Base Flipping in Nucleotide Excision Repair

UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A β-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3′ side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3′ incision by UvrC. UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A β-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3′ side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3′ incision by UvrC. DNA is constantly being threatened by various damaging agents, which can be either exogenous (chemicals or irradiation) or endogenous (reactive metabolites). If left unrepaired the damage could lead to mutations or cell death and therefore several repair mechanisms have evolved to avoid these effects. One of these mechanisms, nucleotide excision repair (NER) 2The abbreviations used are: NER, nucleotide excision repair; 2-AP, 2-aminopurine; CK, creatine kinase; CP, creatine phosphate; ATPγS, adenosine 5′-O-(thiotriphosphate); wt, wild type; AU, arbitrary units. is characterized by the unique feature that it is able to recognize and repair a large variety of structural different damages (1Friedberg E.C. Walker W. Siede W. DNA Repair and Mutagenesis. America Society for Microbiology, Washington, D. C.1995Google Scholar). In bacteria, NER is initiated by three different proteins: UvrA, UvrB, and UvrC (reviewed in Refs. 2Goosen N. Moolenaar G.F. Res. Microbiol. 2001; 152: 401-409Crossref PubMed Scopus (61) Google Scholar and 3Van Houten B. Croteau D.L. DellaVecchia M.J. Wang H. Kisker C. Mutat. Res. 2005; 577: 92-117Crossref PubMed Scopus (115) Google Scholar). First UvrA and UvrB form a complex in solution, which is able to search the DNA for possible damage (4Orren D.K. Sancar A. J. Biol. Chem. 1990; 265: 15796-15803Abstract Full Text PDF PubMed Google Scholar). Once damage has been found, UvrA leaves the complex resulting in a UvrB-DNA preincision complex. Atomic force microscopy (5Verhoeven E.E.A. Wyman C. Moolenaar G.F. Goosen N. EMBO J. 2002; 21: 4196-4205Crossref PubMed Scopus (98) Google Scholar) and bandshift analysis (23Moolenaar G.F. Schut M. Goosen N. DNA Repair. 2005; 4: PubMed Scopus Google Scholar) of this complex have that it UvrB with bound to the and the more The role of this UvrB in the repair to be UvrC to the preincision complex, the UvrB (5Verhoeven E.E.A. Wyman C. Moolenaar G.F. Goosen N. EMBO J. 2002; 21: 4196-4205Crossref PubMed Scopus (98) Google G.F. Schut M. Goosen N. DNA Repair. 2005; 4: PubMed Scopus Google Scholar). UvrC the in the First an incision is at the or 3′ to the damage A. D. Full Text PDF PubMed Scopus Google E.E.A. M. Moolenaar G.F. Goosen N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google which is by an incision at the to the damage Sancar A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The incision is by an incision to the which has been to be the of of the by UvrB G.F. M. Goosen N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The resulting is by which in the and the in the In bacterial a is present that can a 3′ incision at the 3′ to the damage and is to a for UvrC G.F. M. Goosen N. A. 2002; PubMed Scopus Google Scholar). the NER binding and by UvrA and UvrB of the UvrA The role of these in damage is but it has been that by UvrA is for from non-damaged J. Biol. Chem. Full Text PDF PubMed Google an important role in the for The UvrB protein and the in this by UvrA and DNA A. PubMed Scopus Google Scholar). that binding by UvrB of the DNA of the UvrB both in in search of a damage and in bound to a (5Verhoeven E.E.A. Wyman C. Moolenaar G.F. Goosen N. EMBO J. 2002; 21: 4196-4205Crossref PubMed Scopus (98) Google E.E.A. Wyman C. Moolenaar G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google Scholar). The of UvrB has been to be with strand EMBO J. PubMed Scopus Google G.F. M. Goosen N. J. Biol. PubMed Scopus Google M. DellaVecchia M.J. Croteau D.L. Kisker C. Houten B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google which is used to the DNA at a lesion to damage-specific binding. the of UvrB the a to be bound to form a preincision complex that can be by UvrC G.F. Goosen N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of UvrB from M. Houten B. Kisker C. EMBO J. PubMed Scopus Google Scholar) and M. M. J. A. PubMed Scopus Google N. M. J. PubMed Scopus Google Scholar) a feature of a β-hairpin a of residues at base and the of the a protein that is to form a stable preincision complex M. Kisker C. Houten B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). A for DNA binding by UvrB the β-hairpin inserts the of the of the the β-hairpin and of the protein M. Houten B. Kisker C. EMBO J. PubMed Scopus Google Scholar). In to different of base UvrB has been to recognize a in the DNA G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google Scholar). this a for damage UvrB the DNA for the of damage by to of the DNA helix. alterations in base this base flipping and a a flipped of residues and at the base of the β-hairpin with to UvrB that to the cell of stable binding to non-damaged G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google G.F. Schut M. Goosen N. DNA Repair. 2005; 4: PubMed Scopus Google Scholar). This could be by the base flipping for damage In the of flipping is the non-damaged base is in by with This in a with and and of UvrB from the damage is present the base can be flipped of the DNA and residues and the G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google Scholar). flipping is not in DNA of B. PubMed Scopus Google A. PubMed Scopus Google PubMed Scopus Google and A. PubMed Scopus Google Scholar) have that the base in an extrahelical position. is not to the in the structure of it that it is not the lesion but a adenine the that extrahelical M. Full Text PDF PubMed Scopus Google Scholar). In this we have in the structure by the UvrB protein using the fluorescent adenine analogue can be in the DNA using and to the DNA with a with which it can form a base A. PubMed Scopus Google Scholar). of with at a in an of In a DNA fragment this is by base and therefore can be used a for base flipping PubMed Scopus Google Scholar). of has been used to base flipping can be for DNA J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google PubMed Scopus Google B. Res. PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We show that binding of UvrB to a DNA fragment with a cholesterol lesion the base adjacent to the lesion at the 3′ side in an extrahelical position. Also the nucleotide opposite this flipped base is extruded from the DNA in the UvrB-DNA complex. UvrB with in the β-hairpin that residues and not for the base Tyr-95 to be specifically involved in flipping the base in the non-damaged strand and this base flipping to be for the 3′ incision by UvrC. and DNA the UvrB has been G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google Scholar). The and in a using by DNA the G.F. Goosen N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The UvrA M. Moolenaar G.F. J. Biol. Chem. Full Text PDF PubMed Google UvrB G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google UvrC M. Moolenaar G.F. J. Biol. Chem. Full Text PDF PubMed Google and G.F. M. Goosen N. A. 2002; PubMed Scopus Google Scholar) The DNA used in this and in from and from DNA at the side of the strand using M. Moolenaar G.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The DNA with UvrA, UvrB, and UvrC or in of and at for the by using of and of by The incision a DNA with UvrA and UvrB in of for at The a and in M. Moolenaar G.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar). UvrB binding used for the the of DNA with DNA The UvrA, UvrB, and with the DNA in of for at in the of or CP, which a UvrB complex in the used and the at different by the Fluorescence UvrA, UvrB, and in for at in the of or acrylamide to the the The to a and in the of the Fluorescence using a which to a to a the of The at and by from to The and and for by the from the The by in of at the and DNA and in the which at a the of the cofactor or to the at different UvrB the at in the and of a a cholesterol the base with different DNA used and in which the the cholesterol lesion in with The three DNA have three opposite the lesion in with a we a DNA fragment with a the cholesterol First we the of the base incision of the DNA The base is not by the with the not show incision The of and the DNA with the very the of and the The of the of the of this incision the incision is with the has been that the of the lesion can the by which UvrC the DNA E.E.A. M. Moolenaar G.F. Goosen N. Res. 2002; PubMed Scopus Google Scholar). that in this the in incision is by UvrC and not by a in UvrB we the of and using the of UvrC both DNA very that UvrB binding is very UvrB binding more we a bandshift analysis the different with or A and that the of UvrB-DNA complex is for that not or of these UvrB-DNA with the and damage the for Fluorescence a of 2-AP, a DNA of is PubMed Scopus Google J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google B. Res. PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). This is higher the DNA used in the incision and DNA binding we to the protein for of the UvrB-DNA complex at these DNA The of UvrA to and UvrB to but for both and the of UvrB-DNA with DNA with the with DNA for and binding to DNA damage by UvrA and UvrB. at protein and DNA the of and the the this we an in of and this of DNA with UvrA and UvrB to a of UvrB-DNA that these at the of the UvrB has been (23Moolenaar G.F. Schut M. Goosen N. DNA Repair. 2005; 4: PubMed Scopus Google Scholar) that the UvrB which is more with the complex, at UvrB in in A and but is at UvrB in in UvrB-DNA with these at the 3′ of the of a very at in DNA a lesion the is is less when the DNA is in the form but is the of the different the DNA the DNA with UvrA the DNA with UvrA and the at and from by more of the in the of DNA not in not First we the of the lesion the of the in the DNA of in the and is for damage with the cholesterol lesion not the of at and with to the the at and the damage the In the to the damage a a in base The the 3′ to the damage is but less of UvrA to not in change in of of these In for and it is that is higher in the DNA in the fragment and This that damage binding by UvrA not base at the 3′ side of the the the of damage-specific be to be able to conformational UvrA UvrB in the of the UvrB-DNA complex, a change in at the 3′ side of the damage is the at UvrB binding in a that is a of three higher the of the DNA The of this in the UvrB-DNA complex is of the of a of the base This very can be by base the and The at a in binding of UvrB In this the is a of higher the of the DNA that is by of the This can be by a of the UvrB protein the base at the position. the of the base flipping at base flipping at is to at the side of the change in UvrB binding is Fluorescence of very that in the UvrB-DNA complex the at is Also the of at not change UvrB binding of this residue higher in the a of by the this that the of the damage not change in the UvrB-DNA complex. in the of the three opposite the damage is with the non-damaged a of three residues by the damage The is in and the of these higher the and the opposite the lesion and at of the by the cholesterol of UvrA not a change in of the different binding of UvrB the in a that is a of three higher that of the DNA and of the with the base 3′ to the the opposite is in an extrahelical by UvrB. The of of this in the non-damaged strand is less that of the adjacent to the the of UvrB-DNA both and is the A and This that the of the in the strand is either it is extruded from the or by of residues of the UvrB protein. Fluorescence of opposite the lesion or at which in the DNA of the not change UvrB binding and this could that UvrB binding not the of the cholesterol in the DNA helix. not be that in the UvrB-DNA the opposite the lesion and at by the lesion but that the UvrB protein these residues to a of The in the into a of the UvrB of the of in complex with UvrB that the is with the of the base a that the extrahelical residue in the is in an with a J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of the extrahelical 2-AP, using of acrylamide to the base in a of the from to arbitrary of the of acrylamide to the UvrB-DNA complex not of that the extrahelical base is not accessible to the This that the base adjacent to the damage is flipped into a pocket of the UvrB which is to of We with the UvrB-DNA complex of acrylamide in a of the from to a we that the acrylamide not in the of the UvrB-DNA complex not the is of acrylamide of the This that the base in the non-damaged strand is and that it is not by residues of the UvrB protein. by in the UvrB-DNA and to this complex in the at a protein and DNA and First these with in the of the using CP, the a in the it a a in that by a this change of in be to a change in the we the in the of a higher of a but this to which it is by a of the and a the of that of the of CP, which the of a the of the the and of be by a of and therefore the UvrB in the ADP-bound The of is higher with with CP, suggesting that an in the in more UvrB-DNA Also for a change in which to the in the both a that is by a of the or The of the with of the CP, not for but it a that is that of the DNA AU, the is to and of the UvrB-DNA complex we monitored and of these in a and and with UvrA and UvrB and the using different the by the both at a of can be these or of the the of to a both This very to the in of and that the This that the residues in both DNA is to of the UvrB-DNA complex. the of UvrB-DNA the to of This that the in is not of of the