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[]pyrene (B[]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6-4) pyrimidone (6-4PP) photolesion.

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.

Inhibition of RecQ Helicase Activity by Structurally Distinct DNA Lesions: Structure-Function Relationships.

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 RecQ helicase as a model system, and various conformationally defined DNA lesions, the unwinding rate constants = , and processivities = (k) were determined ( unwinding rate constant; , helicase-DNA dissociation rate constant).

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.

Impact of DNA sequences on DNA 'opening' by the Rad4/XPC nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions to initiate nucleotide excision repair (NER). However, NER propensities among lesions vary widely and repair-resistant lesions are persistent and thus highly mutagenic. Rad4 recognizes repair-proficient lesions by unwinding ('opening') the damaged DNA site.

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.

Back to the bones: do muscle area assessment techniques predict functional evolution across a macroevolutionary radiation?

Measures of attachment or accommodation area on the skeleton are a popular means of rapidly generating estimates of muscle proportions and functional performance for use in large-scale macroevolutionary studies. Herein, we provide the first evaluation of the accuracy of these muscle area assessment (MAA) techniques for estimating muscle proportions, force outputs and bone loading in a comparative macroevolutionary context using the rodent masticatory system as a case study. We find that MAA approaches perform poorly, yielding large absolute errors in muscle properties, bite force and particularly bone stress.

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.

Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein.

Biomolecular structural changes upon binding/unbinding are key to their functions. However, characterization of such dynamical processes is difficult as it requires ways to rapidly and specifically trigger the assembly/disassembly as well as ways to monitor the resulting changes over time. Recently, various chemical strategies have been developed to use light to trigger changes in oligonucleotide structures, and thereby their activities.

Evolutionary biomechanics: hard tissues and soft evidence?

Biomechanical modelling is a powerful tool for quantifying the evolution of functional performance in extinct animals to understand key anatomical innovations and selective pressures driving major evolutionary radiations. However, the fossil record is composed predominantly of hard parts, forcing palaeontologists to reconstruct soft tissue properties in such models. Rarely are these reconstruction approaches validated on extant animals, despite soft tissue properties being highly determinant of functional performance.

Tethering-facilitated DNA 'opening' and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein.

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.

The DNA damage-sensing NER repair factor XPC-RAD23B does not recognize bulky DNA lesions with a missing nucleotide opposite the lesion.

The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding.

Variable impact of conformationally distinct DNA lesions on nucleosome structure and dynamics: Implications for nucleotide excision repair.

The packaging of DNA in nucleosomes presents a barrier for biological transactions including replication, transcription and repair. However, despite years of research, how the DNA is freed from the histone proteins and thereby allows the molecular machines to access the DNA remains poorly understood. We are interested in global genomic nucleotide excision repair (GG-NER).

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.

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.

Structure and mechanism of pyrimidine-pyrimidone (6-4) photoproduct recognition by the Rad4/XPC nucleotide excision repair complex.

Failure in repairing ultraviolet radiation-induced DNA damage can lead to mutations and cancer. Among UV-lesions, the pyrimidine-pyrimidone (6-4) photoproduct (6-4PP) is removed from the genome much faster than the cyclobutane pyrimidine dimer (CPD), owing to the more efficient recognition of 6-4PP by XPC-RAD23B, a key initiator of global-genome nucleotide excision repair (NER). Here, we report a crystal structure of a Rad4-Rad23 (yeast XPC-Rad23B ortholog) bound to 6-4PP-containing DNA and 4-μs molecular dynamics (MD) simulations examining the initial binding of Rad4 to 6-4PP or CPD.

Nucleotide Excision Repair and Impact of Site-Specific 5',8-Cyclopurine and Bulky DNA Lesions on the Physical Properties of Nucleosomes.

The nonbulky 5',8-cyclopurine DNA lesions (cP) and the bulky, benzo[ a]pyrene diol epoxide-derived stereoisomeric cis- and trans- N-guanine adducts (BPDE-dG) are good substrates of the human nucleotide excision repair (NER) mechanism. These DNA lesions were embedded at the In or Out rotational settings near the dyad axis in nucleosome core particles reconstituted either with native histones extracted from HeLa cells (HeLa-NCP) or with recombinant histones (Rec-NCP). The cP lesions are completely resistant to NER in human HeLa cell extracts.

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.

Lesion Sensing during Initial Binding by Yeast XPC/Rad4: Toward Predicting Resistance to Nucleotide Excision Repair.

Nucleotide excision repair (NER) excises a variety of environmentally derived DNA lesions. However, NER efficiencies for structurally different DNA lesions can vary by orders of magnitude; yet the origin of this variance is poorly understood. Our goal is to develop computational strategies that predict and identify the most hazardous, repair-resistant lesions from the plethora of such adducts.

Molecular basis for damage recognition and verification by XPC-RAD23B and TFIIH in nucleotide excision repair.

Global genome nucleotide excision repair (GG-NER) is the main pathway for the removal of bulky lesions from DNA and is characterized by an extraordinarily wide substrate specificity. Remarkably, the efficiency of lesion removal varies dramatically and certain lesions escape repair altogether and are therefore associated with high levels of mutagenicity. Central to the multistep mechanism of damage recognition in NER is the sensing of lesion-induced thermodynamic and structural alterations of DNA by the XPC-RAD23B protein and the verification of the damage by the transcription/repair factor TFIIH.

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.

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.

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging.