Fidelity of Uracil-initiated Base Excision DNA Repair in DNA Polymerase β-Proficient and -Deficient Mouse Embryonic Fibroblast Cell Extracts

Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase β (+/+) and (−/−) cells to determine the error frequency and mutational specificity associated with the completed repair process. Form I DNA substrates were constructed with site-specific uracil residues at U·A, U·G, and U·T targets contained within the lacZα gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase β (+/+) and (−/−) cell-free extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced (∼88%) the initial rate of repair in both types of cell-free extracts. In each case, the DNA repair patch size was primarily distributed between 1 and 8 nucleotides in length with 1 nucleotide repair patch constituting ∼20% of the repair events. Addition of p21 peptide or protein to DNA polymerase β (+/+) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by ∼2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U·T) DNA was determined to be 5.7–7.2 × 10−4 when using DNA polymerase β (+/+) and (−/−) cell-free extracts. In these two cases, the error frequency was very similar, but the mutational spectrum was noticeably different. The presence or absence of Ugi did not dramatically influence either the error rate or mutational specificity. In contrast, the combination of Ugi and p21 protein promoted an increase in the mutation frequency associated with repair of M13mp2 (U·G) DNA. Examination of the mutational spectra generated by a forward mutation assay revealed that errors in DNA repair synthesis occurred predominantly at the position of the U·G target and frequently involved a 1-base deletion or incorporation of dTMP. Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase β (+/+) and (−/−) cells to determine the error frequency and mutational specificity associated with the completed repair process. Form I DNA substrates were constructed with site-specific uracil residues at U·A, U·G, and U·T targets contained within the lacZα gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase β (+/+) and (−/−) cell-free extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced (∼88%) the initial rate of repair in both types of cell-free extracts. In each case, the DNA repair patch size was primarily distributed between 1 and 8 nucleotides in length with 1 nucleotide repair patch constituting ∼20% of the repair events. Addition of p21 peptide or protein to DNA polymerase β (+/+) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by ∼2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U·T) DNA was determined to be 5.7–7.2 × 10−4 when using DNA polymerase β (+/+) and (−/−) cell-free extracts. In these two cases, the error frequency was very similar, but the mutational spectrum was noticeably different. The presence or absence of Ugi did not dramatically influence either the error rate or mutational specificity. In contrast, the combination of Ugi and p21 protein promoted an increase in the mutation frequency associated with repair of M13mp2 (U·G) DNA. Examination of the mutational spectra generated by a forward mutation assay revealed that errors in DNA repair synthesis occurred predominantly at the position of the U·G target and frequently involved a 1-base deletion or incorporation of dTMP. base excision repair apurinic/apyrimidinic proliferating cell nuclear antigen bovine serum albumin mouse embryonic fibroblast base pair(s) polymerase polymerase residues in DNA incorporation of in of DNA synthesis by of residues that U·G In the presence of uracil in DNA and these the base excision repair to uracil residues the The of in of and in cells the to each in in to be these uracil-DNA a of of the the uracil base to the DNA in specificity. The uracil-DNA glycosylase activity in cell extracts and uracil residues in DNA and on DNA U·G and base In contrast, glycosylase not uracil in DNA but uracil residues in DNA with the U·G U·T The on U·G in the of or but activity DNA and U·A, or U·T DNA substrates was not the uracil-DNA glycosylase uracil residues DNA to the of with both and DNA was reduced and The uracil-DNA be on to by the and uracil-DNA glycosylase inhibitor (Ugi) protein Ugi a DNA and to a that of Ugi in with the or revealed an and between the The that Ugi these was not because these residues In contrast, and not with to by Ugi addition of Ugi to cell-free extracts to and of of the uracil base and of the a DNA repair by DNA that in either a (1 nucleotide) or or repair patch of a in using the uracil-DNA glycosylase apurinic/apyrimidinic DNA polymerase β and DNA I DNA I in short-patch In the case, addition of protein was to DNA synthesis by β and the nucleotide that short-patch In DNA a and extracts of a in was that the short-patch to that short-patch observed in extracts of mouse embryonic fibroblast cells that or DNA or in the nucleotide DNA polymerase was to an activity to that of β in were conducted that β and with and I using an either a or U·G target of a reduced in a and I In addition of proliferating cell nuclear