Suse Broyde

DNA-peptide cross-links (DpCs) are generated via the proteolytic cleavage of DNA-protein cross-links (DPCs), ubiquitous DNA lesions that block DNA replication and transcription. Translesion synthesis (TLS) DNA polymerases can facilitate replication bypass of DpC adducts in either an error-free or error-prone manner. We have previously demonstrated that local DNA sequence context significantly influences hPol η-mediated replication bypass of 5-formylcytosine (5fC)-mediated DpC lesions. However, the effects of peptide sequence on the efficiency and fidelity of the TLS bypass of 5fC-mediated DpC lesions remained unknown. In the present study, model DpCs containing three different peptides (NH₂-GGGKGLGK*GGA-COOH, NH₂-RPK*PQQFFGLM-COOH, and NH₂-RPKPQQFK*GLM-COOH, K* = oxy-lysine) were subjected to primer extension experiments in the presence of TLS polymerases. We found that in vitro replication of DpC-containing templates by hPol η was more efficient than that catalyzed by hPol l or hPol κ. HPLC-ESI-MS and HPLC-ESI-MS/MS analyses of hPol η primer extension products indicated that the replication bypass of DpC containing NH₂-RPK*PQQFFGLM-COOH was more error-prone than replication of the other two DpCs, leading to targeted C → T transitions, small deletions, and untargeted mutations downstream from the lesion. Steady-state kinetics investigation of hPol η-catalyzed nucleotide incorporation opposite the DpC lesions containing three different peptides revealed that, in all cases, error-free replication was far more efficient than incorporation of incorrect nucleotides. For mutagenic bypass, the catalytic efficiency of hPol η-mediated dAMP misincorporation opposite DpC with peptide NH₂-RPK*PQQFFGLM-COOH was higher than adenine misincorporation across from the other two DpCs and unmodified dC. These steady-state kinetic findings were further explained by molecular modeling and molecular dynamics simulations, revealing that the three different DpC lesions impose varying perturbations to the geometry of the C-G and C-A pairs at the hPol η active site. Collectively, our results reveal that the peptide sequence and conjugation chemistry of DpC lesions can influence the fidelity of lesion bypass by TLS polymerases.

The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5′ → 3′ direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6–4) pyrimidone (6–4PP) photolesion. The experimentally observed unwinding rate constants (k_obs) and processivities (P) were examined. Relative to undamaged DNA, the k_obs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2–5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest k_obs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions’ inhibitory effects than unwinding rate constants.

Ultraviolet light generates cyclobutane pyrimidine dimer (CPD) and pyrimidine 6−4 pyrimidone (6−4PP) photoproducts that cause skin malignancies if not repaired by nucleotide excision repair (NER). While the faster repair of the more distorting 6–4PPs is attributed mainly to more efficient recognition by XPC, the XPD lesion verification helicase may play a role, as it directly scans the damaged DNA strand. With extensive molecular dynamics simulations of XPD-bound single-strand DNA containing each lesion outside the entry pore of XPD, we elucidate strikingly different verification processes for these two lesions that have very different topologies. The open book-like CPD thymines are sterically blocked from pore entry and preferably entrapped by sensors that are outside the pore; however, the near-perpendicular 6−4PP thymines can enter, accompanied by a displacement of the Arch domain toward the lesion, which is thereby tightly accommodated within the pore. This trapped 6−4PP may inhibit XPD helicase activity to foster lesion verification by locking the Arch to other domains. Furthermore, the movement of the Arch domain, only in the case of 6−4PP, may trigger signaling to the XPG nuclease for subsequent lesion incision by fostering direct contact between the Arch domain and XPG, and thereby facilitating repair of 6−4PP.

