Reaction Mechanism of Human DNA Repair Excision Nuclease

Nucleotide excision repair consists of removal of the damaged nucleotide(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation. In humans, 14-16 polypeptides are required for the dual incision step. We have purified the required proteins to homogeneity and reconstituted the dual incision activity (excision nuclease) in a defined enzyme/substrate system. The system was highly efficient, removing >30% of the thymine dimers under optimal conditions. All of the six fractions that constitute the excision nuclease were required for dual incision of the thymine dimer substrate. However, when a cholesterol-substituted oligonucleotide was used as substrate, excision occurred in the absence of the XPC-HHR23B complex, reminiscent of transcription-coupled repair in the XP-C mutant cell line. Replication protein A is absolutely required for both incisions. The XPG subunit is essential to the formation of the preincision complex, but the repair complex can assemble and produce normal levels of 3′-incision in the absence of XPF-ERCC1. Kinetic experiments revealed that the 3′-incision precedes the 5′-incision. Consistent with the kinetic data, uncoupled 5′-incision was never observed in the reconstituted system. Two forms of TFIIH were used in the reconstitution reaction, one containing the CDK7-cyclin H pair and one lacking it. Both forms were equally active in excision. The excised oligomer dissociated from the gapped DNA in a nucleoprotein complex. In total, these results provide a detailed account of the reactions occurring during damage removal by human excision nuclease. Nucleotide excision repair consists of removal of the damaged nucleotide(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation. In humans, 14-16 polypeptides are required for the dual incision step. We have purified the required proteins to homogeneity and reconstituted the dual incision activity (excision nuclease) in a defined enzyme/substrate system. The system was highly efficient, removing >30% of the thymine dimers under optimal conditions. All of the six fractions that constitute the excision nuclease were required for dual incision of the thymine dimer substrate. However, when a cholesterol-substituted oligonucleotide was used as substrate, excision occurred in the absence of the XPC-HHR23B complex, reminiscent of transcription-coupled repair in the XP-C mutant cell line. Replication protein A is absolutely required for both incisions. The XPG subunit is essential to the formation of the preincision complex, but the repair complex can assemble and produce normal levels of 3′-incision in the absence of XPF-ERCC1. Kinetic experiments revealed that the 3′-incision precedes the 5′-incision. Consistent with the kinetic data, uncoupled 5′-incision was never observed in the reconstituted system. Two forms of TFIIH were used in the reconstitution reaction, one containing the CDK7-cyclin H pair and one lacking it. Both forms were equally active in excision. The excised oligomer dissociated from the gapped DNA in a nucleoprotein complex. In total, these results provide a detailed account of the reactions occurring during damage removal by human excision nuclease. INTRODUCTIONDNA repair reactions play an important role in preventing cancer development in humans. Individuals with defects in mismatch repair have high incidence of internal cancers (Kolodner and Alani, 1994; Modrich and Lahue, 1996). Similarly, individuals with defective nucleotide excision repair specific for bulky DNA lesions suffer from xeroderma pigmentosum (XP), ( 1The abbreviations used are: XPxeroderma pigmentosumRPAreplication protein APAGEpolyacrylamide gel electrophoresisCFEcell-free extractATPγSadenosine 5′-O-(thiotriphosphate)CTDcarboxyl-terminal domainCDKcyclin-dependent kinase.) which manifests itself by extremely high incidence of actinic cancers, increased incidence of cancers of internal organs, and mental and neurological abnormalities (Cleaver and Kraemer, 1989; Friedberg et al., 1995). In humans, mutations in seven genes, XPA through XPG, cause XP associated with defective excision repair (Cleaver and Kraemer, 1989). In addition, genetic and biochemical studies with rodent cell lines and radiation-sensitive Saccharomyces cerevisiae mutants and repair assays with cell-free extracts have implicated several other proteins in the excision step of nucleotide excision repair. In particular, it has been found that the general transcription factor TFIIH (five to eight subunits) (Drapkin et al., 1994; Schaeffer et al., 1994; Wang et al., 1994) and the trimeric replication protein A (RPA or HSSB) are absolutely required for the dual incision step of excision repair (Mu et al., 1995).Recently, excision repair has been reconstituted from highly purified repair factors in humans (Mu et al., 1995; Aboussekhra et al., 1995) and in the highly analogous S. cerevisiae system (Guzder et al., 1995). It has been reported that in both systems the following six fractions are necessary and sufficient for the dual incision activity (Mu et al., 1995; Guzder et al., 1995): XPA (Rad14), RPA, TFIIH (five to eight polypeptides including XPB (Rad25) and XPD (Rad3)), XPC-HHR23B (Rad4-Rad23), XPG (Rad2), and XPF-ERCC1 (Rad1-Rad10). In this study, we have obtained the six fractions of human excision nuclease (excinuclease) free of contaminants and have used synthetic substrates