Variable Inhibition of DNA Unwinding Rates Catalyzed by the SARS-CoV-2 Helicase Nsp13 by Structurally Distinct Single DNA Lesions.

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 (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs 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 kobs 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.

Differing structures and dynamics of two photolesions portray verification differences by the human XPD helicase.

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.

Mechanism of lesion verification by the human XPD helicase in nucleotide excision repair.

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.

Molecular dynamics simulations reveal how H3K56 acetylation impacts nucleosome structure to promote DNA exposure for lesion sensing.

The first order of DNA packaging is the nucleosome with the DNA wrapped around the histone octamer. This leaves the nucleosomal DNA with access restrictions, which impose a significant barrier to repair of damaged DNA. The efficiency of DNA repair has been related to nucleosome structure and chromatin status, which is modulated in part by post-translational modifications (PTMs) of histones. Numerous studies have suggested a role for acetylation of lysine at position 56 of the H3 histone (H3K56ac) in various DNA transactions, including the response to DNA damage and its association with human cancer. Biophysical studies have revealed that H3K56ac increases DNA accessibility by facilitating spontaneous and transient unwrapping motions of the DNA ends. However, how this acetylation mark modulates nucleosome structure and dynamics to promote accessibility to the damaged DNA for repair factors and other proteins is still poorly understood. Here, we utilize approximately 5-6 microseconds of atomistic molecular dynamics simulations to delineate the impact of H3K56 acetylation on the nucleosome structure and dynamics, and to elucidate how these nucleosome properties are further impacted when a bulky benzo[a]pyrene-derived DNA lesion is placed near the acetylation site. Our findings reveal that H3K56ac alone induces considerable disturbance to the histone-DNA/histone-histone interactions, and amplifies the distortions imposed by the presence of the lesion. Our work highlights the important role of H3K56 acetylation in response to DNA damage and depicts how access to DNA lesions by the repair machinery can be facilitated within the nucleosome via a key acetylation event.

Translesion Synthesis Past 5-Formylcytosine-Mediated DNA-Peptide Cross-Links by hPolη Is Dependent on the Local DNA Sequence.

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.

5-Formylcytosine-induced DNA-peptide cross-links reduce transcription efficiency, but do not cause transcription errors in human cells.

5-Formylcytosine (5fC) is an endogenous epigenetic DNA mark introduced via enzymatic oxidation of 5-methyl-dC in DNA. We and others recently reported that 5fC can form reversible DNA-protein conjugates with histone proteins, likely contributing to regulation of nucleosomal organization and gene expression. The protein component of DNA-protein cross-links can be proteolytically degraded, resulting in smaller DNA-peptide cross-links. Unlike full-size DNA-protein cross-links that completely block replication and transcription, DNA-peptide cross-links can be bypassed by DNA and RNA polymerases and can potentially be repaired via the nucleotide excision repair (NER) pathway. In the present work, we constructed plasmid molecules containing reductively stabilized, site-specific 5fC-polypeptide lesions and employed a quantitative MS-based assay to assess their effects on transcription in cells. Our results revealed that the presence of DNA-peptide cross-link significantly inhibits transcription in human HEK293T cells but does not induce transcription errors. Furthermore, transcription efficiency was similar in WT and NER-deficient human cell lines, suggesting that the 5fC-polypeptide lesion is a weak substrate for NER. This finding was confirmed by in vitro NER assays in cell-free extracts from human HeLa cells, suggesting that another mechanism is required for 5fC-polypeptide lesion removal. In summary, our findings indicate that 5fC-mediated DNA-peptide cross-links dramatically reduce transcription efficiency, are poor NER substrates, and do not cause transcription errors.

Transcriptional Bypass of DNA-Protein and DNA-Peptide Conjugates by T7 RNA Polymerase.

DNA-protein cross-links (DPCs) are unusually bulky DNA adducts that block the access of proteins to DNA and interfere with gene expression, replication, and repair. We previously described DPC formation at the N7-guanine position of DNA in human cells treated with antitumor nitrogen mustards and platinum compounds and have shown that DPCs can form endogenously at DNA epigenetic mark 5-formyl-dC. However, insufficient information is available about the effects of these structurally distinct DPCs on transcription. In the present work, we employ a combination of in vitro assays, mass spectrometry, and molecular dynamics simulations to examine the ability of phage T7 RNA polymerase to bypass DPCs conjugated to the C7 position of 7-deaza-dG and the C5 position of dC. These model adducts represent endogenous DPCs induced by exposure to antitumor drugs and formed at epigenetics DNA marks, respectively. Our results reveal that DPCs containing full-length proteins significantly inhibit in vitro transcription by T7 RNA polymerase, while short DNA-peptide cross-links (DpCs) are bypassed. DpCs conjugated to the C7 position of 7-deaza-dG are transcribed with high fidelity, while the same polypeptides attached to the C5 position of dC induce transcription errors. Molecular dynamics simulations of DpCs conjugated either to the C5 atom of dC or the C7 position of 7-deaza-dG on the template strand in T7 RNA polymerase explain how the conjugated peptide can be accommodated in the narrow major groove of the DNA-RNA hybrid and how the modified dC can form a stable mismatch with the incoming ATP in the polymerase active site, allowing for transcriptional mutagenesis.

