9cih
Crystal structure of human polymerase eta with incoming dCMPnPP nucleotide across O4-methyl threofuranosyl thymidine in DNA template at extension stage
Structural highlights
DiseasePOLH_HUMAN Defects in POLH are the cause of xeroderma pigmentosum variant type (XPV) [MIM:278750; also designated as XP-V. Xeroderma pigmentosum (XP) is an autosomal recessive disease due to deficient nucleotide excision repair. It is characterized by hypersensitivity of the skin to sunlight, followed by high incidence of skin cancer and frequent neurologic abnormalities. XPV shows normal nucleotide excision repair, but an exaggerated delay in recovery of replicative DNA synthesis. Most XPV patients do not develop clinical symptoms and skin neoplasias until a later age. Clinical manifestations are limited to photo-induced deterioration of the skin and eyes.[1] [2] [3] [4] [5] FunctionPOLH_HUMAN DNA polymerase specifically involved in DNA repair. Plays an important role in translesion synthesis, where the normal high fidelity DNA polymerases cannot proceed and DNA synthesis stalls. Plays an important role in the repair of UV-induced pyrimidine dimers. Depending on the context, it inserts the correct base, but causes frequent base transitions and transversions. May play a role in hypermutation at immunoglobulin genes. Forms a Schiff base with 5'-deoxyribose phosphate at abasic sites, but does not have lyase activity. Targets POLI to replication foci.[6] [7] [8] [9] [10] Publication Abstract from PubMedalpha-l-(3'-2')-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase eta (hPol eta) opposite a TNA-modified template strand without and in combination with O(4)-alkyl thymine lesions. Across TNA-T (tT), hPol eta inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol eta inserted dAMP opposite O(4)-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol eta complexes with template tT and O(4)-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3' --> 2' versus 5' --> 3', respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3'-OH of the primer's terminal thymine was positioned at 3.4 A on average from the alpha-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol eta. Conversely, the crystal structure of a ternary hPol eta.DNA.tTTP complex revealed that the primer's terminal 3'-OH was too distant from the tTTP alpha-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-a-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate. DNA Replication across alpha-l-(3'-2')-Threofuranosyl Nucleotides Mediated by Human DNA Polymerase eta.,Tomar R, Ghodke PP, Patra A, Smyth E, Pontarelli A, Copp W, Guengerich FP, Chaput JC, Wilds CJ, Stone MP, Egli M Biochemistry. 2024 Sep 11. doi: 10.1021/acs.biochem.4c00387. PMID:39259676[11] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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