig . 2 b. ( c ) distribution of mutations along the pre - switch sequence .
mutations from pol-deficient mice are listed above the sequence , and mutations from double - deficient mice are listed below the sequence . 
pattern of nucleotide changes in jh4 intronic sequences of normal , pol- , and msh2-deficient mice values in bold represent significant differences from controls discussed in the text .
this study ( 122 mutations ) . from a larger sample ( 334 mutations ; reference 36 ) .
data from mice deficient in the msh2-dependent pathway ( msh2 , exo1 , msh2 ) were pooled ( 326 mutations obtained by pfu polymerase ; references 8 , 11 , 12 ) . from ref .
these profiles appear extremely similar , either in their distribution along the sequence ( shown for the pre - switch sequence in fig .
3 c ) , or in their base substitution characteristics ( figs . 2 a and 3 a ) .
the lack of phenotype of pol-deficient mice on ig gene hypermutation is , therefore , not due to a compensatory role of pol in this specific strain .
all further analyses were , therefore , performed on pooled data from both types of mutant mice , allowing us to compare larger databases ( 366 mutations for the jh4 intron and 277 for the pre - switch sequence ) .
the targeting was slightly more pronounced for c than for g , in particular for the pre - switch sequence .
such an increase has been described in switch junctions from xp - v patients ( 18 ) .
however , among the three types of sequences that we have analyzed in similar patients , we could not observe this bias in either switch junctions or jh4 introns , the specific increase in c mutations being only significant in pre - switch sequences from xp - v individuals ( 17 ) .
the general relevance of this observation is , therefore , difficult to assess at this stage .
moreover , among g / c mutations , no difference from wild type is noticeable in the relative proportion of transitions and transversions ( table ii ) .
msh2 deficiency , together with defects in msh6 and exo1 , results in a similar bias toward g / c mutagenesis ( 713 ) .
we , therefore , wanted to compare the mutation pattern in the jh4 intronic sequence between msh2 and polh animals .
this pattern was established using data from both msh2- and exo1-deficient animals , as well as msh2-atpase mutant mice ( 326 mutations ) , taking into account only data obtained using pfu polymerase , to exclude any contribution of less accurate enzymes whose intrinsic error spectrum might specifically impinge on the a / t pattern . at g
/ c base pairs , these mutant mice show a strong bias toward transitions , which has been interpreted as an increased replicative bypass of uracils , the absence of the mismatch - binding complex leading to an increased fraction of deaminated bases escaping detection and repair .
polh animals , in contrast , show a transition / transversion distribution at g / c bases similar to controls .
strikingly , the mutation pattern at a / t bases pairs shows the reverse situation .
msh2 deficiency shows a distribution similar to wild type , whereas pol deficiency results in a strong transversion bias .
this transversion bias is consistent enough to emerge from both types of sequences analyzed ( jh4 and pre - switch ) , and in both mouse and human pol deficiencies ( table ii ) .
moreover , among transversions at a / t bases , most of them are a to c or t to g changes , which represent more than half of all a / t mutations .
such a mutation bias has been described for dna polymerase  ( 26 , 27 ) .
it is thus possible that , in the absence of pol , the mismatch binding complex is able to recruit a different translesional enzyme , which , due to its specific misincorporation pattern , would be less mutagenic at a / t base pairs , leading to a lower frequency of a / t mutations with a specific transversion bias . because pol-deficient animals do not harbor the slightest alteration of their a / t mutation pattern ( 28 ) ,
it is probable that this enzyme is not a normal actor of the ig gene mutation process , being on duty only in case of absence of the regular partner of the msh2msh6 complex ( i.e. , pol ) .
however , contrary to pol and pol , its preferred misincorporation is at copying a , and not t. it is therefore unlikely , in the mouse at least , that pol could generate the a over t bias of mutations in jh4 sequences , present in control mice and conserved in pol-deficient animals .
ung and msh2msh6 are the only two repair pathways handling uracils generated by aid during ig gene hypermutation .