antigen was observed to the repair and DNA synthesis two to nucleotides was to was in the In extracts of mouse embryonic fibroblast both and were to be dependent on in was to be dependent on the of the in DNA between and a of involved in DNA I and a of and to the of p21 a inhibitor of DNA that with at the the the of p21 to in the protein a p21 peptide the was to be of in DNA and repair the p21 peptide and protein to be with to the of in DNA repair the of both and the and mutational specificity associated with not in to determine the of the DNA synthesis DNA an of the mutation frequency associated with completed to be In the M13mp2 lacZα to the of using mouse embryonic fibroblast the mutational and patch size of in extracts of and cells in the presence and absence of the uracil-DNA glycosylase inhibitor In the influence of p21 peptide and protein on the patch and of was and DNA and in the of M13mp2op14 (U·T) and by the base in the M13mp2 and M13mp2op14 by the position of the site-specific base by the length of the and were and were to M13mp2op14 and uracil target was nucleotides on the of the base of the DNA and M13mp2 and (U·G) were by and and were of was M13mp2 DNA polymerase or mouse embryonic fibroblast cell were DNA polymerase β and polymerase β were by of of and PBS-2 DNA and DNA and The p21 peptide that was by and in p21 peptide 1 by The p21 was by and p21 protein was p21 was in cells to cells were by at × in of 1 1 with 1 and at on cells were to at and p21 protein were by at × The was and the was with and the the was in of 8 and to The was to a × in and with of by of the p21 protein was the by the addition of of (1 were and p21 protein determined by were and at 1 1 The was to a × in with of and with a to 1 in were and p21 were p21 1 1 using and at were determined by the using the with the M13mp2op14 DNA an at nucleotide of lacZα gene was constructed and by M13mp2 DNA was using the DNA was to the and the DNA was to a DNA were to and I DNA was and 1 The DNA was I determined by cells β were in with bovine and each of and were at and and to in were using 1 and in extracts were by and 1 and and in The protein of the cell-free extracts was determined using were by with a with and to a using a at the was in β serum was in and with the 1 at with was in and with the The was and with and the were to of uracil-DNA glycosylase activity in β (+/+) cell-free extracts. In the contained M13mp2op14 (U·T) (1 1 and Ugi at the were by at with and and the DNA by The DNA each was in of and a was to with and 1 at were by at each was with (1 at the were by the was and the associated with the and each was using a The uracil-DNA glycosylase activity in each was determined the of the associated with the by the of associated with both and In these were a of the of uracil-DNA glycosylase activity observed in the absence of the of by a Ugi the of uracil-DNA glycosylase activity was In the uracil-DNA glycosylase inhibitor uracil-DNA glycosylase activity was by of DNA. glycosylase contained 1 1 DNA activity of β (+/+) cell-free and Ugi at were on by the addition of of was using a in by and contained 1 each of and of the M13mp2 DNA and cell-free at the were by to and at was to and the were at was to and at and the M13mp2 DNA was and in of by and of the M13mp2 DNA base excision DNA repair were with at to uracil residues that repair the the uracil-DNA glycosylase by Ugi the were with (1 at to was by at and I DNA that was to the was DNA by The of I and DNA was determined by the at of the DNA to that of I and DNA The was with a and using the were that a was in of each of the M13mp2op14 (U·T) DNA was the The was constructed of and the of DNA Repair in and contained a nucleotides of the target uracil and nucleotides of the on the of the lacZα gene the DNA were and in of were with of 1 at The was at and were in the absence or the presence of of 1 at each was at and the was with of 1 at of was and were by the was and the of associated with each was using a and the I DNA was and Form I DNA was by using an was conducted at in 1 The I DNA was using a and cells were with DNA with and and 1 and on the either or the mutation frequency was the of the of to were in reversion and and in forward mutation and (U·G) of each was conducted in of the lacZα gene and nucleotide that was conducted by the and and an M13mp2op14 reversion assay to in cell-free extracts and determine the base substitution error frequency associated with the completed repair and the with the lacZα was of DNA was that contained a uracil in the at the position of the to a U·T at position excision of the target uracil to the 1 DNA synthesis was and either or at position reversion of the in lacZα and a DNA an base at position was constructed to the of reversion of the in the cell-free 1 using assay with M13mp2op14 lacZα (U·T) DNA and DNA substrates the and mutational specificity of the was using DNA polymerase (+/+) and DNA polymerase (−/−) mouse embryonic fibroblast M13mp2op14 (U·T) DNA was with β (+/+) cell-free and the were by In a Ugi was to the to uracil-DNA glycosylase activity