complex, but to a in the of the residue the UvrB or a different of the UvrB-DNA complex for the complex stable the when it is to be in the ATP-bound but it in it is in the ADP-bound of the UvrB monitored in the to be at a in the This can be by the both and UvrB that has from the DNA can the is the of the UvrB-DNA in the can be when of the UvrB is in the ADP-bound The in of the and be by the of the a of in which a different of UvrB with the DNA the the of the cofactor the in the DNA we the with the of and and ATPγS, or at different the of the of to of the the the of with the of the The of at the in a of the which of UvrB the the of is to be very be and therefore the to the of the ADP-bound UvrB. The of can be to of the complex. the the in that it is the that is for these alterations of the The of to of ATPγS, that in the ATP-bound form the UvrB-DNA complex is very stable. this the UvrB-DNA complex has been is for the base to more that the in the of is of the ADP-bound form of UvrB, we to the at this is left in the that of the to we a large of a the more which the of the of and this is the is left in the and of the be to the that is is an of the and ATP-bound the is the and with or of the when the is to be to of the in an of the The with is higher with ATPγS, which be by the of a of in the the of but with the that of and a of of the This that the of the of the and ADP-bound of UvrB, Tyr-95 in in the the of different UvrB base flipping in and UvrB at of in the of and First we used a that in This has an in the Walker A of the resulting in a protein capable of with UvrA but to A. PubMed Scopus Google Scholar). not form UvrB-DNA and and is incision by UvrC or and and with the for UvrA the in the of UvrA and is higher the of UvrA but with UvrA and This that base flipping of both residues can be UvrB has been the of UvrB the of and The DNA for at with UvrA and UvrB in the of the using The at and at is the of at in a UvrB can to the DNA damage a with with the and the more (5Verhoeven E.E.A. Wyman C. Moolenaar G.F. Goosen N. EMBO J. 2002; 21: 4196-4205Crossref PubMed Scopus (98) Google G.F. Schut M. Goosen N. DNA Repair. 2005; 4: PubMed Scopus Google Scholar). the involved in UvrB and a the UvrB from the UvrB-DNA complex (5Verhoeven E.E.A. Wyman C. Moolenaar G.F. Goosen N. EMBO J. 2002; 21: 4196-4205Crossref PubMed Scopus (98) Google G.F. Schut M. Goosen N. DNA Repair. 2005; 4: PubMed Scopus Google Scholar). The of UvrB preincision by with wild type and and by and by not and the an important G.F. Goosen N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in the of UvrA to that of the wild type for both DNA the This that the UvrB is not for of the extrahelical The β-hairpin of UvrB plays an important role in damage-specific binding by UvrB M. Kisker C. Houten B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google H. 4196-4205Crossref PubMed Scopus Google Scholar). can therefore be that residues in this structure involved in the base flipping that we We have three different with in the residues of the The has been G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google G.F. Schut M. Goosen N. DNA Repair. 2005; 4: PubMed Scopus Google Scholar). of the residues in a protein that can form stable with the is capable of the The of and by UvrC or in the of the with in the of wild type UvrB and and G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google Scholar) the UvrB of the is more with the complex with the DNA the damage and therefore the bandshift the complex and complex of the is to that of The of a stable UvrB with of a very both This that the UvrB not base the of the protein the show that residues and not involved in either of the in an extrahelical position. We that the capable of damage-specific binding G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google Scholar). of these β-hairpin residues is involved in base flipping we have the and very in UvrB-DNA complex and In incision by UvrC and or and is Fluorescence of both and not by the that by to be able to change in in with the by M. DellaVecchia M.J. Croteau D.L. Kisker C. Houten B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the of B. UvrB is not capable of a stable UvrB-DNA complex. very UvrB-DNA and for the these in the that the the incision of the is very for the wild type and In UvrC incision is and is in the 3′ incision that 3′ incision a at the 3′ the incision with is wild type The 3′ incision be to the more stable UvrB the in which the UvrB is incision by UvrC. is more that the protein to a in the DNA that is for UvrC this be from the the of is with that by that base flipping adjacent to the damage when residue Tyr-95 is with not the of or This that Tyr-95 is involved in the base in the non-damaged strand. In the of this base be important for the UvrC incision with with UvrB G.F. Goosen N. EMBO J. 2001; PubMed Scopus Google G.F. Schut M. Goosen N. DNA Repair. 