containing either a thymine dimer or a a to the of the excision nuclease. results that in with the other factors that are necessary for excision is required for excision of the thymine but for excision of a In this study, it is that the 3′-incision by XPG and 1994; et al., 1994; et al., 1995) precedes the 5′-incision by XPF-ERCC1 et al., 1995) and that the and that are in TFIIH as with the repair activity of TFIIH and are required for repair. the dual the excised et al., are in a complex with repair the gapped DNA with a of repair of Nucleotide of were from and the cell-free was to et The in this was from (Mu et al., 1995) in that and were used following the step. The step was as by et TFIIH and were found in the to the XPG, RPA, and a of XPF-ERCC1 were in the fractions with by The of XPF-ERCC1 the and was purified to homogeneity as et al., The purified complex in reconstituted excision nuclease when with other However, to the of the XPF-ERCC1 of the experiments were with the XPF-ERCC1 from the step of et al., XPG, the fractions were an A of in was used to this and which in the XPG was found to by fractions were to XPG from et to TFIIH and the fractions from the containing these factors were the and with a of in was from TFIIH as by was obtained the of et TFIIH was to (Mu et al., that was as the step. was a of in and fractions were was an in and XPA with the was in and and purified by a of and The DNA the human was obtained from of and the of homogeneity was from to et for was a pair containing either a or a thymine dimer and in the strand and The nucleotide of the has been et al., 1995). However, the nucleotide the thymine dimer is from that of the substrate, and the is as in on the of we used or substrates were from six substrates were from either of or the six used for et al., 1995). Two of were used in this was from and was from thymine oligomer was obtained from by the of and used in this and the of thymine All of these lesions were pair as under in A and were and and of the substrates for the thymine dimer and and the used for excision assays that the excised oligomer by dual of the human DNA repair excision nuclease were as et al., and 1994; et al., 1995; et al., 1995). The excision was for The of purified repair factor for a excision is as XPG, and RPA, of the excised of and the to incision was by the gel a of and of of was with the of was to a for with either reconstituted excision nuclease or cell-free The DNA the proteins that with the was a and with of excision the excised by the both the and were and reactions to preincision complex were with the substrates of mutant cell the that repair proteins were with of were as DNA for the the was in the in to complex. the was to a excision in the of either a mutant cell of a or the purified repair The of this excision were with has been reported that the of highly purified fractions containing RPA, XPG, and XPF-ERCC1 was of DNA lesions in from synthetic substrates (Mu et al., 1995) or DNA et al., 1995). However, of the fractions used in these and the for proteins (Mu et al., 1995). In it was that an factor that with XPF-ERCC1 was for specific et al., 1995). was observed in the absence of et al., the that was essential for but a In addition, in both reconstitution of the lesions were one to that factors were for optimal the by these we have purified six repair factors to proteins of high as in The of these fractions excised both thymine dimer and of lesions from DNA in the of with high Both lesions were excised and to the in it that the of the defined excision repair system is to the in reaction, that it is that factors are required for optimal excision. The excision obtained in reconstitution to the of the we have found that the of XPC-HHR23B in the from to can excision of and thymine dimer by reconstituted human excision nuclease. of the The of the that was either or excised is the excision of and the thymine was excised in the of the thymine dimer was excised in the of The DNA and the following of the repair XPG, and for in we have that six factors are required for excision (Mu et al., 1995). However, that the of or incision when one or of these six factors are the of Aboussekhra et that a of damaged was observed when was that either the or the can we assays the defined excision nuclease system and a pair with a thymine dimer under the one of factors was from the system. In this 3′-incision is to in the of uncoupled 3′-incision is to in et al., et al., 1995). The results of this are in In the the excision to a the to uncoupled 3′-incision these of the were to 5′-incision. of with the of both and of XPF-ERCC1 to incision the of the the of this uncoupled 3′-incision in the absence of XPF-ERCC1 was to the of the excision uncoupled 3′-incision in the system that 3′-incision a normal in the absence of the protein for 5′-incision. A in is that in the absence of was of the in and excision or uncoupled or that is required for both for excision of thymine dimer and excision of The of the excision and uncoupled 3′-incision are was and of the uncoupled 3′-incision in the system In the XPF-ERCC1 of the an uncoupled excision of The of the excision are by a The of excision in the by the was in the system and in the lacking in for of XP-C mutant cell lines a biochemical XP-C mutants repair thymine dimers of the strand of the a normal et al., et al., 1995). these it was either that a for in the of excision nuclease or that the of damaged DNA in the complex the for the we a in to the thymine dimer in reconstitution a was in with the thymine the was excised in the absence of However, the of excision was that of the and of of the the of of the other Similarly, of and XPF-ERCC1 to excision with the that in the absence of 3′-incision was results that is required for either the or the 5′-incision with this substrate. the of the DNA as as the were by the nuclease activity of XPG or XPF-ERCC1. The of excision of lesion, to the the excision of a substrate, on the of these it is to excision of is However, the that this is excised in the absence of and of transcription as for the that in transcription-coupled the of the DNA in the complex the excision nuclease to assemble and damage from the strand an normal these results were we the that in other factors for the excision nuclease activity reconstituted The of other factors XPG, and was for by an of of in was to the of that the excision of by reconstitution from these is that with substrates or with substrates in as during is for excision nuclease of the of the excision nuclease in have to for the of this complex system and 1994; 1995; et al., for these was The in that the 3′-incision that is by XPG and 1994; et al., 1994; et al., 1995) occurred to the normal that the other fractions can assemble the XPF-ERCC1. XPF-ERCC1 the factor to the 5′-incision was observed in the absence of XPG Two were for this XPG to the preincision and the 5′-incision by XPF-ERCC1 on the of the 3′-incision by XPG, but on the of XPG in the preincision complex. studies have that of 3′-incision by with the that the formation of 5′-incision is on the of the et al., 1995). the to 5′-incision in the absence of XPG to the that XPG in the preincision complex to XPF-ERCC1 to and the 5′-incision. this we the following from an or mutant cell was with that the the and preincision on the were from the proteins or cell-free was to the from or The results that of the or purified XPF-ERCC1 to the complex from the excision In of to the from the in the in excision these data, we that XPG the preincision complex and that XPG is required for of the XPF-ERCC1 nuclease in the of complex formation with lacking but with in extracts from or a cell defective in XPG were with with a The preincision complex was by the as under and with either a mutant or purified repair the cell-free was used as a the in with was in as that purified XPF-ERCC1 of the cell-free was to the complex. the following with by was with The of the excision are to the of the of that XPG, with other factors can the 3′-incision the that the 3′-incision the 5′-incision. However, in a study, it was found that 3′-incision a on both and to the et al., 1995). are with 5′-incision and necessary for the of incision a kinetic was to the of the incisions. The of the results of this and the the of uncoupled and excision that the to on substrate. and to 5′-incision was that 3′-incision the uncoupled to dual incision to The results of kinetic experiments a of the were as in the of uncoupled the dual incision to the the and to the of excision the in and the of 3′-incision of 5′-incision has been analogous to the excision nuclease and we that in human excision the 3′-incision precedes the of are of the in the thymine dimer was with the reconstituted excision nuclease. were the with of and on an The uncoupled 3′-incision the excision which required both and 5′-incision is have of in of TFIIH and in general transcription factor TFIIH is an essential general excision repair factor (Drapkin et al., 1994; Schaeffer et al., 1994; Wang et al., from to on the et al., and and 1995). The of TFIIH in excision nuclease is of XPB and have that are to important for the and the nuclease to it has been that excision is absolutely on et al., However, it was both and required this we an incision with DNA in a with an of that the with and both and the that uncoupled incision to the that TFIIH of DNA is required for the nuclease and to the is required for both and the excision containing were the of and the was for with substrate. in the CDK7-cyclin H pair was found to a of TFIIH and to for activity et al., 1994; et al., 1995; et al., 1995). it was reported that of to excision repair in et al., to the that was essential for excision repair. TFIIH of the and H However, in the a of TFIIH of and H was obtained A and as by and H and a to the of a of the of that the TFIIH has of the activity of the TFIIH with the that CDK7-cyclin H is for the activity of that CDK7-cyclin H was in other repair factors XPG, and we the on both factors and a of the and found of activity the forms were for reconstitution of excision nuclease was the containing CDK7-cyclin H and the it we that the CDK7-cyclin H pair is necessary for with the repair of of TFIIH in excision. of the step by of the fractions by a of and H with and TFIIH of fractions and of TFIIH fractions and of protein as by the protein was as by et excision assays with and The of the excision are in of and the dual incision in excision of the repair the excised and the gapped DNA associated in a complex. complex is by repair which the repair gap and the et al., the we to in humans the excision nuclease a of the excised and the gapped DNA in a complex to the by repair with a was with either or human excision and the and gapped DNA were from the by Both the and the were on a that in this