the each was with and to uracil residues that excision the and to the the of the uracil-DNA to DNA In contrast, DNA that uracil-DNA repair uracil DNA and DNA were to and I DNA. was to or DNA of the that I DNA in a in both and the of I DNA increased in the β (+/+) by a the of I DNA in the Ugi was and that the of Ugi to the was to the uracil-DNA glycosylase two were In the the uracil-DNA glycosylase activity in the β (+/+) was in the presence of of Ugi using a contained base The that of Ugi was to but of uracil-DNA glycosylase activity and that Ugi did not In the uracil-DNA glycosylase activity was using an M13mp2op14 (U·T) DNA The that of Ugi was to of uracil-DNA glycosylase activity these that of Ugi was to the uracil-DNA glycosylase activity in the β cell-free extracts. The of uracil-DNA glycosylase activity was to initial that in β (+/+) extracts was in by the of repair at in the was in both β (+/+) and β (−/−) cell-free extracts In were conducted to determine the of Ugi on the repair The that in both cell extracts the of were in the absence of the rate and of were in both β (+/+) and β (−/−) cell of both cell extracts was conducted to the of the two cell of the that DNA polymerase β protein was in the β (+/+) but not in the β (−/−) cell Ugi was to the β (+/+) and β (−/−) the initial rate of was reduced by and and The patch size of DNA repair synthesis associated with of M13mp2op14 (U·T) in β (+/+) and β (−/−) cell-free extracts was determined using the by and by on the incorporation of the to the DNA to in by in the uracil target was on the nucleotides and nucleotides to the and site-specific was to the uracil target at position were generated to in the patch a was by the M13mp2op14 (U·T) with and and the by was generated the uracil and the was by and the was by The to the to DNA synthesis and the of the repair determine the of a repair M13mp2op14 (U·T) was with and in the to with DNA of the was to the the of the repair with a the length of the patch size size was conducted in β (+/+) and β (−/−) cell-free extracts both in the presence and absence of Ugi a M13mp2op14 (U·T) was each of the the not with by of the with and or and of a repair patch was not the were not the in the absence of cell-free each of the with were to the of DNA of the repair patch size was using a and the patch size each was a of the The that repair were the patch size in both β (+/+) and β (−/−) constituting and of repair of the repair in both β (+/+) and β (−/−) were to 8 nucleotides in The that Ugi addition did not the of patch size in either cell-free Repair patch size were conducted to the influence of the p21 peptide and protein on of M13mp2op14 (U·T) in β (+/+) cell-free extracts the two 1 and and and were and by of the revealed that the p21 influence on patch size and In contrast, addition of p21 peptide or p21 protein a increase in the repair patch and a in repair and of the with the of p21 peptide or protein in of repair and The were and the p21 peptide and protein in and Addition of p21 peptide increased the of the repair patch to the and p21 protein promoted a patch addition of p21 peptide and with p21 protein the of DNA synthesis was reduced and to in the presence of p21 peptide and p21 protein that a in the patch size associated with The frequency of of M13mp2op14 (U·T) DNA was determined using the M13mp2op14 reversion the reversion frequency of M13mp2op14 DNA was in extracts of β (+/+) and β (−/−) cells reversion frequency × was observed both cell extracts. the reversion frequency associated with of M13mp2op14 (U·T) DNA was determined to be × 10−4 and × 10−4 conducted with β (+/+) and β (−/−) cell-free Ugi was to and the reversion associated with the were to be × 10−4 β (+/+) and × β (−/−) cell-free extracts the error frequency of was by the addition of Ugi and did not to with the gene of by of M13mp2op14 DNA in mouse embryonic fibroblast DNA polymerase β (+/+) and (−/−) cell-free the of mutation frequency each were by the of by the of and The reversion frequency by the and nucleotide at the target β β were that contained of β (+/+) or β (−/−) cell-free 1 of M13mp2op14 or and Ugi at the were and DNA and I DNA to was by cells were with the I and the M13mp2 lacZα reversion assay was the of mutation frequency each were by the of by the of and The reversion frequency by the The and nucleotide at the target in a were that contained of β (+/+) or β (−/−) cell-free 1 of M13mp2op14 or and Ugi at the were and DNA and I DNA to was by cells were with the I and the M13mp2 lacZα reversion assay was The mutational spectrum of M13mp2op14 DNA was determined to the and of base by with β (+/+) and β (−/−) cell-free extracts and Examination of the mutational spectrum in β (+/+) that of the reversion occurred at the position of the of the occurred at the position of the The spectrum observed in β (−/−) cell-free extracts revealed that of occurred at the position of the and at the position these a position in the error The mutational spectrum of of M13mp2op14 (U·T) DNA in β (+/+) cell in of was at the position of the the uracil target was The