Rotational and translational positions determine the structural and dynamic impact of a single ribonucleotide incorporated in the nucleosome.

Ribonucleotides misincorporated by replicative DNA polymerases are by far the most common DNA lesion. The presence of ribonucleotides in DNA is associated with genome instability, causing replication stress, chromosome fragility, gross chromosomal rearrangements, and other mutagenic events. Furthermore, nucleosome and chromatin assembly as well as nucleosome positioning are affected by the presence of ribonucleotides. Notably, nucleosome formation is significantly reduced by a single ribonucleotide. Single ribonucleotides are primarily removed from DNA by the ribonucleotide excision repair (RER) pathway via the RNase H2 enzyme, which incises the DNA backbone on the 5'-side of the ribonucleotide. While the structural implications of a single ribonucleotide in free duplex DNA have been well studied, how a single ribonucleotide embedded in nucleosomal DNA impacts nucleosome structure and dynamics, and the possible consequent impact on RER, have not been explored. We have carried out 3.5 µs molecular dynamics simulations of a single ribonucleotide incorporated at various translational and rotational positions in a nucleosome core particle. We find that the presence of the 2'-OH group on the ribose impacts the local conformation and dynamics of both the ribonucleotide and nearby DNA nucleotides as well as their interactions with histones; the nature of these disturbances depends on the rotational and translational setting, including whether the ribose faces toward or away from the histones. The ribonucleotide's preferred C3'-endo pucker is stabilized by interactions with the histones, and furthermore the ribonucleotide can cause dynamic local duplex disturbance involving an abnormal C3'-endo population of the adjacent deoxyribose pucker, minor groove opening, ruptured Watson-Crick pairing, and duplex unwinding that are governed by translation-dependent histone-nucleotide interactions. Possible effects of these disturbances on RER are considered.

5-Formylcytosine mediated DNA-protein cross-links block DNA replication and induce mutations in human cells.

5-Formylcytosine (5fC) is an epigenetic DNA modification introduced via TET protein-mediated oxidation of 5-methyl-dC. We recently reported that 5fC form reversible DNA-protein conjugates (DPCs) with histone proteins in living cells (Ji et al. (2017) Angew. Chem. Int. Ed., 56:14130-14134). We now examined the effects of 5fC mediated DPCs on DNA replication. Synthetic DNA duplexes containing site-specific DPCs between 5fC and lysine-containing proteins and peptides were subjected to primer extension experiments in the presence of human translesion synthesis DNA polymerases η and κ. We found that DPCs containing histones H2A or H4 completely inhibited DNA replication, but the replication block was removed when the proteins were subjected to proteolytic digestion. Cross-links to 11-mer or 31-mer peptides were bypassed by both polymerases in an error-prone manner, inducing targeted C→T transitions and -1 deletions. Similar types of mutations were observed when plasmids containing 5fC-peptide cross-links were replicated in human embryonic kidney (HEK) 293T cells. Molecular simulations of the 11-mer peptide-dC cross-links bound to human polymerases η and κ revealed that the peptide fits well on the DNA major groove side, and the modified dC forms a stable mismatch with incoming dATP via wobble base pairing in the polymerase active site.

Synergistic effects of H3 and H4 nucleosome tails on structure and dynamics of a lesion-containing DNA: Binding of a displaced lesion partner base to the H3 tail for GG-NER recognition.

How DNA lesions in nucleosomes are recognized for global genome nucleotide excision repair (GG-NER) remains poorly understood, and the roles that histone tails may play remains to be established. Histone H3 and H4 N-terminal tails are of particular interest as their acetylation states are important in regulating nucleosomal functions in transcription, replication and repair. In particular the H3 tail has been the focus of recent attention as a site for the interaction with XPC, the GG-NER lesion recognition factor. Here we have investigated how the structure and dynamics of the DNA lesion cis-B[a]P-dG, derived from the environmental carcinogen benzo[a]pyrene (B[a]P), is impacted by the presence of flanking H3 and H4 tails. This lesion is well-repaired by GG-NER, and adopts a base-displaced/intercalated conformation in which the lesion partner C is displaced into the major groove. We used molecular dynamics simulations to obtain structural and dynamic characterizations for this lesion positioned in nucleosomal DNA so that it is bracketed by the H3 and H4 tails. The H4 tail was studied in unacetylated and acetylated states, while the H3 tail was unacetylated, its state when it binds XPC (Kakumu, Nakanishi et al., 2017). Our results reveal that upon acetylation, the H4 tail is released from the DNA surface; the H3 tail then forms a pocket that induces flipping and capture of the displaced lesion partner base C. This reveals synergistic effects of the behavior of the two tails. We hypothesize that the dual capability of the H3 tail to sense the displaced lesion partner base and to bind XPC could foster recognition of this lesion by XPC for initiation of GG-NER in nucleosomes.

