Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

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Background: Arg-61 and Gln-38 of human DNA polymerase (hpol) play important roles in the catalytic reaction. Results: Mutations R61M or Q38A/R61A dramatically disrupt the activity of hpol . Conclusion: Polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. Significance: The positioning and positive charge of Arg-61 synergistically contribute to the activity of hpol , with additional effects of Gln-38.

Section: Methodsmentioning

confidence: 99%

“…Thus, Arg-61 can adopt different conformations during each stage of the catalytic reaction cycle. In addition, the reported structures have also indicated that another highly conserved residue near the active site, Gln-38, may play an important role in stabilizing the template base in the nucleotidyl transfer reaction (10,11,21,30,34,35,37).…”

mentioning

confidence: 99%

Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2′-deoxyguanosine

Patra

Harp

et al. 2015

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“…Specifically, we invoke a "purine rule," meaning that dATP and dGTP are preferred over dCTP and dTTP by this polymerase opposite an abasic lesion (48). Structures of the preferred dNTPs opposite the abasic site in ternary hpol complexes offered insight into this preference, in that the longer purine bases could be linked to the phosphate group of the abasic residue via water bridges.…”

Section: Tablementioning

confidence: 99%

Structural and Kinetic Analysis of Miscoding Opposite the DNA Adduct 1,N6-Ethenodeoxyadenosine by Human Translesion DNA Polymerase η

Patra¹,

Su²,

Zhang³

et al. 2016

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1,N 6-Ethenodeoxyadenosine (1,N 6 -⑀dA) is the major etheno lesion formed in the reaction of DNA with epoxides substituted with good leaving groups (e.g. vinyl chloride epoxide). This lesion is also formed endogenously in DNA from lipid oxidation. Recombinant human DNA polymerase (hpol ) can replicate oligonucleotide templates containing 1,N 6 -⑀dA. In steady-state kinetic analysis, hpol preferred to incorporate dATP and dGTP, compared with dTTP. Mass spectral analysis of incorporation products also showed preferred purine (A, G) incorporation and extensive ؊1 frameshifts, suggesting pairing of the inserted purine and slippage before further replication. Five x-ray crystal structures of hpol ternary complexes were determined, three at the insertion and two at the extension stage. Two insertion complexes revealed incoming non-hydrolyzable dATP or dGTP analogs not pairing with but instead in a staggered configuration relative to 1,N 6 -⑀dA in the anti conformation, thus opposite the 5-T in the template, explaining the proclivity for frameshift misincorporation. In another insertion complex, dTTP was positioned opposite 1,N 6 -⑀dA, and the adduct base was in the syn conformation, with formation of two hydrogen bonds. At the extension stage, with either an incorporated dA or dT opposite 1,N 6 -⑀dA and 2-deoxythymidine-5-[(␣,␤)-imido-]triphosphate opposite the 5-A, the 3-terminal nucleoside of the primer was disordered, consistent with the tendency not to incorporate dTTP opposite 1,N 6 -⑀dA. Collectively, the results show a preference for purine pairing opposite 1,N 6 -⑀dA and for ؊1 frameshifts.

“…pol catalyzes phosphodiester bond formation very rapidly, with k pol values reported from 50 to 190 s Ϫ1 (48,49). The elemental sulfur effect is small for both correct (1.6) and incorrect (2.5) base pair formation (48) indicating that the conformational changes are the primary rate-limiting steps.…”

mentioning

confidence: 99%

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

Gowda

Moldovan

Spratt

2015