of the DNA was and from by the and the were in this it was found that of the excised oligomer associated with the gapped is in with studies that that incision the and and the excised oligomer in the complex with the gapped DNA et al., et al., with the human excision nuclease of the excised oligomer was in the that the excised oligomer is the gapped DNA the in humans, the excised oligomer is the of repair excision nuclease excised from gapped The in and 1994; et al., was with or human excision nuclease. The DNA was with and the and fractions were on a in and DNA in and by and with the and with the human excision nuclease. The of and human excision are The excision with the and proteins was as by et or the excised oligomer and the gapped DNA were from the excision nuclease or or we an excision a of and The were on in were observed in to free The DNA these was on that the DNA and DNA with an uncoupled The the excised but gapped as by the of from the DNA by 5′-incision the complex the gapped DNA and excised oligomer that was gapped DNA with to the was with to by in under these were the DNA and a of on a gel revealed that it the gapped as by the of the in a of gapped of of with a of and substrates was and the were on a DNA excision excision with gel were as by et of the DNA in the on in from the in The of the excision and of the 5′-incision are that to the of as a these experiments that the excised oligomer is from the gapped both the excised oligomer and the gapped DNA are with we the of the proteins in these However, it is important that the excision gap is by repair proteins from repair the excision repair factors purified to we have reconstituted human excision repair nuclease and the of the and the of in the excision We have used synthetic substrates with either a or an a for these the of repair factors of high and of has to the in In reconstitution studies of both human (Mu et al., 1995; Aboussekhra et al., 1995) and (Guzder et al., 1995) excision the of the reconstituted systems was of the was the that the reconstituted systems factors required for optimal The in factors are required for the excision sufficient of of the repair we were to the of in the that the excision and were to the in we the results obtained with this system to to excision repair in In the following we to the of the in of results in this and studies and a with on human nucleotide excision for of human excision nuclease. is by the of followed by the of TFIIH and XPC-HHR23B to the DNA the in an The of XPG through with TFIIH and the preincision complex and results in the 3′-incision of XPF-ERCC1 by XPA to the incision complex the 5′-incision the by XPF-ERCC1 the the excised oligomer is dissociated from the gapped DNA and by protein the gapped DNA by from of and cell-free it was found that was required for the repair step of excision repair et al., it was reported that was absolutely required for excision of DNA damage (Mu et al., 1995). However, reported that in the absence of RPA, of a to the system et al., that either one of the or that both with the other but that the The results that is absolutely required for both the and In with this it has been found that the incision activity of XPF-ERCC1 and the activity of ( and the that to XPA et al., 1995; et al., 1995; et al., 1995) and that both and are absolutely on RPA, we that RPA, in with as a damage complex and the nuclease to the incision by specific The results obtained with reconstituted excision nuclease are in with this The highly purified excision nuclease system was found to absolutely on for of (Guzder et al., of humans, the strand of is et al., XP mutants are defective in repair as as in repair However, XP-C mutants are mutants repair the strand of a normal lacking in repair of et al., 1995). It was that can by either protein or a DNA in a complex. The results in this that with excision can in the absence of In of these we that the of DNA of the transcription complex itself the role in repair. of of the of human excision repair the the and et al., and in to XP-C mutants are defective in repair et al., Friedberg et al., 1995). The role of is However, this protein to DNA with high and et al., 1994) ( and and with TFIIH with (Drapkin et al., in reconstitution of to of the damaged but the strand by the of XPG et al., a for is that it to the damaged strand in the preincision complex and the by the dual DNA from the nuclease of excision nuclease as as other of is to the damage by XPA et al., to in it DNA the of both and by that the of this factor et al., is important for both incisions. The activity is to the XPB and XPD et al., et al., et al., The and that have et al., 1994) play a role in DNA important TFIIH is or CDK7-cyclin is required for repair et reported that of excision repair in and that as was for repair. In an it was reported that in forms of TFIIH one and and one with these proteins and activity et al., 1994; et al., 1995). was that the TFIIH the repair activity to cell-free extracts of mutants et al., 1995). In humans, we found that TFIIH with or the subunit was equally active in excision repair a repair system of highly purified The that the with the through a to that the of with the is with we that the during the excision to the in of excision repair by it as that of the TFIIH is in the in and that of the with the repair complex to that or activity is required for excision of XPG and are the nuclease of