Structure-dependent bypass of DNA interstrand crosslinks by translesion synthesis polymerases

DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen mustards. Multiple complex repair pathways of ICLs exist in humans that share translesion synthesis (TLS) past a partially processed ICL as a common step. We have generated site-specific major groove ICLs and studied the ability of Y-family polymerases and Pol zeta to bypass ICLs that induce different degrees of distortion in DNA. Two main factors influenced the efficiency of ICL bypass: the length of the dsDNA flanking the ICL and the length of the crosslink bridging two bases. Our study shows that ICLs can readily be bypassed by TLS polymerases if they are appropriately processed and that the structure of the ICL influences which polymerases are able to read through itope

XPG: Its products and biological roles

Xeroderma pigmetosum patients of the complementation group G are rare. One group of XP-G patients displays a rather mild and typical XP phenotype. Mutations in these patients interfere with the function of XPG in the nucleotide excision repair, where it has a structural role in the assembly of the preincision complex and a catalytic role in making the incision 3' to the damaged site in DNA. Another set of XP-G patient is much more severely affected, displaying combined symptoms of xeroderma pigmentosum and Cockayne syndrome, referred to as XP/CS complex. Although the molecular basis leading to the XP/CS complex has not yet been fully established, current evidence suggests that these patients suffer from a mild defect in transcription in addition to a repair defect. Here, the history of how the XPG gene was discovered, the biochemical properties of the XPG protein and the molecular defects found in XP-G patients and mouse models are reviewed.clos

Multistep damage recognition, pathway coordination and connections to transcription, damage signaling, chromatin structure, cancer and aging: Current perspectives on the nucleotide excision repair pathway Preface

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Alkyltransferase-like Proteins: Brokers Dealing with Alkylated DNA Bases

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Structure-Specific Endonucleases in DNA Repair

Nucleotide excision repair (NER) is an essential DNA repair pathway that addresses a variety of DNA lesions formed by UV light, environmental mutagens and agents used in cancer chemotherapy. NER involves the concerted action of over 30 proteins and operates by the sequential assembly of the involved factors at sites of damage. NER culminates in the removal of an oligonucleotide containing the damage by two incision reactions 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG. ERCC1-XPF and XPG are structure-specific endonucleases, a large class of enzymes involved in a variety of DNA repair, recombination and replication pathways. This review focuses on some of our recent work on how the activities of the ERCC1-XPF and XPG proteins are regulated to incise DNA only at defined times during the NER pathway, thereby preventing the formation of unwanted and cytotoxic DNA breaks by random incision of DNA. The implications of this work for our understanding of endonuclease enzymology, carcinogenesis and anti-tumor therapy are discussedclos

Using synthetic DNA interstrand crosslinks to elucidate repair pathways and identify new therapeutic targets for cancer chemotherapy

Many cancer chemotherapeutic agents form DNA interstrand crosslinks (ICLs), extremely cytotoxic lesions that form covalent bonds between two opposing DNA strands, blocking DNA replication and transcription. However, cellular responses triggered by ICLs can cause resistance in tumor cells, limiting the efficacy of such treatment. Here we discuss recent advances in our understanding of the mechanisms of ICL repair that cause this resistance. The recent development of strategies for the synthesis of site-specific ICLs greatly contributed to these insights. Key features of repair are similar for all ICLs, but there is increasing evidence that the specifics of lesion recognition and synthesis past ICLs by DNA polymerases are dependent upon the structure of ICLs. These new insights provide a basis for the improvement of antitumor therapy by targeting DNA repair pathways that lead to resistance to treatment with crosslinking agents.clos

New Synthetic Analogs of Nitrogen Mustard DNA Interstrand Cross-Links and Their Use to Study Lesion Bypass by DNA Polymerases

Nitrogen mustards are a widely used class of antitumor agents that exert their cytotoxic effects through the formation of DNA interstrand cross-links (ICLs). Despite being among the first antitumor agents used, the biological responses to NM ICLs remain only partially understood. We have previously reported the generation of NM ICL mimics by incorporation of ICL precursors into DNA using solid-phase synthesis at defined positions, followed by a double reductive amination reaction. However, the structure of these mimics deviated from the native NM ICLs. Using further development of our approach, we report a new class of NM ICL mimics that only differ from their native counterpart by substitution of dG with 7-deaza-dG at the ICL. Importantly, this approach allows for the synthesis of diverse NM ICLs, illustrated here with a mimic of the adduct formed by chlorambucil. We used the newly generated ICLs in reactions with replicative and translesion synthesis DNA polymerase to demonstrate their stability and utility for functional studies. These new NM ICLs will allow for the further characterization of the biological responses to this important class of antitumor agents

Synthesis and Molecular Modeling of a Nitrogen Mustard DNA Interstrand Crosslink

Nitrogen mustard reloaded: Over 60 years after nitrogen mustards (NMs) were the first agents used to treat tumors by chemotherapy, we provide a method to generate the main DNA adduct formed by NMs and validate them by using molecular dynamics simulations (see graphic). We are able to provide amounts that permit extensive structural and biological studies.clos

Crosslinking of nucleotide excision repair proteins with DNA containing photoreactive damages

Photoreactive DNA duplexes mimicking substrates of nucleotide excision repair (NER) system were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA. Photoreactive groups in one strand of DNA duplex (arylazido-dCMP or 4-thio-dUMP) were combined with anthracenyl-dCMP residue at the opposite strand to analyze contacts of NER factors with damaged and undamaged strands. Crosslinking of XPC-HR23B complex with photoreactive 48-mers results in modification of XPC subunit. XPC-HR23B did not crosslink with DNA duplex bearing bulky residues in both strands while this modification does not prevent interaction of DNA with XPA. The data on crosslinking of XPA and RPA with photoreactive DNA duplexes containing bulky group in one of the strands are in favor of XPA preference to interact with the damaged strand and RPA preference for the undamaged strand. The results support the understanding and set the stage for dynamically oriented experiments of how the pre-incision complex is formed in the early stage of NER.clos