Translesion DNA Synthesis: Little Fingers Teach …
T1 - CRL4Cdt2 E3 Ubiquitin Ligase Monoubiquitinates PCNA to Promote Translesion DNA Synthesis
Quantitative Measurement of Translesion DNA Synthesis …
AB - The Rev1-Polζ pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes. While it is widely believed that Rev1 plays a non-catalytic function in translesion synthesis, the role of its dCMP transferase activity remains uncertain. To determine the relevance of its catalytic function in translesion synthesis, we separated the Rev1 dCMP transferase activity from its non-catalytic function in yeast. This was achieved by mutating two conserved amino acid residues in the catalytic domain of Rev1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact. In this mutant strain, whereas translesion synthesis and mutagenesis of UV radiation were fully functional, those of a site-specific 1,N6-ethenoadenine were severely deficient. Specifically, the predominant A→G mutations resulting from C insertion opposite the lesion were abolished. Therefore, translesion synthesis and mutagenesis of 1,N6-ethenoadenine require the catalytic function of the Rev1 dCMP transferase, in contrast to those of UV lesions, which only require the non-catalytic function of Rev1. These results show that the catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.
AB - The mechanism and dynamics of translesion DNA synthesis were evaluated using primer/ templates containing a tetrahydrofuran moiety designed to mimic an abasic site. Steady-state kinetic analysis reveals that the T4 DNA polymerase preferentially incorporates dATP across from the abasic site with 100-fold higher efficiency than the other nucleoside triphosphates. Under steady-state conditions, the catalytic efficiency of dATP incorporation across from an abasic site is only 220-fold lower than that across from T. Surprisingly, misincorporation across from T is favored 4-6-fold versus replication across an abasic site, suggesting that the dynamics of the polymerization cycle are differentially affected by formation of aberrant base pairs as opposed to the lack of base-pairing capabilities afforded by the abasic site. Linear pre-steady-state time courses were obtained for the incorporation of any dNTP across from an abasic site, indicating that chemistry or a step prior to chemistry is rate-limiting for the polymerization cycle. Low elemental effects (
Translesion DNA Polymerases | SpringerLink
Although these adducts block DNA replication, cells have a mechanism allowing to continue replication by bypassing these adducts: translesion DNA synthesis (TLS).
Meindert Lamers (Leiden University Medical Center)
'STRUCTURAL FEATURES OF HIGH FIDELITY DNA REPLICATION'
Susan Lees-Miller (Arnie Charbonneau Cancer Institute)
'NON-REPAIR FUNCTIONS OF DNA-PKcs'
Karolin Luger (University of Colorado at Boulder)
'POLY (ADP-RIBOSE) POLYMERASE 1 IN DNA DAMAGE RECOGNITION'
Mauro Modesti (Cancer Research Center of Marseille)
'DYNAMICS AND MECHANICS OF DNA TETHERING BY CORE c-NHEJ FACTORS AND BY THE MRE11/RAD50/NBS1 COMPLEX'
Zachary Nagel (Harvard University)
'COMPREHENSIVE ANALYSIS OF DNA REPAIR CAPACITY IN NORMAL AND CANCEROUS CELLS'
Mark O'Connor (AstraZeneca)
'TARGETING THE REPLICATION STRESS RESPONSE IN CANCER'
John Pascal (Université de Montréal)
'STRUCTURAL BIOLOGY OF POLY(ADP-ribose) POLYMERASE ENZYMES'
Tanya Paull (University of Texas at Austin)
'THE Mre11-Rad50-Nbs1 COMPLEX AND REGULATION OF DNA REPAIR'
Phoebe Rice (University of Chicago)
'AN MCM-RELATED HELICASE AND OTHER REPLICATION-RELATED MACHINERY FOUND ON THE MOBILE ELEMENT BEHIND THE MRSA EPIDEMIC'
Andrej Sali (University of California, San Francisco)
'INTEGRATIVE STRUCTURE MODELING OF DNA-PKcs'
Orlando Scharer (Ulsan National Institute of Science and Technology)
'REPLICATIVE AND TRANSLESION SYNTHESIS DNA POLYMERASES IN ICL REPAIR'
Katharina Schlacher (MD Anderson Cancer Center)
'RAD51C IN DNA FORK PROTECTION AND STABILITY'
David Schriemer (University of Calgary)
'PROTEOMICS-GRADE STRATEGIES FOR INTEGRATIVE STRUCTURAL BIOLOGY'
Titia Sixma (NKI)
'STRUCTURAL BIOLOGY OF DNA MISMATCH REPAIR'
Maria Spies (University of Iowa)
'INHIBITORS OF THE RAD52-ssDNA INTERACTION'
Terence Strick (Institut Jacques Monod)
'SINGLE-MOLECULE ANALYSIS OF MULTICOMPONENT DNA REPAIR SYSTEMS'
Madalena Tarsounas (The CR-UK/MRC Oxford Institute for Radiation Oncology)
'WNT/b-CATENIN SIGNALING IN BRCA-DEFICIENT CELLS'
Translesion DNA synthesis in the context of cancer research
The replicative bypass of base damage in DNA (translesion DNA synthesis [TLS]) is a ubiquitous mechanism for relieving arrested DNA replication. The process requires multiple polymerase switching events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery. This review addresses recent advances in our understanding of DNA polymerase switching during TLS in bacteria such as E. coli and in lower and higher eukaryotes.
Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical posttranslational modification essential for DNA repair by translesion DNA synthesis (TLS). The Rad18 E3 ubiquitin ligase cooperates with the E2 Rad6 to monoubiquitinate PCNA in response to DNA damage. How PCNA is monoubiquitinated in unperturbed cells and whether this plays a role in the repair of DNA associated with replication is not known. We show that the CRL4Cdt2 E3 ubiquitin ligase complex promotes PCNA monoubiqutination in proliferating cells in the absence of external DNA damage independent of Rad18. PCNA monoubiquitination via CRL4Cdt2 is constitutively antagonized by the action of the ubiquitin-specific protease 1 (USP1). In vitro, CRL4Cdt2 monoubiquitinates PCNA at Lys164, the same residue that is monoubiquitinated by Rad18. Significantly, CRL4Cdt2 is required for TLS in nondamaged cells via a mechanism that is dependent on PCNA monoubiquitination. We propose that CRL4Cdt2 regulates PCNA-dependent TLS associated with stresses accompanying DNA replication.
structure and mechanism of DNA synthesis by Polη ..
Error-Prone Translesion DNA Synthesis by Escherichia …
Translesion DNA Synthesis: ..
This process is termed translesion DNA synthesis ..
Specialised translesion synthesis DNA polymerases can replicate DNA over such lesions to …
Translesion DNA synthesis-assisted non-homologous …
Translesion DNA synthesis-assisted non-homologous end-joining of complex ..
ASMscience | Translesion DNA Synthesis
Monoubiquitination of PCNA at lys 164 (PCNA-Ub) appears to be necessary to make it capable of stimulating TLS by translesion pols in the cell . Therefore we investigated whether monoubiquitination of PCNA could affect gap directed TLS of an AP site by pol λ in the presence of pol ε, RPA and RFC. We directly compared TLS by this polymerase on the 4 nucleotides gapped circular DNA when increasing amount of either unmodified or monoubiquitinated PCNA were present. The result of such experiment is shown in and quantified in 5B. As expected, efficient TLS by pol λ is achieved already in absence of PCNA (lane 4). As it can be seen, addition of either increasing amount of unmodified or monoubiquitinated PCNA did not affect the extent of the TLS reaction (compare lanes 5–7 to lanes 8–10), indicating that monoubiquitination of PCNA does not play a significant role in the TLS of gapped circular DNA by pol λ.
The latter process is referred to as Translesion DNA Synthesis ..
Crystallographic analysis has captured a Y-family polymerase synthesizing across an abasic site, providing insight into the mechanisms of DNA damage tolerance and mutation.
Mechanism of Mammalian Translesion DNA synthesis
AB - Anti-cancer agents exert therapeutic effects by damaging DNA. Unfortunately, DNA polymerases can effectively replicate the formed DNA lesions to cause drug resistance and create more aggressive cancers. To understand this process at the cellular level, we developed an artificial nucleoside that visualizes the replication of damaged DNA to identify cells that acquire drug resistance through this mechanism. Visualization is achieved using "click" chemistry to covalently attach azide-containing fluorophores to the ethynyl group present on the nucleoside analog after its incorporation opposite damaged DNA. Flow cytometry and microscopy techniques demonstrate that the extent of nucleotide incorporation into genomic DNA is enhanced by treatment with DNA damaging agents. In addition, this nucleoside analog inhibits translesion DNA synthesis and synergizes the therapeutic activity of certain anticancer agents such as temozolomide. The combined diagnostic and therapeutic activities of this synthetic nucleoside analog represent a new paradigm in personalized medicine.
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