DNA helicase unwinding activity can be inhibited by small molecules and by covalently bound DNA lesions. Little is known about the relationships between the structural features of DNA lesions and their impact on unwinding rates and processivities. Employing E.coli RecQ helicase as a model system, and various conformationally defined DNA lesions, the unwinding rate constants k_obs = k_U + k_D, and processivities P = (k_U/(k_U + k_D) were determined (k_U, unwinding rate constant; k_D, helicase-DNA dissociation rate constant). The highest k_obs values were observed in the case of intercalated benzo[a]pyrene (BP)-derived adenine adducts, while k_obs values of guanine adducts with minor groove or base-displaced intercalated adduct conformations were ~10–20 times smaller. Full unwinding was observed in each case with the processivity P = 1.0 (100% unwinding). The k_obs values of the non-bulky lesions T(6−4)T, CPD cyclobutane thymine dimers, and a guanine oxidation product, spiroiminodihydantoin (Sp), are up to 20 times greater than some of the bulky adduct values; their unwinding efficiencies are strongly inhibited with processivities P = 0.11 (CPD), 0.062 (T(6−4)T), and 0.63 (Sp). These latter observations can be accounted for by correlated decreases in unwinding rate constants and enhancements in the helicase DNA complex dissociation rate constants.

In nucleotide excision repair (NER), the xeroderma pigmentosum D helicase (XPD) scans DNA searching for bulky lesions, stalls when encountering such damage to verify its presence, and allows repair to proceed. Structural studies have shown XPD bound to its single-stranded DNA substrate, but molecular and dynamic characterization of how XPD translocates on undamaged DNA and how it stalls to verify lesions remains poorly understood. Here, we have performed extensive all-atom MD simulations of human XPD bound to undamaged and damaged ssDNA, containing a mutagenic pyrimidine (6−4) pyrimidone UV photoproduct (6−4PP), near the XPD pore entrance. We characterize how XPD responds to the presence of the DNA lesion, delineating the atomistic-scale mechanism that it utilizes to discriminate between damaged and undamaged nucleotides. We identify key amino acid residues, including FeS residues R112, R196, H135, K128, Arch residues E377 and R380, and ATPase lobe 1 residues 215−221, that are involved in damage verification and show how movements of Arch and ATPase lobe 1 domains relative to the FeS domain modulate these interactions. These structural and dynamic molecular depictions of XPD helicase activity with unmodified DNA and its inhibition by the lesion elucidate how the lesion is verified by inducing XPD stalling.

DNA–protein cross-links (DPCs) are unusually bulky DNA lesions that form when cellular proteins become trapped on DNA following exposure to ultraviolet light, free radicals, aldehydes, and transition metals. DPCs can also form endogenously when naturally occurring epigenetic marks [5-formyl cytosine (5fC)] in DNA react with lysine and arginine residues of histones to form Schiff base conjugates. Our previous studies revealed that DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA–peptide conjugates. We have shown that 5fC-conjugated DNA–peptide cross-links (DpCs) placed within the CXA sequence (X = DpC) can be bypassed by human translesion synthesis (TLS) polymerases η and κ in an error-prone manner. However, the local nucleotide sequence context can have a strong effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex among the DNA template, polymerase, and the incoming dNTP. In this work, we investigated polymerase bypass of 5fC–DNA–11-mer peptide cross-links placed in seven different sequence contexts (CXC, CXG, CXT, CXA, AXA, GXA, and TXA) in the presence of human TLS polymerase η. Primer extension products were analyzed by gel electrophoresis, and steady-state kinetics of the misincorporation of dAMP opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase η activity in the absence and presence of a DpC lesion. Molecular dynamics simulations were used to structurally explain the experimental findings. Our results suggest a possible role of local DNA sequence in promoting TLS-related mutational hot spots in the presence and absence of DpC lesions.

Rad4/XPC DNA damage sensor protein specifically binds to a photocleavable NPOM-DNA adduct, and this recognition is abolished upon photo-cleavage of NPOM.

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively ‘opening’ these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a ‘kinetic gating’ mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how ‘opening’ is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed ‘open’ undamaged DNA in solution and that such ‘opening’ can largely occur without one or the other of the β-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound ‘open’ DNA adopts multiple conformations in solution notwithstanding the DNA’s original structure or the β-hairpins. Molecular dynamics simulations reveal compensatory roles of the β-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA ‘opening’ of undamaged sites and the dynamic nature of ‘open’ DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.