2005; 4: PubMed Scopus Google is that the UvrB protein the DNA for the of damage by to of the helix. base is a of the of flipping be to of a stable UvrB-DNA complex. The not of the of the damage UvrB but we show that the base adjacent to the damage at the 3′ side is flipped into an extrahelical by UvrB. could be the for flipping of this non-damaged One possible be that the of the lesion used in these that the adjacent base is of the helix. The cholesterol damage used is to the DNA a G.F. M. Goosen N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and is or base present at this position. of a UvrB the base that is not which is the base adjacent to the the the in the of acrylamide that the extrahelical base adjacent to the damage inserts into a pocket of the UvrB protein that is to of the is not very that a pocket the of a base that a large variety of flipping of the adjacent base be a for damage of a base it can be that the the flipping and to damage in the DNA used in this the of the cholesterol lesion not be DNA binding is by the of the residues to the lesion and in the non-damaged strand opposite the lesion and at In three the lesion of these residues in the UvrB the of these residues not which could that the cholesterol is in the not flipped in an extrahelical The base in the non-damaged strand is UvrB binding. The that for this residue base flipping is less suggesting that with is has been that a residue opposite an is less in a Res. PubMed Scopus Google Scholar). The of the base in the non-damaged strand that we could therefore be the of flipping the base adjacent to the the of this flipping an the we could show that of the base flipping of the residue adjacent to the damage wild of the base in the non-damaged strand Tyr-95 therefore to be involved in of this base in the non-damaged either by it or by it to to position. and H. PubMed Scopus Google Scholar) have using of the in a that binding of UvrB to DNA residue Tyr-95 less exposed. to of this residue in DNA can be that a residue can an extruded base in a a The preincision by can be by with the the wild type The 3′ incision by is is not that residue Tyr-95 is involved in binding to UvrC this residue into not the incision H. 4196-4205Crossref PubMed Scopus Google Scholar). it is the extruded base in the non-damaged strand or conformational in the UvrB-DNA complex by the that form an important for the UvrC protein. The cofactor have that the base in the non-damaged strand is more when UvrB is bound to or when it is in the ADP-bound In with this we have in a that the 3′ incision by UvrC is higher when the UvrB preincision complex or with G.F. Goosen N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The with show that these residues not for flipping of the base adjacent to the for being involved in the flipped configuration of this damage base is in with this but this to the to form a stable preincision complex. One in that it is very that a that base flipping stable UvrB binding. a for the flipped in that of the with the a of UvrB binding the of the of the in the to be is not from this is of the UvrB that or to the that for the UvrB in the ADP-bound either by the or by it for in The of of the residues varies with or Fluorescence of the base adjacent to the damage higher when UvrB is in the ADP-bound this the of the base flipping that of UvrB. UvrB it G.F. Goosen N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). this UvrB is in the ADP-bound in which the base is extrahelical but not into the protein In the ADP-bound the complex is not stable and the this can lead to of the protein from the In a the is with that of the base into the protein resulting in a more stable UvrB-DNA complex has been by M. Houten B. Kisker C. EMBO J. PubMed Scopus Google Scholar) that UvrB to the DNA a of the DNA the β-hairpin and of the protein. If we in of this it that UvrB binding to a when has been and UvrB is in the ADP-bound this is the DNA to from the and of the complex. The of with the resulting in a very stable complex, in which the base in the non-damaged strand is extruded to incision by UvrC.

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