( 5 ) posits that g / c mutations are introduced by replication over the uracils or the abasic sites generated by uracil glycosylase , both events occurring on the dna strand opposed to the lesion and without repair .
replication over the abasic site would involve translesional dna polymerases in their  classical  role of damage bypass . effectively , in the aid - dependent mutations observed in the chicken cell line dt40 that are almost entirely restricted to g / c bases
, rev1 has been proposed to be the major enzyme involved ( 30 ) . as the mutation pattern of dt40 is more biased toward g to c and c to g transversions ( the hallmark of rev1 ) than it is in the mouse , it is so far unclear how many translesional polymerases are involved in the g / c mutation pattern in mouse ( and human ) b cells .
mutations at a / t bases are more difficult to explain on a strict replication mode within this model . in another scenario ,
the msh2msh6 complex would recruit pol for an error - prone repair of the lesion ( i.e. , on the same dna strand ) that would remove the uracil or the abasic site and create mismatches at nearby a / t bases because this enzyme is inherently more mutagenic at copying ts . along this line ,
specific modifications of aid in hypermutating b cells might also actively recruit ung at the site of the lesion ( 31 ) , driving the g / c mutagenesis to proceed differently from a strict saturation of the normal repair of uracils .
in such a scheme , the dichotomy between translesion bypass and error - prone repair for the g / c versus a / t mutation pathways might have to be reassessed . the absence of phenotype of the pol mutation of the 129 strains obviously questions the relevance of the pol-dependent mutagenic process that we described in the bl2 burkitt 's lymphoma cell line ( 20 ) .
in fact , it has been reported that such a pol-dependent mutagenesis can be induced at the ig locus in b cell lines infected by the hepatitis c virus ( 32 ) .
these mutations would be related to the specific metabolic alterations brought upon infection by this oncogenic virus , alterations that may be shared by many b cell lymphomas ( 33 ) .
alternatively , inactivation of pol in the 129/ola mouse strain might show leakiness , in particular in activated b cells , by either read - trough of the stop codon or alternative splicing .
a classical inactivation of the poli gene and the analysis of mice cumulating several deficiencies in the activities involved in hypermutation might contribute to address these issues .
three fragments from the polh locus were amplified and cloned in the pflox vector ( 34 ) : fragment 1 , 5 flank ; fragment 2 , exon 4 ; and fragment 3 , 3 flank .
construction of the targeting vector and screening of recombinant clones are described in supplemental materials and methods ( available at http://www.jem.org/cgi/content/full/jem.20050292/dc1 ) .
one recombinant clone was obtained out of 287 es clones , and subjected to cre - mediated excision to generate exon 4deleted clones .
screening of gene targeted mice was performed by pcr around exon 4 ( described in fig .
1 ) using the following primers : 5-screen , 5-gtctccccagatgtatctcc-3 , and 3-screen , 5-gcattgtacacctgtggtcc-3 ( 45 s at 94c , 30 s at 59c , 3 min at 72c , 40 cycles with taq polymerase ; biolabs ) .
the pol mutation of the 129/ola background was screened by direct sequencing of a 540-bp genomic fragment encompassing exon 2 ( amplified by iota - exon 2 - 5 , 5-ttaaagcaggactgaagacc-3 ; iota - exon 2 - 3 , 5-cacatttactctcggttgca-3 , for 15 s at 98c , 30 s at 60c , 15 s at 72c , 40 cycles with phusion dna polymerase ; finnzymes ) .
pol mrna expression was analyzed by rt - pcr ( stratascript ; stratagene ) , using splenic rna extracted by the qiagen rneasy kit and pcr primers located in exons 2 and 6 ( exon 2 , 5-gggcagaatcgagtggttgc-3 ; exon 6 reverse , 5-gcggttgggcttatttagtcc-3 , 30 s at 95c , 90 s at 68c , 40 cycles with advantage ii polymerase mix ; clontech laboratories , inc . ) .
b220 pna b cells from peyer 's patches were isolated from 34-mo - old animals as described previously ( 8) .
amplification of the jh4 intronic sequence flanking rearranged vh genes was performed using a mixture of primers amplifying most vh gene families described ( compiled from the mouse v gene imgt database , http://imgt.cines.fr ) : v1-fr3 , 5-gaggactctgcrgtctattwctgtgc-3 ; v5-fr3 , 5-gaggacacrgccatgtattactgtgc-3 ; v3-fr3 , 5-gaggacacacccacatattactgtgc-3 ; v7-fr3 , 5-gaggacagtgccacttattactgtgc-3 ; and v9-fr3 , 5-atgaggacatggctacatatttctgt-3 , respectively , in a 6:3:1:1:1 ratio , and jh3 , 5-tgagaccgaggctagatgcc-3. pcr was performed on five aliquots of 500 or 1,000 cells ( 15 s 98c , 30 s 64c , 30 s 72c for 50 cycles using phusion dna polymerase ) and 480 bp sequences were determined using the jh3 primer .
a 735-bp fragment upstream from the s core repeat sequence was amplified using primers s-5 , 5-gttgaggtactgatgctgtc-3 and s-3 , 5-ccagcctagtttagcttagc-3 ( 45 s at 94c , 30 s at 58c , 2 min at 72c , 40 cycles with pfu turbo ) , and a 560-bp sequence was determined using primer 5-ctattctggctcttcttaagc-3. sequences were obtained with an abi prism 3100 genetic analyzer after cloning in the zero blunt vector ( invitrogen ) .