mutation was to by to and to In β (−/−) cell-free of base occurred at the to of with Ugi a on the specificity on in β (+/+) in β (−/−) cell-free the increased to of was using a uracil target contained within a base of the 1 The frequency of of M13mp2op14 DNA was to be × 10−4 and × 10−4 conducted with β (+/+) and β (−/−) cell-free Ugi was to the the error associated with β (+/+) and β (−/−) cell-free extracts were × 10−4 and × of the mutation using the at the position of the with the U·T target at the position that the frequency of reversion did not β (+/+) cell the β (−/−) cell extracts that repair was U·T of by of or M13mp2op14 DNA in mouse embryonic fibroblast DNA polymerase β (+/+) and (−/−) cell-free the of mutation frequency each were by the of by the of and The reversion frequency by the and nucleotide at the target β β β were that contained of β (+/+) or β (−/−) cell-free 1 of M13mp2op14 or and Ugi at the were and DNA and I DNA to was by cells were with the I and the M13mp2 lacZα reversion assay was the of mutation frequency each were by the of by the of and The reversion frequency by the The and nucleotide at the target in a were that contained of β (+/+) or β (−/−) cell-free 1 of M13mp2op14 or and Ugi at the were and DNA and I DNA to was by cells were with the I and the M13mp2 lacZα reversion assay was The mutational spectrum of of M13mp2op14 DNA in β (+/+) cell in The base substitution mutation was of the uracil was Addition of Ugi to the did not to the mutational specificity of the mutational spectrum of on the DNA but β (−/−) cell in In of with The addition of Ugi to the β (−/−) the specificity of reversion that of of was the of at the of the error frequency of on the of the uracil excision was using M13mp2op14 DNA 1 In the uracil target (U·T) was nucleotides on the of the base of the excision of the uracil DNA synthesis nucleotides to the base substitution errors in reversion The reversion frequency of M13mp2op14 DNA extracts of β (−/−) cells was × 10−4 and × 10−4 in the presence and absence of were the observed M13mp2op14 DNA that the of DNA synthesis was the mutational specificity associated with the DNA the reversion spectrum of the DNA. at in DNA occurred frequently at the position at the position 8 reversion in DNA occurred predominantly at the position to the position of In the specificity of DNA synthesis errors with that using the (U·T) and errors at the of uracil excision predominantly of incorporation of and not observed in the DNA synthesis associated with repair of (U·G) a forward mutation assay was using M13mp2 (U·G) DNA that contained a of lacZα 1 In the lacZα gene to were because of a site-specific uracil at position of the of of base errors at of base nucleotides of the uracil and of base substitution errors nucleotides of the In in lacZα were using the forward mutation the error frequency using the M13mp2 DNA 1 was determined to be × 10−4 and × 10−4 β (+/+) and β (−/−) cell extracts to the mutation frequency of the M13mp2op14 reversion the of the forward assay was the mutation frequency associated with of M13mp2 (U·G) DNA was determined to be × 10−4 and × 10−4 in β (+/+) or β (−/−) cell the of Ugi on the error frequency associated with of M13mp2 (U·G) DNA was and determined to be × 10−4 and × 10−4 β (+/+) and β (−/−) the mutation frequency of M13mp2 (U·G) DNA with p21 protein was determined to be × 10−4 β (+/+) and × × 10−4 β (−/−) cell extracts. when were with both Ugi and the error frequency increased to × × 10−4 β (+/+) and β (−/−) the addition of Ugi and p21 protein a on the mutation frequency in both the of conducted in the presence of p21 protein was reduced not of by of M13mp2 DNA in mouse embryonic fibroblast DNA polymerase β (+/+) and (−/−) cell-free the of mutation frequency each were by the of by the of and The reversion frequency by the and nucleotide at the target β β were that contained of β (+/+) or β (−/−) cell-free 1 of M13mp2 or and Ugi or p21 protein at the were and DNA and I DNA to was by cells were with the I and the M13mp2 lacZα reversion assay was the of mutation frequency each were by the of by the of and The reversion frequency by the The and nucleotide at the target in a were that contained of β (+/+) or β (−/−) cell-free 1 of M13mp2 or and Ugi or p21 protein at the were and DNA and I DNA to was by cells were with the I and the M13mp2 lacZα reversion assay was The mutational spectrum of M13mp2 DNA was to the specificity and of base substitution and within the patch size that of uracil-DNA repair synthesis occurred in of nucleotides that occurred at to were to be the of the M13mp2 DNA β (+/+) cell-free occurred within the target the that occurred the deletion of a in a to occurred the were nucleotides to were a deletion of an the at to and an of a in the at to the error spectrum of M13mp2 DNA in β (+/+) cell-free extracts primarily of and The mutational spectrum of M13mp2 DNA β (−/−) in The DNA of was of these within the of of the deletion at to of and deletion of the at to of on these in β (−/−) cell-free primarily of The mutational spectra of M13mp2 (U·G) DNA β (+/+) or β (−/−) cell-free extracts in and each were of β and β within the of the spectra that the mutation in both was a 1-base deletion to at the of the uracil deletion by a the nucleotide at position in the be that the nucleotide position was that by the uracil on the because that the DNA 1-base deletion occurred by a or or to be the the β (+/+) occurred at position of to base that excision of uracil and of the spectrum of β (−/−) cell-free of and base to at position In an of in the to to and a deletion of at position were The of Ugi on the mutational specificity of of M13mp2 (U·G) DNA was In β (+/+) cell-free and in the target were deletion of and 8 were incorporation β (−/−) were with in the target were of and 8 were incorporation of not the of mouse embryonic fibroblast cell-free either or in to of U·A, U·G, and U·T DNA the to of in cell-free extracts of In of the patch size associated with in the presence and absence of β was the of p21 on patch The that the of in cell-free extracts of β (−/−) cells was with that observed β (+/+) cell-free of by Ugi reduced but did not the patch size associated with β (+/+) and (−/−) cell was addition of p21 to the reduced the of in extracts of both cell and the patch size to and the error of in cell-free extracts was conducted with not that repair of each of The in extracts of β (−/−) of I DNA was in extracts of β (+/+) In both a of I DNA was observed the of the size that the repair occurred frequently patch repair of length occurred in of the repair in both cell extracts because patch size was conducted on DNA to on the patch size with that repair in the by repair at that short-patch of an in extracts of β (−/−) cells was short-patch of the in extracts of β (+/+) were a extracts of β (−/−) repair was observed The of to the was in and by were by addition of short-patch to I DNA. a in the of I DNA was observed in that contained either p21 protein or that to the in the a in an M13mp2 DNA that repair in I because DNA was to the In that of be to DNA I to of in DNA In β to with the of DNA I with DNA I that p21 to with the I and the of The addition of Ugi to in of of uracil-DNA glycosylase and the rate of in the presence of Ugi was and with the that the uracil-DNA glycosylase activity in extracts of cells and that uracil-DNA repair primarily the a by uracil-DNA to in to that observed in and cells Ugi addition did not in a increase in the mutation frequency associated with of M13mp2 lacZα DNA the in cells not to be the was not the in extracts of that of in a increase in mutation In two gene and to uracil-DNA glycosylase activity In contrast, cells to uracil-DNA glycosylase and and of these and or in the of uracil in DNA was with that of these in uracil-DNA in to be the of the activity or the in β (+/+) and (−/−) Examination of the mutational spectra of both β (+/+) and β (−/−) that errors in DNA synthesis associated with occurred predominantly at the position of the uracil In when the mutation spectra were of of the the error frequency associated with of M13mp2op14 in the uracil was nucleotides of the was to that of the M13mp2op14 DNA. In β (+/+) of M13mp2op14 were by of with the by that the of by β in a was the of the observed a in DNA synthesis by In β (−/−) of M13mp2op14 of and were The of Ugi on the specificity of at the target was in that of was largely in of in both the of Ugi on DNA repair synthesis was to incorporation of the In addition of Ugi to M13mp2op14 (U·T) DNA in an increase in and to at the Ugi of M13mp2op14 did not to influence reversion specificity. that in of M13mp2op14 (U·T) the frequency of be to the nucleotide substitution rate and using β or DNA synthesis in cell-free the that not the a the mutational specificity observed in of M13mp2op14 (U·T) DNA. the mutational specificity of of M13mp2op14 DNA in β cell-free extracts was to of the of the of or was a of DNA to be The error frequency associated with of M13mp2 (U·G) DNA was determined using a forward mutation assay and to be to the error frequency of the M13mp2 DNA. The mutation frequency was not because the target size of the forward mutation assay and base substitution be at of nucleotide In lacZα the error frequency associated with β (+/+) and β (−/−) cells was similar, the mutational specificity was different. The mutational spectrum of M13mp2 DNA in β (+/+) was by base in β (−/−) the spectrum was by in of to both was a deletion that occurred of revealed the a at of the observed of a U·G at position in a of of occurred at position two types of were a base at the of the and of the mutational spectrum of in both β (+/+) and (−/−) M13mp2 (U·G) DNA was the uracil target in was the in a of a be to the of the base deletion mutation at deletion at position of lacZα was observed by the base in DNA synthesis by of the mutational spectrum that the base deletion observed in the at position the of The mutation observed at position of was to be with of or of the mutational specificity associated with the DNA and in in to be be to the that mutation and the to and by DNA and the DNA at the and of and of