Nucleosome Histone Tail Conformation and Dynamics: Impacts of Lysine Acetylation and a Nearby Minor Groove Benzo[a]pyrene-Derived Lesion.

Histone tails in nucleosomes play critical roles in regulation of many biological processes, including chromatin compaction, transcription, and DNA repair. Moreover, post-translational modifications, notably lysine acetylation, are crucial to these functions. While the tails have been intensively studied, how the structures and dynamics of tails are impacted by the presence of a nearby bulky DNA lesion is a frontier research area, and how these properties are impacted by tail lysine acetylation remains unexplored. To obtain molecular insight, we have utilized all atom 3 µs molecular dynamics simulations of nucleosome core particles (NCPs) to determine the impact of a nearby DNA lesion, 10S (+)-trans-anti-B[a]P-N2-dG-the major adduct derived from the procarcinogen benzo[a]pyrene-on H2B tail behavior in unacetylated and acetylated states. We similarly studied lesion-free NCPs to investigate the normal properties of the H2B tail in both states. In the lesion-free NCPs, charge neutralization upon lysine acetylation causes release of the tail from the DNA. When the lesion is present, it stably engulfs part of the nearby tail, impairing the interactions between DNA and tail. With the tail in an acetylated state, the lesion still interacts with part of it, although unstably. The lesion's partial entrapment of the tail should hinder the tail from interacting with other nucleosomes, and other proteins such as acetylases, deacetylases, and acetyl-lysine binding proteins, and thus disrupt critical tail-governed processes. Hence, the lesion would impede tail functions modulated by acetylation or deacetylation, causing aberrant chromatin structures and impaired biological transactions such as transcription and DNA repair.

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases.

DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases η and κ. DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50-100-fold lower than for native G. Both human polymerase (hPol) κ and hPol η inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol η. Molecular dynamics simulation of an hPol κ ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.

Entrapment of a Histone Tail by a DNA Lesion in a Nucleosome Suggests the Lesion Impacts Epigenetic Marking: A Molecular Dynamics Study.

Errors in epigenetic markings are associated with human diseases, including cancer. We have used molecular dynamics simulations of a nucleosome containing the 10S (+)-trans-anti-B[a]P-N(2)-dG lesion, derived from the environmental pro-carcinogen benzo[a]pyrene, to elucidate the impact of the lesion on the structure and dynamics of a nearby histone N-terminal tail. Our results show that a lysine-containing part of this H2B tail that is subject to post-translational modification is engulfed by the enlarged DNA minor groove imposed by the lesion. The tail entrapment suggests that epigenetic markings could be hampered by this lesion, thereby impacting critical cellular functions, including transcription and repair.

Dynamic Water-Mediated Hydrogen Bonding in a Collagen Model Peptide.

In the canonical (G-X-Y)(n) sequence of the fibrillar collagen triple helix, stabilizing direct interchain hydrogen bonding connects neighboring chains. Mutations of G can disrupt these interactions and are linked to connective tissue diseases. Here we integrate computational approaches with nuclear magnetic resonance (NMR) to obtain a dynamic view of hydrogen bonding distributions in the (POG)(4)(-)(POA)-(POG)(5) peptide, showing that the solution conformation, dynamics, and hydrogen bonding deviate from the reported X-ray crystal structure in many aspects. The simulations and NMR data provide clear evidence of inequivalent environments in the three chains. Molecular dynamics (MD) simulations indicate direct interchain hydrogen bonds in the leading chain, water bridges in the middle chain, and nonbridging waters in the trailing chain at the G → A substitution site. Theoretical calculations of NMR chemical shifts using a quantum fragmentation procedure can account for the unusual downfield NMR chemical shifts at the substitution sites and are used to assign the resonances to the individual chains. The NMR and MD data highlight the sensitivity of amide shifts to changes in the acceptor group from peptide carbonyls to water. The results are used to interpret solution NMR data for a variety of glycine substitutions and other sequence triplet interruptions to provide new connections between collagen sequences, their associated structures, dynamical behavior, and their ability to recognize collagen receptors.