construction of the targeting vector for inactivation of the polh gene , as well as conditions for analysis of the recombinant es clones are described online .
three fragments from the polh locus were amplified and cloned in the pflox vector ( 34 ) : fragment 1 , 5 flank ; fragment 2 , exon 4 ; and fragment 3 , 3 flank .
construction of the targeting vector and screening of recombinant clones are described in supplemental materials and methods ( available at http://www.jem.org/cgi/content/full/jem.20050292/dc1 ) .
one recombinant clone was obtained out of 287 es clones , and subjected to cre - mediated excision to generate exon 4deleted clones .
screening of gene targeted mice was performed by pcr around exon 4 ( described in fig .
1 ) using the following primers : 5-screen , 5-gtctccccagatgtatctcc-3 , and 3-screen , 5-gcattgtacacctgtggtcc-3 ( 45 s at 94c , 30 s at 59c , 3 min at 72c , 40 cycles with taq polymerase ; biolabs ) .
the pol mutation of the 129/ola background was screened by direct sequencing of a 540-bp genomic fragment encompassing exon 2 ( amplified by iota - exon 2 - 5 , 5-ttaaagcaggactgaagacc-3 ; iota - exon 2 - 3 , 5-cacatttactctcggttgca-3 , for 15 s at 98c , 30 s at 60c , 15 s at 72c , 40 cycles with phusion dna polymerase ; finnzymes ) .
pol mrna expression was analyzed by rt - pcr ( stratascript ; stratagene ) , using splenic rna extracted by the qiagen rneasy kit and pcr primers located in exons 2 and 6 ( exon 2 , 5-gggcagaatcgagtggttgc-3 ; exon 6 reverse , 5-gcggttgggcttatttagtcc-3 , 30 s at 95c , 90 s at 68c , 40 cycles with advantage ii polymerase mix ; clontech laboratories , inc . ) .
b220 pna b cells from peyer 's patches were isolated from 34-mo - old animals as described previously ( 8) .
amplification of the jh4 intronic sequence flanking rearranged vh genes was performed using a mixture of primers amplifying most vh gene families described ( compiled from the mouse v gene imgt database , http://imgt.cines.fr ) : v1-fr3 , 5-gaggactctgcrgtctattwctgtgc-3 ; v5-fr3 , 5-gaggacacrgccatgtattactgtgc-3 ; v3-fr3 , 5-gaggacacacccacatattactgtgc-3 ; v7-fr3 , 5-gaggacagtgccacttattactgtgc-3 ; and v9-fr3 , 5-atgaggacatggctacatatttctgt-3 , respectively , in a 6:3:1:1:1 ratio , and jh3 , 5-tgagaccgaggctagatgcc-3. pcr was performed on five aliquots of 500 or 1,000 cells ( 15 s 98c , 30 s 64c , 30 s 72c for 50 cycles using phusion dna polymerase ) and 480 bp sequences were determined using the jh3 primer .
a 735-bp fragment upstream from the s core repeat sequence was amplified using primers s-5 , 5-gttgaggtactgatgctgtc-3 and s-3 , 5-ccagcctagtttagcttagc-3 ( 45 s at 94c , 30 s at 58c , 2 min at 72c , 40 cycles with pfu turbo ) , and a 560-bp sequence was determined using primer 5-ctattctggctcttcttaagc-3. sequences were obtained with an abi prism 3100 genetic analyzer after cloning in the zero blunt vector ( invitrogen ) .
construction of the targeting vector for inactivation of the polh gene , as well as conditions for analysis of the recombinant es clones are described online .
 the mutation pattern of immunoglobulin genes was studied in mice deficient for dna polymerase  , a translesional polymerase whose inactivation is responsible for the xeroderma pigmentosum variant ( xp - v ) syndrome in humans . 
 mutations show an 85% g / c biased pattern , similar to that reported for xp - v patients . 
 breeding these mice with animals harboring the stop codon mutation of the 129/olain background in their dna polymerase  gene did not alter this pattern further . 
 although this g / c biased mutation profile resembles that of mice deficient in the msh2 or msh6 components of the mismatch repair complex , the residual a / t mutagenesis of pol-deficient mice differs markedly . 
 this suggests that , in the absence of pol , the msh2msh6 complex is able to recruit another dna polymerase that is more accurate at copying a / t bases , possibly pol , to assume its function in hypermutation . 
Results and discussion
Materials and Methods
Construction of targeting vectors.
Analysis of gene-targeted mice.
Analysis of mutations in the Ig locus.
Online supplemental material.