the excision nuclease system. XPG is a with for to DNA for the strand the in the to et al., The protein has a to activity et al., experiments that XPG is the nuclease that the 3′-incision et al., with the of XPF-ERCC1 is a et al., with by for the the to DNA in the to with the that XPF-ERCC1 the 5′-incision et al., 1995). The experiments in this that XPG is required for the formation of a preincision complex. in to nuclease XPG has a role in a preincision complex, which XPF-ERCC1. an mutant cell-free a complex can with from a mutant cell or purified to excision. The of the 3′-incision observed in the defined system XPF-ERCC1 is to that of uncoupled excision by the system and that the complex in the absence of XPF-ERCC1 is to the one in the of XPF-ERCC1. In this the human excision nuclease system from the system and of subunits) the systems et al., Friedberg et al., 1995): in uncoupled 3′-incision in the absence of the complex, which is the and of XPF-ERCC1 (Guzder et al., of the XPA and XPF-ERCC1 et al., 1994; and 1994) and it is to that XPA in damage but as an to XPF-ERCC1 to the 5′-incision In other this that XPA an of the excision complex from the step of damage to the step to mutants a these repair damage of the normal but excision to with other XP mutants the of are (Cleaver and Kraemer, 1989). from these mutants are defective in excision nuclease activity as by an of of normal excision activity et al., In of the results in this that XPF-ERCC1 is required for an can for this of the other factors of the excision nuclease assemble and the which to damage removal by to the with the normal excision. an in an important is the uncoupled 3′-incision a normal during the of of human and it for a of the XPF-ERCC1 complex to the and the excision was in was a the of in to of the of dual incision by several The of excision repair proteins in a complex as has been for et al., 1995). XPF-ERCC1 is to the damage the to in this as protein can 3′-incision by of this protein by both and in a but experiments are to these the the the nuclease and the gapped DNA in a complex that is by repair in human following the dual the excised oligomer is free from the gapped However, both the excised oligomer and the gapped DNA are to specific repair as by the of following dual is important for of gapped DNA from by which produce strand we the of the proteins associated with either the excised oligomer or the gapped However, of is the that the excised complex has the complex of the gapped the of human et al., it is that the gapped DNA is associated with and for gap filling by DNA or et al., and The excised oligomer with it in the reactions excision is to the and the of excision nuclease INTRODUCTIONDNA repair reactions play an important role in preventing cancer development in humans. Individuals with defects in mismatch repair have high incidence of internal cancers (Kolodner and Alani, 1994; Modrich and Lahue, 1996). Similarly, individuals with defective nucleotide excision repair specific for bulky DNA lesions suffer from xeroderma pigmentosum (XP), ( 1The abbreviations used are: XPxeroderma pigmentosumRPAreplication protein APAGEpolyacrylamide gel electrophoresisCFEcell-free extractATPγSadenosine 5′-O-(thiotriphosphate)CTDcarboxyl-terminal domainCDKcyclin-dependent kinase.) which manifests itself by extremely high incidence of actinic cancers, increased incidence of cancers of internal organs, and mental and neurological abnormalities (Cleaver and Kraemer, 1989; Friedberg et al., 1995). In humans, mutations in seven genes, XPA through XPG, cause XP associated with defective excision repair (Cleaver and Kraemer, 1989). In addition, genetic and biochemical studies with rodent cell lines and radiation-sensitive Saccharomyces cerevisiae mutants and repair assays with cell-free extracts have implicated several other proteins in the excision step of nucleotide excision repair. In particular, it has been found that the general transcription factor TFIIH (five to eight subunits) (Drapkin et al., 1994; Schaeffer et al., 1994; Wang et al., 1994) and the trimeric replication protein A (RPA or HSSB) are absolutely required for the dual incision step of excision repair (Mu et al., 1995).Recently, excision repair has been reconstituted from highly purified repair factors in humans (Mu et al., 1995; Aboussekhra et al., 1995) and in the highly analogous S. cerevisiae system (Guzder et al., 1995). It has been reported that in both systems the following six fractions are necessary and sufficient for the dual incision activity (Mu et al., 1995; Guzder et al., 1995): XPA (Rad14), RPA, TFIIH (five to eight polypeptides including XPB (Rad25) and XPD (Rad3)), XPC-HHR23B (Rad4-Rad23), XPG (Rad2), and XPF-ERCC1 (Rad1-Rad10). In this study, we have obtained the six fractions of human excision nuclease (excinuclease) free of contaminants and have used synthetic substrates containing either a thymine dimer or a a to the of the excision nuclease. results that in with the other factors that are necessary for excision is required for excision of the thymine but for excision of a In this study, it is that the 3′-incision by XPG and 1994; et al., 1994; et al., 1995) precedes the 5′-incision by XPF-ERCC1 et al., 1995) and that the and that are in TFIIH as with the repair activity of TFIIH and are required for repair. the dual the excised et al., are in a complex with repair the gapped DNA with a of repair