Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

Abstract

1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG1,8·dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism.

Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases

“…As with 20/26-mer-dG C8- N -ABA , a similar biphasic kinetic trend and a dNTP concentration-dependent increase in the rate constants were observed with 21/26-mer-dG C8- N -ABA during the extension step (Figure 3B and Table 5). Interestingly, the dNTP concentration-dependent increase in the rate constants ( k f and k s ) had been observed previously with Dpo4 when it bypassed the dG C8- N -ABA 11 and dG 1,8 lesions 30 . However, our current study is the first one to report this concentration-dependence for lesion bypass catalyzed by a human Y-family DNA polymerase.…”

Investigating the trade-off between folding and function in a multidomain Y-family DNA polymerase

“…For example, DPO4 binds DNA containing benzo[ a ]pyrene-deoxyguanosine and allows the bulky lesion to be flipped/looped out of the DNA helix into a structural gap between the F and LF domains ( Bauer et al, 2007 ). In addition, the structure of DPO4 with DNA containing 8-(deoxyguanosine- N 2 -yl)-1-aminopyrene (dG1,8) reveals that the dG moiety of the bulky lesion projects into the cleft between the F and LF domains of DPO4 ( Vyas et al, 2015 ). These structural characteristics, differing from those of DPO4 binding to undamaged DNA, provide the dynamic basis for TLS and are probably favored by the fluctuating F and T domain conformations when binding to DNA with backtracking.…”

“…These expectations can be verified through future FRET experiments. Furthermore, the structural determination of DPO4 in complex with a damaged DNA substrate and an incoming nucleotide ( Ling et al, 2001 ; Vaisman et al, 2005 ; Ling et al, 2004b ; Bauer et al, 2007 ; Ling et al, 2004a ; Vyas et al, 2015 ) can provide insights into the effects of backtracking caused by weakening intradomain interactions via mutations, which disrupt the structures of the ternary complexes.…”

Conformational Insights into the Mechanism of Acetylaminofluorene-dG-Induced Frameshift Mutations in the NarI Mutational Hotspot

“…33 These SMIs exist in a mixture of B- and S-SMI conformers, with the population of the S conformer and the thermodynamic stability being in the order of −1 > −2 > −3. Vyas and co-workers 37 have recently obtained crystals of the complex of Dpo4 and DNA containing templating of the 1,8-diaminopyrene dG lesion in the presence of dCTP. The ternary structure showed that the bulky planar pyrene is sandwiched between the nascent and junction base pairs, while the modified guanine is exposed in the major groove.…”