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Berkeley MCELLBI 110 - Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase

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30. Beclin, C., Boutet, S., Waterhouse, P. & Vaucheret, H. A branched pathway for transgene-inducedRNA silencing in plants. Curr. Biol. 12, 684–688 (2002).Supplementary Information accompanies the paper on www.nature.com/nature.Acknowledgements This research was supported by grants from AIST and by a Grant-in-Aid forScientific Research and for the 21st Century COE programmes, Center for Integrated BrainMedical Science and Human-Friendly Materials based on Chemistry, from the Ministry ofEducation, Culture, Sports, Science and Culture (MEXT) of Japan.Competing interests statement The authors declare that they have no competing financialinterests.Correspondence and requests for materials should be addressed to K.T.([email protected]) or H.K. ([email protected])...............................................................Error-prone replication ofoxidatively damaged DNA bya high-fidelity DNA polymeraseGerald W. Hsu1, Matthias Ober2, Thomas Carell2& Lorena S. Beese11Department of Biochemistry, Duke University Medical Center, Durham, NorthCarolina 27710, USA2Department of Chemistry and Biochemistry, Ludwig Maximilians UniversityMunich, Butenandtstrasse 5-13, D 81377 Munich, Germany.............................................................................................................................................................................Aerobic respiration generates reactive oxygen species thatcan damage guanine residues and lead to the production of8-oxoguanine (8oxoG), the major mutagenic oxidative lesion inthe genome1. Oxidative damage is implicated in ageing2andcancer, and its prevalence presents a constant challenge to DNApolymerases that ensure accurate transmission of genomic infor-mation. When these polymerases encounter 8oxoG, they fre-quently catalyse misincorporation of adenine in preference toaccurate incorporation of cytosine3. This results in the propa-gation of G to T transversions, which are commonly observedsomatic mutations associated with human cancers4,5. Here, wepresent sequential snapshots of a high-fidelity DNA polymeraseduring bo th accurate and mutagenic replication of 8oxoG.Comparison of these cr ystal structures reveals that 8oxoGinduces an inversion of the mismatch recognition mechanismsthat normally proofread DNA, such that the 8oxoGzadeninemismatch mimics a cognate base pair whereas the 8oxoGzcyto-sine base pair behaves as a mismatch. These studies reveal afundamental mechanism of error-prone replication and showhow 8oxoG, and DNA lesions in general, can form mismatchesthat evade polymerase error-detection mechanisms, potentiallyleading to the stable incorporation of lethal mutations.Many DNA damage lesions stall or block DNA replication;however, 8oxoG is bypassed efficiently and inaccurately by high-fidelity polymerases3,6–8. 8oxoG retains the ability to engage incorrect Watson–Crick base pairs with C, but oxidation of G (atC8) converts a hydrogen bond acceptor (N7) to a hydrogen bonddonor, allowing a stable Hoogsteen base pair to form between8oxoG and A, which is not possible in undamaged DNA (Fig. 1). Inthe absence of accessory factors such as proliferating cell nuclearantigen (PCNA), preferential mutagenic translesion replicationof 8oxoG by the major replicative DNA polymerasesaanddin vitro indicates that 8oxoG is not recognized as a DNA lesion.Instead, the Az8oxoG mismatch evades the intrinsic mechanismsthat cause DNA polymerases to achieve high-fidelity replication ofundamaged DNA.Insights into the mechanisms of faithful replication by high-fidelity polymerases9and mismatch recognition10have been pro-vided by capturing structures of the replication cycle of the DNApolymerase I fragment from a thermostable strain of Bacillusstearothermophilus (BF) in a polymerase crystal that retains theability to replicate DNA11. Throughout the replicative cycle of BF,there are many points at which recognition of DNA mismatches orlesions can occur. First, during the transition of the template strandfrom a pre-insertion site that sequesters the template base beforenucleotide incorporation to an insertion site where the templatebase interacts with the incoming nucleotide, the conformations ofthe template base and the incoming nucleotide are both tightlyregulated to ensure that hydrogen bonding interactions are limitedto the Watson–Crick faces of the nascent base pair. Second, beforecovalent incorporation of the paired dNTP at the insertion site, thepolymerase selects for base pairs that exhibit the shape andgeometry of cognate Watson–Crick base pairs in preference toones that do not12,13. Third, after covalent incorporation, the newbase pair moves to a post-insertion site where residues Arg 615 andGln 797 form hydrogen bonds to the DNA minor groove of correctlyformed base pairs. At this step, DNA mismatches induce distortionswithin the polymerase active site that cause the polymerase to stalland dissociate10. Last, covalently incorporated mismatches orlesions continue to impede replication from up to four base pairsaway14, inducing distortions to the active site as they translocatealong the surface of the polymerase10. In this way, a molecular‘memory’ of the mismatch is retained by the polymerase.To investigate whether BF behaves analogously to replicativepolymerasesaanddwith respect to replication of 8oxoG, primerextension assays were performed (Fig. 2) and steady-state kineticparameters were determined for incorporation of dCTP or dATPopposite 8oxoG (Table 1). Comparison of the specificity constants(kcat/Km; where kcatis the turnover number and Kmis the Michaelisconstant) for incorporation of these nucleotides opposite 8oxoGindicates that misincorporation of dATP is ninefold more efficientthan dCTP incorporation, whereas in undamaged DNA, accuratedCTP incorporation opposite an unmodified guanine is 106-foldmore efficient than misincorporation of dATP. After incorporationof either dATP or dCTP, extension past the newly formed 8oxoGbase pair is readily observed (Fig. 2a, b) and proceeds to the end ofthe template in the presence of a full complement of dNTPs(Supplementary Fig. 1). These results indicate that BF, as withreplicative polymerasesaandd, preferentially misincorporatesdATP opposite 8oxoG. BF can therefore serve as a model systemto address mutagenic 8oxoG replication.To determine the effects of 8oxoG on the structural mechanismsFigure 1 Modes of base pairing for 8oxoG. a, Oxidation of guanine at C8


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